99 results on '"Matthew J. Belousoff"'
Search Results
2. A Structurally Characterized Staphylococcus aureus Evolutionary Escape Route from Treatment with the Antibiotic Linezolid
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Laura Perlaza-Jiménez, Kher-Shing Tan, Sarah J. Piper, Rachel M. Johnson, Rebecca S. Bamert, Christopher J. Stubenrauch, Alexander Wright, David Lupton, Trevor Lithgow, and Matthew J. Belousoff
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antimicrobial resistance ,MRSA ,antibiotics ,ribosomes ,cryoEM ,Staphylococcus aureus ,Microbiology ,QR1-502 - Abstract
ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen that presents great health concerns. Treatment requires the use of last-line antibiotics, such as members of the oxazolidinone family, of which linezolid is the first member to see regular use in the clinic. Here, we report a short time scale selection experiment in which strains of MRSA were subjected to linezolid treatment. Clonal isolates which had evolved a linezolid-resistant phenotype were characterized by whole-genome sequencing. Linezolid-resistant mutants were identified which had accumulated mutations in the ribosomal protein uL3. Multiple clones which had two mutations in uL3 exhibited resistance to linezolid, 2-fold higher than the clinical breakpoint. Ribosomes from this strain were isolated and subjected to single-particle cryo-electron microscopic analysis and compared to the ribosomes from the parent strain. We found that the mutations in uL3 lead to a rearrangement of a loop that makes contact with Helix 90, propagating a structural change over 15 Å away. This distal change swings nucleotide U2504 into the binding site of the antibiotic, causing linezolid resistance. IMPORTANCE Antibiotic resistance poses a critical problem to human health and decreases the utility of these lifesaving drugs. Of particular concern is the “superbug” methicillin-resistant Staphylococcus aureus (MRSA), for which treatment of infection requires the use of last-line antibiotics, including linezolid. In this paper, we characterize the atomic rearrangements which the ribosome, the target of linezolid, undergoes during its evolutionary journey toward becoming drug resistant. Using cryo-electron microscopy, we describe a particular molecular mechanism which MRSA uses to become resistant to linezolid.
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- 2022
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3. Structure and dynamics of the active Gs-coupled human secretin receptor
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Maoqing Dong, Giuseppe Deganutti, Sarah J. Piper, Yi-Lynn Liang, Maryam Khoshouei, Matthew J. Belousoff, Kaleeckal G. Harikumar, Christopher A. Reynolds, Alisa Glukhova, Sebastian G. B. Furness, Arthur Christopoulos, Radostin Danev, Denise Wootten, Patrick M. Sexton, and Laurence J. Miller
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Science - Abstract
The class B secretin GPCR (SecR) has broad physiological effects, with target potential for treatment of metabolic and cardiovascular disease. Here, authors present a cryo-EM structure and biochemical studies of secretin binding to the SecR:Gs complex which show that interactions between peptide and receptor were dynamic.
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- 2020
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4. The architecture and stabilisation of flagellotropic tailed bacteriophages
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Joshua M. Hardy, Rhys A. Dunstan, Rhys Grinter, Matthew J. Belousoff, Jiawei Wang, Derek Pickard, Hariprasad Venugopal, Gordon Dougan, Trevor Lithgow, and Fasséli Coulibaly
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Science - Abstract
Flagellotropic phages spin down flagella to reach the bacterial surface and must withstand remarkable drag forces. Here authors show how two nested sets of chainmail stabilise the viral head and a beta-hairpin regulates the formation of the robust yet pliable tail, characteristic of siphoviruses.
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- 2020
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5. Mechanistic Insights into the Capsule-Targeting Depolymerase from a Klebsiella pneumoniae Bacteriophage
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Rhys A. Dunstan, Rebecca S. Bamert, Matthew J. Belousoff, Francesca L. Short, Christopher K. Barlow, Derek J. Pickard, Jonathan J. Wilksch, Ralf B. Schittenhelm, Richard A. Strugnell, Gordon Dougan, and Trevor Lithgow
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Klebsiella ,bacteriophages ,capsular polysaccharide ,cryo-EM ,depolymerase ,Microbiology ,QR1-502 - Abstract
ABSTRACT The production of capsular polysaccharides by Klebsiella pneumoniae protects the bacterial cell from harmful environmental factors such as antimicrobial compounds and infection by bacteriophages (phages). To bypass this protective barrier, some phages encode polysaccharide-degrading enzymes referred to as depolymerases to provide access to cell surface receptors. Here, we characterized the phage RAD2, which infects K. pneumoniae strains that produce the widespread, hypervirulence-associated K2-type capsular polysaccharide. Using transposon-directed insertion sequencing, we have shown that the production of capsule is an absolute requirement for efficient RAD2 infection by serving as a first-stage receptor. We have identified the depolymerase responsible for recognition and degradation of the capsule, determined that the depolymerase forms globular appendages on the phage virion tail tip, and present the cryo-electron microscopy structure of the RAD2 capsule depolymerase at 2.7-Å resolution. A putative active site for the enzyme was identified, comprising clustered negatively charged residues that could facilitate the hydrolysis of target polysaccharides. Enzymatic assays coupled with mass spectrometric analyses of digested oligosaccharide products provided further mechanistic insight into the hydrolase activity of the enzyme, which, when incubated with K. pneumoniae, removes the capsule and sensitizes the cells to serum-induced killing. Overall, these findings expand our understanding of how phages target the Klebsiella capsule for infection, providing a framework for the use of depolymerases as antivirulence agents against this medically important pathogen. IMPORTANCE Klebsiella pneumoniae is a medically important pathogen that produces a thick protective capsule that is essential for pathogenicity. Phages are natural predators of bacteria, and many encode diverse “capsule depolymerases” which specifically degrade the capsule of their hosts, an exploitable trait for potential therapies. We have determined the first structure of a depolymerase that targets the clinically relevant K2 capsule and have identified its putative active site, providing hints to its mechanism of action. We also show that Klebsiella cells treated with a recombinant form of the depolymerase are stripped of capsule, inhibiting their ability to grow in the presence of serum, demonstrating the anti-infective potential of these robust and readily producible enzymes against encapsulated bacterial pathogens such as K. pneumoniae.
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- 2021
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6. Structure and dynamics of semaglutide- and taspoglutide-bound GLP-1R-Gs complexes
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Xin Zhang, Matthew J. Belousoff, Yi-Lynn Liang, Radostin Danev, Patrick M. Sexton, and Denise Wootten
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glucagon-like peptide-1 receptor ,GLP-1R ,cryoelectron microscopy ,GPCRs ,GPCR dynamics ,semaglutide ,Biology (General) ,QH301-705.5 - Abstract
Summary: The glucagon-like peptide-1 receptor (GLP-1R) regulates insulin secretion, carbohydrate metabolism, and appetite and is an important target for treatment of type 2 diabetes and obesity. Multiple GLP-1R agonists have entered into clinical trials, with some, such as semaglutide, progressing to approval. Others, including taspoglutide, failed due to the high incidence of side effects or insufficient efficacy. GLP-1R agonists have a broad spectrum of signaling profiles, but molecular understanding is limited by a lack of structural information on how different agonists engage with the GLP-1R. Here, we report cryoelectron microscopy (cryo-EM) structures and cryo-EM 3D variability analysis of semaglutide- and taspoglutide-bound GLP-1R-Gs protein complexes. These reveal similar peptide interactions to GLP-1 but different motions within the receptor and bound peptides, providing insights into the molecular determinants of GLP-1R peptide engagement.
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- 2021
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7. Global Trends in Proteome Remodeling of the Outer Membrane Modulate Antimicrobial Permeability in Klebsiella pneumoniae
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Andrea Rocker, Jake A. Lacey, Matthew J. Belousoff, Jonathan J. Wilksch, Richard A. Strugnell, Mark R. Davies, and Trevor Lithgow
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antimicrobial resistance ,porin ,OmpK37 ,beta-barrel ,carbapenem ,carbapenems ,Microbiology ,QR1-502 - Abstract
ABSTRACT In Gram-negative bacteria, the permeability of the outer membrane governs rates of antibiotic uptake and thus the efficacy of antimicrobial treatment. Hydrophilic drugs like β-lactam antibiotics depend on diffusion through pore-forming outer membrane proteins to reach their intracellular targets. In this study, we investigated the distribution of porin genes in more than 2,700 Klebsiella isolates and found a widespread loss of OmpK35 functionality, particularly in those strains isolated from clinical environments. Using a defined set of outer-membrane-remodeled mutants, the major porin OmpK35 was shown to be largely responsible for β-lactam permeation. Sequence similarity network analysis characterized the porin protein subfamilies and led to discovery of a new porin family member, OmpK38. Structure-based comparisons of OmpK35, OmpK36, OmpK37, OmpK38, and PhoE showed near-identical pore frameworks but defining differences in the sequence characteristics of the extracellular loops. Antibiotic sensitivity profiles of isogenic Klebsiella pneumoniae strains, each expressing a different porin as its dominant pore, revealed striking differences in the antibiotic permeability characteristics of each channel in a physiological context. Since K. pneumoniae is a nosocomial pathogen with high rates of antimicrobial resistance and concurrent mortality, these experiments elucidate the role of porins in conferring specific drug-resistant phenotypes in a global context, informing future research to combat antimicrobial resistance in K. pneumoniae. IMPORTANCE Klebsiella pneumoniae is a pathogen of humans with high rates of mortality and a recognized global rise in incidence of carbapenem-resistant K. pneumoniae (CRKP). The outer membrane of K. pneumoniae forms a permeability barrier that modulates the ability of antibiotics to reach their intracellular target. OmpK35, OmpK36, OmpK37, OmpK38, PhoE, and OmpK26 are porins in the outer membrane of K. pneumoniae, demonstrated here to have a causative relationship to drug resistance phenotypes in a physiological context. The data highlight that currently trialed combination treatments with a carbapenem and β-lactamase inhibitors could be effective on porin-deficient K. pneumoniae. Together with structural data, the results reveal the role of outer membrane proteome remodeling in antimicrobial resistance of K. pneumoniae and point to the role of extracellular loops, not channel parameters, in drug permeation. This significant finding warrants care in the development of phage therapies for K. pneumoniae infections, given the way porin expression will be modulated to confer phage-resistant—and collateral drug-resistant—phenotypes in K. pneumoniae.
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- 2020
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8. Structural Basis of Type 2 Secretion System Engagement between the Inner and Outer Bacterial Membranes
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Iain D. Hay, Matthew J. Belousoff, and Trevor Lithgow
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Pseudomonas aeruginosa ,T2SS ,protein secretion ,protein secretion system ,secretin ,Microbiology ,QR1-502 - Abstract
ABSTRACT Sophisticated nanomachines are used by bacteria for protein secretion. In Gram-negative bacteria, the type 2 secretion system (T2SS) is composed of a pseudopilus assembly platform in the inner membrane and a secretin complex in the outer membrane. The engagement of these two megadalton-sized complexes is required in order to secrete toxins, effectors, and hydrolytic enzymes. Pseudomonas aeruginosa has at least two T2SSs, with the ancestral nanomachine having a secretin complex composed of XcpQ. Until now, no high-resolution structural information was available to distinguish the features of this Pseudomonas-type secretin, which varies greatly in sequence from the well-characterized Klebsiella-type and Vibrio-type secretins. We have purified the ~1-MDa secretin complex and analyzed it by cryo-electron microscopy. Structural comparisons with the Klebsiella-type secretin complex revealed a striking structural homology despite the differences in their sequence characteristics. At 3.6-Å resolution, the secretin complex was found to have 15-fold symmetry throughout the membrane-embedded region and through most of the domains in the periplasm. However, the N1 domain and N0 domain were not well ordered into this 15-fold symmetry. We suggest a model wherein this disordering of the subunit symmetry for the periplasmic N domains provides a means to engage with the 6-fold symmetry in the inner membrane platform, with a metastable engagement that can be disrupted by substrate proteins binding to the region between XcpP, in the assembly platform, and the XcpQ secretin. IMPORTANCE How the outer membrane and inner membrane components of the T2SS engage each other and yet can allow for substrate uptake into the secretin chamber has challenged the protein transport field for some time. This vexing question is of significance because the T2SS collects folded protein substrates in the periplasm for transport out of the bacterium and yet must discriminate these few substrate proteins from all the other hundred or so folded proteins in the periplasm. The structural analysis here supports a model wherein substrates must compete against a metastable interaction between XcpP in the assembly platform and the XcpQ secretin, wherein only structurally encoded features in the T2SS substrates compete well enough to disrupt XcpQ-XcpP for entry into the XcpQ chamber, for secretion across the outer membrane.
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- 2017
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9. Structural Basis for Linezolid Binding Site Rearrangement in the Staphylococcus aureus Ribosome
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Matthew J. Belousoff, Zohar Eyal, Mazdak Radjainia, Tofayel Ahmed, Rebecca S. Bamert, Donna Matzov, Anat Bashan, Ella Zimmerman, Satabdi Mishra, David Cameron, Hans Elmlund, Anton Y. Peleg, Shashi Bhushan, Trevor Lithgow, and Ada Yonath
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staphylococcus ,antibiotic resistance ,ribosomal mutations ,Microbiology ,QR1-502 - Abstract
ABSTRACT An unorthodox, surprising mechanism of resistance to the antibiotic linezolid was revealed by cryo-electron microscopy (cryo-EM) in the 70S ribosomes from a clinical isolate of Staphylococcus aureus. This high-resolution structural information demonstrated that a single amino acid deletion in ribosomal protein uL3 confers linezolid resistance despite being located 24 Å away from the linezolid binding pocket in the peptidyl-transferase center. The mutation induces a cascade of allosteric structural rearrangements of the rRNA that ultimately results in the alteration of the antibiotic binding site. IMPORTANCE The growing burden on human health caused by various antibiotic resistance mutations now includes prevalent Staphylococcus aureus resistance to last-line antimicrobial drugs such as linezolid and daptomycin. Structure-informed drug modification represents a frontier with respect to designing advanced clinical therapies, but success in this strategy requires rapid, facile means to shed light on the structural basis for drug resistance (D. Brown, Nat Rev Drug Discov 14:821–832, 2015, https://doi.org/10.1038/nrd4675 ). Here, detailed structural information demonstrates that a common mechanism is at play in linezolid resistance and provides a step toward the redesign of oxazolidinone antibiotics, a strategy that could thwart known mechanisms of linezolid resistance.
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- 2017
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10. Cholesterol-dependent dynamic changes in the conformation of the type 1 cholecystokinin receptor affect ligand binding and G protein coupling.
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Kaleeckal G Harikumar, Peishen Zhao, Brian P Cary, Xiaomeng Xu, Aditya J Desai, Maoqing Dong, Jesse I Mobbs, Chirine Toufaily, Sebastian G B Furness, Arthur Christopoulos, Matthew J Belousoff, Denise Wootten, Patrick M Sexton, and Laurence J Miller
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Biology (General) ,QH301-705.5 - Abstract
Development of optimal therapeutics for disease states that can be associated with increased membrane cholesterol requires better molecular understanding of lipid modulation of the drug target. Type 1 cholecystokinin receptor (CCK1R) agonist actions are affected by increased membrane cholesterol, enhancing ligand binding and reducing calcium signaling, while agonist actions of the closely related CCK2R are not. In this work, we identified a set of chimeric human CCK1R/CCK2R mutations that exchange the cholesterol sensitivity of these 2 receptors, providing powerful tools when expressed in CHO and HEK-293 model cell lines to explore mechanisms. Static, low energy, high-resolution structures of the mutant CCK1R constructs, stabilized in complex with G protein, were not substantially different, suggesting that alterations to receptor dynamics were key to altered function. We reveal that cholesterol-dependent dynamic changes in the conformation of the helical bundle of CCK receptors affects both ligand binding at the extracellular surface and G protein coupling at the cytosolic surface, as well as their interrelationships involved in stimulus-response coupling. This provides an ideal setting for potential allosteric modulators to correct the negative impact of membrane cholesterol on CCK1R.
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- 2024
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11. Pharmacological hallmarks of allostery at the M4 muscarinic receptor elucidated through structure and dynamics
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Ziva Vuckovic, Jinan Wang, Vi Pham, Jesse I Mobbs, Matthew J Belousoff, Apurba Bhattarai, Wessel AC Burger, Geoff Thompson, Mahmuda Yeasmin, Vindhya Nawaratne, Katie Leach, Emma T van der Westhuizen, Elham Khajehali, Yi-Lynn Liang, Alisa Glukhova, Denise Wootten, Craig W Lindsley, Andrew Tobin, Patrick Sexton, Radostin Danev, Celine Valant, Yinglong Miao, Arthur Christopoulos, and David M Thal
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allostery ,drug discovery ,molecular dynamics ,molecular pharmacology ,muscarinic acetylcholine receptors ,structural biology ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Allosteric modulation of G protein-coupled receptors (GPCRs) is a major paradigm in drug discovery. Despite decades of research, a molecular-level understanding of the general principles that govern the myriad pharmacological effects exerted by GPCR allosteric modulators remains limited. The M4 muscarinic acetylcholine receptor (M4 mAChR) is a validated and clinically relevant allosteric drug target for several major psychiatric and cognitive disorders. In this study, we rigorously quantified the affinity, efficacy, and magnitude of modulation of two different positive allosteric modulators, LY2033298 (LY298) and VU0467154 (VU154), combined with the endogenous agonist acetylcholine (ACh) or the high-affinity agonist iperoxo (Ipx), at the human M4 mAChR. By determining the cryo-electron microscopy structures of the M4 mAChR, bound to a cognate Gi1 protein and in complex with ACh, Ipx, LY298-Ipx, and VU154-Ipx, and applying molecular dynamics simulations, we determine key molecular mechanisms underlying allosteric pharmacology. In addition to delineating the contribution of spatially distinct binding sites on observed pharmacology, our findings also revealed a vital role for orthosteric and allosteric ligand–receptor–transducer complex stability, mediated by conformational dynamics between these sites, in the ultimate determination of affinity, efficacy, cooperativity, probe dependence, and species variability. There results provide a holistic framework for further GPCR mechanistic studies and can aid in the discovery and design of future allosteric drugs.
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- 2023
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12. Pharmacological hallmarks of allostery at the M4 muscarinic receptor elucidated through structure and dynamics
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Jesse I Mobbs, Vi Pham, Jinan Wang, Ziva Vuckovic, Matthew J Belousoff, Apurba Bhattarai, Wessel AC Burger, Geoff Thompson, Mahmuda Yeasmin, Vindhya Nawaratne, Katie Leach, Emma T van der Westhuizen, Elham Khajehali, Yi-Lynn Liang, Alisa Glukhova, Denise Wootten, Craig W Lindsley, Andrew Tobin, Patrick Sexton, Radostin Danev, Celine Valant, Yinglong Miao, Arthur Christopoulos, and David M Thal
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General Immunology and Microbiology ,General Neuroscience ,General Medicine ,General Biochemistry, Genetics and Molecular Biology - Abstract
Allosteric modulation of G protein-coupled receptors (GPCRs) is a major paradigm in drug discovery. Despite decades of research, a molecular-level understanding of the general principles that govern the myriad pharmacological effects exerted by GPCR allosteric modulators remains limited. The M4 muscarinic acetylcholine receptor (M4 mAChR) is a validated and clinically relevant allosteric drug target for several major psychiatric and cognitive disorders. In this study, we rigorously quantified the affinity, efficacy, and magnitude of modulation of two different positive allosteric modulators, LY2033298 (LY298) and VU0467154 (VU154), combined with the endogenous agonist acetylcholine (ACh) or the high-affinity agonist iperoxo (Ipx), at the human M4 mAChR. By determining the cryo-electron microscopy structures of the M4 mAChR, bound to a cognate Gi1 protein and in complex with ACh, Ipx, LY298-Ipx, and VU154-Ipx, and applying molecular dynamics simulations, we determine key molecular mechanisms underlying allosteric pharmacology. In addition to delineating the contribution of spatially distinct binding sites on observed pharmacology, our findings also revealed a vital role for orthosteric and allosteric ligand–receptor–transducer complex stability, mediated by conformational dynamics between these sites, in the ultimate determination of affinity, efficacy, cooperativity, probe dependence, and species variability. There results provide a holistic framework for further GPCR mechanistic studies and can aid in the discovery and design of future allosteric drugs.
- Published
- 2023
13. Molecular insights into peptide agonist engagement with the PTH receptor
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Brian P. Cary, Elliot J. Gerrard, Matthew J. Belousoff, Madeleine M. Fletcher, Yan Jiang, Isabella C. Russell, Sarah J. Piper, Denise Wootten, and Patrick M. Sexton
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Structural Biology ,Molecular Biology - Published
- 2023
14. Dynamics of GLP-1R peptide agonist engagement are correlated with kinetics of G protein activation
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Lachlan Clydesdale, Hari Venugopal, Peishen Zhao, Patrick M. Sexton, Alisa Glukhova, Arthur Christopoulos, Christopher A. Reynolds, Yi Lynn Liang, Xin Zhang, Tin T. Truong, Andrew N. Keller, Maryam Khoshouei, Matthew J. Belousoff, Karen J. Gregory, Giuseppe Deganutti, Radostin Danev, Denise Wootten, and Katie Leach
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Protein Conformation, alpha-Helical ,Gene Expression ,General Physics and Astronomy ,Peptide ,Ligands ,chemistry.chemical_compound ,Receptor pharmacology ,G protein-coupled receptors ,Glucagon-Like Peptide 1 ,Cloning, Molecular ,Receptor ,chemistry.chemical_classification ,Multidisciplinary ,digestive, oral, and skin physiology ,Recombinant Proteins ,Transmembrane domain ,Oxyntomodulin ,Baculoviridae ,hormones, hormone substitutes, and hormone antagonists ,Protein Binding ,Agonist ,endocrine system ,medicine.drug_class ,G protein ,Science ,Genetic Vectors ,Allosteric regulation ,Molecular Dynamics Simulation ,Glucagon-Like Peptide-1 Receptor ,Article ,General Biochemistry, Genetics and Molecular Biology ,Structure-Activity Relationship ,Allosteric Regulation ,Electron microscopy ,medicine ,Humans ,Protein Interaction Domains and Motifs ,Binding Sites ,Cryoelectron Microscopy ,Mutagenesis ,General Chemistry ,Kinetics ,HEK293 Cells ,chemistry ,Mutation ,Biophysics ,Exenatide ,Protein Conformation, beta-Strand - Abstract
The glucagon-like peptide-1 receptor (GLP-1R) has broad physiological roles and is a validated target for treatment of metabolic disorders. Despite recent advances in GLP-1R structure elucidation, detailed mechanistic understanding of how different peptides generate profound differences in G protein-mediated signalling is still lacking. Here we combine cryo-electron microscopy, molecular dynamics simulations, receptor mutagenesis and pharmacological assays, to interrogate the mechanism and consequences of GLP-1R binding to four peptide agonists; glucagon-like peptide-1, oxyntomodulin, exendin-4 and exendin-P5. These data reveal that distinctions in peptide N-terminal interactions and dynamics with the GLP-1R transmembrane domain are reciprocally associated with differences in the allosteric coupling to G proteins. In particular, transient interactions with residues at the base of the binding cavity correlate with enhanced kinetics for G protein activation, providing a rationale for differences in G protein-mediated signalling efficacy from distinct agonists., The glucagon-like peptide-1 receptor (GLP-1R) can be targeted in the treatment of diabetes, obesity and other metabolic disorders. Here, the authors assess the molecular mechanisms of peptide agonists binding to GLP-1R and the responses elucidated by these ligands, including distinct kinetics of G protein activation.
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- 2022
15. Pharmacological hallmarks of allostery at the M4 muscarinic receptor elucidated through structure and dynamics
- Author
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Ziva Vuckovic, Jinan Wang, Vi Pham, Jesse I. Mobbs, Matthew J. Belousoff, Apurba Bhattarai, Wessel A.C. Burger, Geoff Thompson, Mahmuda Yeasmin, Katie Leach, Emma T. van der Westhuizen, Elham Khajehali, Yi-Lynn Liang, Alisa Glukhova, Denise Wootten, Craig W. Lindsley, Andrew B. Tobin, Patrick M. Sexton, Radostin Danev, Celine Valant, Yinglong Miao, Arthur Christopoulos, and David M. Thal
- Abstract
Allosteric modulation of G protein-coupled receptors (GPCRs) is a major paradigm in drug discovery. Despite decades of research, a molecular level understanding of the general principals that govern the myriad pharmacological effects exerted by GPCR allosteric modulators remains limited. The M4 muscarinic acetylcholine receptor (M4 mAChR) is a well-validated and clinically relevant allosteric drug target for several major psychiatric and cognitive disorders. Here, we present high-resolution cryo-electron microscopy structures of the M4 mAChR bound to a cognate Gi1 protein and the high affinity agonist, iperoxo, in the absence and presence of two different positive allosteric modulators, LY2033298 or VU0467154. We have also determined the structure of the M4 mAChR-Gi1 complex bound to its endogenous agonist, acetylcholine (ACh). Structural comparisons, together with molecular dynamics, mutagenesis, and pharmacological validations, have provided in-depth insights into the role of structure and dynamics in orthosteric and allosteric ligand binding, global mechanisms of receptor activation, cooperativity, probe-dependence, and species variability; all key hallmarks underpinning contemporary GPCR drug discovery.
- Published
- 2022
16. Molecular insights into peptide agonist engagement with the PTH1 receptor
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Brian P. Cary, Elliot J. Gerrard, Matthew J. Belousoff, Madeleine M. Fletcher, Yan Jiang, Isabella C. Russell, Sarah J. Piper, Denise Wootten, and Patrick M. Sexton
- Abstract
The parathyroid hormone (PTH) 1 receptor (PTH1R) is a class B1 G protein-coupled receptor (GPCR) that critically regulates skeletal development and calcium homeostasis. Despite extensive study, the molecular underpinnings of PTH1R stimulation by its cognate hormones, as well as by therapeutic agents, remain unclear. Here, we describe cryo-EM structures of the PTH1R in complex with active fragments of the two hormones, PTH and parathyroid hormone related protein (PTHrP), the peptidic drug abaloparatide, as well as the engineered tool compounds, long-acting PTH (LA-PTH) and the truncated peptide, M-PTH(1-14). We found that the N-terminus of each agonist that is critical for activity, engages the transmembrane bundle in a topologically similar fashion, which reflects similarities in measures of Gαs activation. The full-length peptides bind the extracellular domain (ECD) using a shared interface but induce subtly different ECD orientations relative to the transmembrane domain (TMD). In the structure bound to M-PTH, an agonist which only binds the TMD, the ECD is completely unresolved, demonstrating that the ECD is highly dynamic when unconstrained by a peptide. High resolutions enabled identification of water molecules near the peptide and G protein binding sites, some of which are structurally conserved with other class B1 GPCRs. Our results shed light on the action of orthosteric agonists of the PTH1R and provide a foundation for structure based-drug design.
- Published
- 2022
17. A Structurally Characterized
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Laura, Perlaza-Jiménez, Kher-Shing, Tan, Sarah J, Piper, Rachel M, Johnson, Rebecca S, Bamert, Christopher J, Stubenrauch, Alexander, Wright, David, Lupton, Trevor, Lithgow, and Matthew J, Belousoff
- Subjects
Methicillin-Resistant Staphylococcus aureus ,Staphylococcus aureus ,Cryoelectron Microscopy ,Linezolid ,Humans ,Microbial Sensitivity Tests ,Staphylococcal Infections ,Anti-Bacterial Agents - Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen that presents great health concerns. Treatment requires the use of last-line antibiotics, such as members of the oxazolidinone family, of which linezolid is the first member to see regular use in the clinic. Here, we report a short time scale selection experiment in which strains of MRSA were subjected to linezolid treatment. Clonal isolates which had evolved a linezolid-resistant phenotype were characterized by whole-genome sequencing. Linezolid-resistant mutants were identified which had accumulated mutations in the ribosomal protein uL3. Multiple clones which had two mutations in uL3 exhibited resistance to linezolid, 2-fold higher than the clinical breakpoint. Ribosomes from this strain were isolated and subjected to single-particle cryo-electron microscopic analysis and compared to the ribosomes from the parent strain. We found that the mutations in uL3 lead to a rearrangement of a loop that makes contact with Helix 90, propagating a structural change over 15 Å away. This distal change swings nucleotide U2504 into the binding site of the antibiotic, causing linezolid resistance.
- Published
- 2022
18. A structural basis for amylin receptor phenotype
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Jianjun Cao, Matthew J. Belousoff, Yi-Lynn Liang, Rachel M. Johnson, Tracy M. Josephs, Madeleine M. Fletcher, Arthur Christopoulos, Debbie L. Hay, Radostin Danev, Denise Wootten, and Patrick M. Sexton
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Multidisciplinary ,Amylin Receptor Agonists ,Phenotype ,Protein Conformation ,Salmon ,Cryoelectron Microscopy ,Animals ,Humans ,Protein Multimerization ,Receptors, Islet Amyloid Polypeptide - Abstract
Amylin receptors (AMYRs) are heterodimers of the calcitonin (CT) receptor (CTR) and one of three receptor activity–modifying proteins (RAMPs), AMY 1 R, AMY 2 R, and AMY 3 R. Selective AMYR agonists and dual AMYR/CTR agonists are being developed as obesity treatments; however, the molecular basis for peptide binding and selectivity is unknown. We determined the structure and dynamics of active AMYRs with amylin, AMY 1 R with salmon CT (sCT), AMY 2 R with sCT or human CT (hCT), and CTR with amylin, sCT, or hCT. The conformation of amylin-bound complexes was similar for all AMYRs, constrained by the RAMP, and an ordered midpeptide motif that we call the bypass motif. The CT-bound AMYR complexes were distinct, overlapping the CT-bound CTR complexes. Our findings indicate that activation of AMYRs by CT-based peptides is distinct from their activation by amylin-based peptides. This has important implications for the development of AMYR therapeutics.
- Published
- 2022
19. Thermo Scientific™ Glacios Cryo-TEM: A Versatile 200 kV Tool for Structure-Based Drug Discovery
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Evgeniya V. Pechnikova, Rachel M. Johnson, Ieva Drulyte, Denise Wootten, Adrian Koh, Patrick M. Sexton, S. Masiulis, Xin Zhang, Matthew J. Belousoff, and Sebastian Unger
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Cryo tem ,Materials science ,Drug discovery ,Structure based ,Nanotechnology ,Instrumentation - Published
- 2021
20. Structure of the PCBP2/stem–loop IV complex underlying translation initiation mediated by the poliovirus type I IRES
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Jacqueline A. Wilce, Bert L. Semler, Neelam Shah, Mehdi Y Matak, Matthew J Belousoff, Hans Elmlund, Hariprasad Venugopal, Naveen Vankadari, Joseph H. C. Nguyen, Matthew C.J. Wilce, and Simone A. Beckham
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Small Angle ,Models, Molecular ,AcademicSubjects/SCI00010 ,Protein Conformation ,Biology ,Cleavage (embryo) ,Tetraloop ,Scattering ,Protein structure ,Eukaryotic translation ,X-Ray Diffraction ,Models ,Information and Computing Sciences ,Scattering, Small Angle ,Genetics ,RNA and RNA-protein complexes ,Viral ,Peptide Chain Initiation, Translational ,Translational ,Cryoelectron Microscopy ,RNA ,Molecular ,RNA-Binding Proteins ,Biological Sciences ,Stem-loop ,Cell biology ,Internal ribosome entry site ,Poliovirus ,Peptide Chain Initiation ,Nucleic Acid Conformation ,RNA, Viral ,Linker ,Environmental Sciences ,Developmental Biology - Abstract
The poliovirus type I IRES is able to recruit ribosomal machinery only in the presence of host factor PCBP2 that binds to stem–loop IV of the IRES. When PCBP2 is cleaved in its linker region by viral proteinase 3CD, translation initiation ceases allowing the next stage of replication to commence. Here, we investigate the interaction of PCBP2 with the apical region of stem–loop IV (SLIVm) of poliovirus RNA in its full-length and truncated form. CryoEM structure reconstruction of the full-length PCBP2 in complex with SLIVm solved to 6.1 Å resolution reveals a compact globular complex of PCBP2 interacting with the cruciform RNA via KH domains and featuring a prominent GNRA tetraloop. SEC-SAXS, SHAPE and hydroxyl-radical cleavage establish that PCBP2 stabilizes the SLIVm structure, but upon cleavage in the linker domain the complex becomes more flexible and base accessible. Limited proteolysis and REMSA demonstrate the accessibility of the linker region in the PCBP2/SLIVm complex and consequent loss of affinity of PCBP2 for the SLIVm upon cleavage. Together this study sheds light on the structural features of the PCBP2/SLIV complex vital for ribosomal docking, and the way in which this key functional interaction is regulated following translation of the poliovirus genome.
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- 2020
21. Structure and Dynamics of Adrenomedullin Receptors AM1 and AM2 Reveal Key Mechanisms in the Control of Receptor Phenotype by Receptor Activity-Modifying Proteins
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Madeleine M. Fletcher, Xin Zhang, Debbie L. Hay, Matthew J. Belousoff, Patrick M. Sexton, Giuseppe Deganutti, Arthur Christopoulos, Yi Lynn Liang, Denise Wootten, Maryam Khoshouei, Radostin Danev, Laurence J. Miller, Christopher A. Reynolds, Sebastian G.B. Furness, and Cassandra Koole
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Pharmacology ,Receptor activity-modifying protein ,RAMP2 ,Chemistry ,RAMP1 ,RAMP3 ,Pharmacology (medical) ,Receptor Activity-Modifying Protein 1 ,CALCRL ,Receptor ,Cell biology ,G protein-coupled receptor - Abstract
Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) receptors are critically important for metabolism, vascular tone, and inflammatory response. AM receptors are also required for normal lymphatic and blood vascular development and angiogenesis. They play a pivotal role in embryo implantation and fertility and can provide protection against hypoxic and oxidative stress. CGRP and AM receptors are heterodimers of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) (CGRPR), as well as RAMP2 or RAMP3 (AM1R and AM2R, respectively). However, the mechanistic basis for RAMP modulation of CLR phenotype is unclear. In this study, we report the cryo-EM structure of the AM1R in complex with AM and Gs at a global resolution of 3.0 A, and structures of the AM2R in complex with either AM or intermedin/adrenomedullin 2 (AM2) and Gs at 2.4 and 2.3 A, respectively. The structures reveal distinctions in the primary orientation of the extracellular domains (ECDs) relative to the receptor core and distinct positioning of extracellular loop 3 (ECL3) that are receptor-dependent. Analysis of dynamic data present in the cryo-EM micrographs revealed additional distinctions in the extent of mobility of the ECDs. Chimeric exchange of the linker region of the RAMPs connecting the TM helix and the ECD supports a role for this segment in controlling receptor phenotype. Moreover, a subset of the motions of the ECD appeared coordinated with motions of the G protein relative to the receptor core, suggesting that receptor ECD dynamics could influence G protein interactions. This work provides fundamental advances in our understanding of GPCR function and how this can be allosterically modulated by accessory proteins.
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- 2020
22. Structural and Dynamic Mechanisms of Allostery at the M4 Muscarinic Acetylcholine Receptor
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Ziva Vuckovic, Jinan Wang, Vi Pham, Jesse I. Mobbs, Matthew J. Belousoff, Apurba Bhattarai, Wessel A.C. Burger, Geoff Thompson, Mahmuda Yeasmin, Vindhya Nawaratne, Katie Leach, Emma T. van der Westhuizen, Elham Khajehali, Yi-Lynn Liang, Alisa Glukhova, Denise Wootten, Craig W. Lindsley, Andrew Tobin, Patrick M. Sexton, Radostin Danev, Celine Valant, Yinglong Miao, Arthur Christopoulos, and David Thal
- Published
- 2022
23. Structural and Functional Diversity among Agonist-Bound States of the GLP-1 Receptor
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Brian P. Cary, Giuseppe Deganutti, Peishen Zhao, Tin T. Truong, Sarah J. Piper, Xinyu Liu, Matthew J. Belousoff, Radostin Danev, Patrick M. Sexton, Denise Wootten, and Samuel H. Gellman
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Protein Domains ,Glucagon-Like Peptide 1 ,Exenatide ,Cell Biology ,Peptides ,Molecular Biology ,Article ,Glucagon-Like Peptide-1 Receptor - Abstract
Recent advances in G protein-coupled receptor (GPCR) structural elucidation have strengthened previous hypotheses that multi-dimensional signal propagation mediated by these receptors depends, in part, on their conformational mobility. However, the relationship between receptor function and static structures is inherently uncertain. Here we examine the contribution of peptide agonist conformational plasticity to activation of the glucagon-like peptide-1 receptor (GLP-1R), an important clinical target. We employ variants of the peptides GLP-1 and exendin-4 to explore the interplay between helical propensity near the agonist N-terminus and the ability to bind to and activate the receptor. Cryo-EM analysis of a complex involving an exendin-4 analogue, the GLP-1R and G(s) heterotrimer revealed two receptor conformers with distinct modes of peptide-receptor engagement. Our functional and structural data, along with molecular dynamics simulations, suggest that receptor conformational dynamics associated with flexibility of the peptide N-terminal activation domain may be a key determinant of agonist efficacy.
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- 2021
24. Discovery and biosynthesis of persiathiacins: Unusual polyglycosylated thiopeptides active against multi-drug resistant tuberculosis
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Isolda Romero-Canelón, Fatemeh Mohammadipanah, Anthony Vocat, Boyke Bunk, Christopher D. Fage, Cathrin Spröer, Daniel Zabala, Stewart T. Cole, Gregory L. Challis, Matthew J Belousoff, Jörg Overmann, and Yousef Dashti
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Actinokineospora ,biology ,medicine.drug_class ,Antibiotics ,Antimicrobial ,biology.organism_classification ,Mycobacterium tuberculosis ,chemistry.chemical_compound ,Biochemistry ,Biosynthesis ,chemistry ,Gene cluster ,medicine ,Protein biosynthesis ,Antibacterial activity - Abstract
Thiopeptides are ribosomally biosynthesized and post-translationally modified peptides (RiPPs) that potently inhibit the growth of Gram-positive bacteria by targeting multiple steps in protein biosynthesis. The poor pharmacological properties of thiopeptides, in particular their low aqueous solubility, has hindered their development into clinically useful antibiotics. Antimicrobial activity screens of a library of Actinobacterial extracts led to discovery of the novel polyglycosylated thiopeptides persiathiacins A and B from Actinokineospora sp. UTMC 2475 and Actinokineospora sp. UTMC 2448. Persiathiacin A is active against methicillin-resistant Staphylococcus aureus (MRSA) and several Mycobacterium tuberculosis strains, including drug-resistant and multidrug-resistant clinical isolates, and does not significantly affect the growth of ovarian cancer cells at concentrations up to 400 μM. In vitro translation assays showed that, like other thiopeptide antibiotics, persiathiacin A targets protein biosynthesis. Polyglycosylated thiopeptides are extremely rare and nothing is known about their biosynthesis. Sequencing and analysis of the Actinokineospora sp. UTMC 2448 genome enabled identification of the putative persiathiacin biosynthetic gene cluster. A cytochrome P450 encoded by this gene cluster catalyses the hydroxylation of nosiheptide in vitro and in vivo, consistent with the proposal that the cluster directs persiathiacin biosynthesis. Several genes in the cluster encode homologues of enzymes known to catalyse the assembly and attachment of deoxysugars during the biosynthesis of other classes of glycosylated natural products. The discovery of the persiathiacins and their biosynthetic gene cluster thus provides the basis for the development of biosynthetic engineering approaches to the creation of novel (poly)glycosylated thiopeptide derivatives with enhanced pharmacological properties.
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- 2021
25. Mechanistic Insights into the Capsule-Targeting Depolymerase from a Klebsiella pneumoniae Bacteriophage
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Richard A. Strugnell, Trevor Lithgow, Francesca L. Short, Rebecca S. Bamert, Rhys A. Dunstan, Jonathan J. Wilksch, Christopher K. Barlow, Matthew J Belousoff, Gordon Dougan, Derek Pickard, and Ralf B. Schittenhelm
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Microbiology (medical) ,Klebsiella ,bacteriophages ,Physiology ,Klebsiella pneumoniae ,Virulence ,Microbiology ,Bacterial cell structure ,Bacteriophage ,Viral Proteins ,Genetics ,Pathogen ,Bacterial Capsules ,Polysaccharide-Lyases ,chemistry.chemical_classification ,General Immunology and Microbiology ,Ecology ,biology ,Chemistry ,Cryoelectron Microscopy ,Cell Biology ,biology.organism_classification ,capsular polysaccharide ,QR1-502 ,Infectious Diseases ,Enzyme ,cryo-EM ,depolymerase ,Bacteria ,Research Article - Abstract
The production of capsular polysaccharides by Klebsiella pneumoniae protects the bacterial cell from harmful environmental factors such as antimicrobial compounds and infection by bacteriophages (phages). To bypass this protective barrier, some phages encode polysaccharide-degrading enzymes referred to as depolymerases to provide access to cell surface receptors. Here, we characterized the phage RAD2, which infects K. pneumoniae strains that produce the widespread, hypervirulence-associated K2-type capsular polysaccharide. Using transposon-directed insertion sequencing, we have shown that the production of capsule is an absolute requirement for efficient RAD2 infection by serving as a first-stage receptor. We have identified the depolymerase responsible for recognition and degradation of the capsule, determined that the depolymerase forms globular appendages on the phage virion tail tip, and present the cryo-electron microscopy structure of the RAD2 capsule depolymerase at 2.7-Å resolution. A putative active site for the enzyme was identified, comprising clustered negatively charged residues that could facilitate the hydrolysis of target polysaccharides. Enzymatic assays coupled with mass spectrometric analyses of digested oligosaccharide products provided further mechanistic insight into the hydrolase activity of the enzyme, which, when incubated with K. pneumoniae, removes the capsule and sensitizes the cells to serum-induced killing. Overall, these findings expand our understanding of how phages target the Klebsiella capsule for infection, providing a framework for the use of depolymerases as antivirulence agents against this medically important pathogen. IMPORTANCE Klebsiella pneumoniae is a medically important pathogen that produces a thick protective capsule that is essential for pathogenicity. Phages are natural predators of bacteria, and many encode diverse “capsule depolymerases” which specifically degrade the capsule of their hosts, an exploitable trait for potential therapies. We have determined the first structure of a depolymerase that targets the clinically relevant K2 capsule and have identified its putative active site, providing hints to its mechanism of action. We also show that Klebsiella cells treated with a recombinant form of the depolymerase are stripped of capsule, inhibiting their ability to grow in the presence of serum, demonstrating the anti-infective potential of these robust and readily producible enzymes against encapsulated bacterial pathogens such as K. pneumoniae.
- Published
- 2021
26. BonA from Acinetobacter baumannii Forms a Divisome-Localized Decamer That Supports Outer Envelope Function
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Trevor Lithgow, Rhys A. Dunstan, Jian Li, Sachith D. Gunasinghe, Pok Man Leung, Anton Y. Peleg, Ashleigh Kropp, Simone Beckham, Rhys Grinter, Matthew J Belousoff, Chris Greening, Eva Heinz, Faye C. Morris, and Nichollas E. Scott
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Acinetobacter baumannii ,cell division ,0303 health sciences ,cell envelope ,biology ,Architecture domain ,Cell division ,030306 microbiology ,Chemistry ,biology.organism_classification ,Microbiology ,QR1-502 ,Cell biology ,03 medical and health sciences ,chemistry.chemical_compound ,Virology ,outer membrane proteins ,Peptidoglycan ,Cell envelope ,Bacterial outer membrane ,Function (biology) ,Bacteria ,030304 developmental biology - Abstract
Acinetobacter baumannii is a high-risk pathogen due to the rapid global spread of multidrug-resistant lineages. Its phylogenetic divergence from other ESKAPE pathogens means that determinants of its antimicrobial resistance can be difficult to extrapolate from other widely studied bacteria. A recent study showed that A. baumannii upregulates production of an outer membrane lipoprotein, which we designate BonA, in response to challenge with polymyxins. Here, we show that BonA has limited sequence similarity and distinct structural features compared to lipoproteins from other bacterial species. Analyses through X-ray crystallography, small-angle X-ray scattering, electron microscopy, and multiangle light scattering demonstrate that BonA has a dual BON (Bacterial OsmY and Nodulation) domain architecture and forms a decamer via an unusual oligomerization mechanism. This analysis also indicates this decamer is transient, suggesting dynamic oligomerization plays a role in BonA function. Antisera recognizing BonA shows it is an outer membrane protein localized to the divisome. Loss of BonA modulates the density of the outer membrane, consistent with a change in its structure or link to the peptidoglycan, and prevents motility in a clinical strain (ATCC 17978). Consistent with these findings, the dimensions of the BonA decamer are sufficient to permeate the peptidoglycan layer, with the potential to form a membrane-spanning complex during cell division. IMPORTANCE The pathogen Acinetobacter baumannii is considered an urgent threat to human health. A. baumannii is highly resistant to treatment with antibiotics, in part due to its protective cell envelope. This bacterium is only distantly related to other bacterial pathogens, so its cell envelope has distinct properties and contains components distinct from those of other bacteria that support its function. Here, we report the discovery of BonA, a protein that supports A. baumannii outer envelope function and is required for cell motility. We determine the atomic structure of BonA and show that it forms part of the cell division machinery and functions by forming a complex, features that mirror those of distantly related homologs from other bacteria. By improving our understanding of the A. baumannii cell envelope this work will assist in treating this pathogen.
- Published
- 2021
27. The architecture and stabilisation of flagellotropic tailed bacteriophages
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Derek Pickard, Jiawei Wang, Trevor Lithgow, Joshua M. Hardy, Gordon Dougan, Matthew J. Belousoff, Rhys Grinter, Rhys A. Dunstan, Fasséli Coulibaly, Hariprasad Venugopal, Hardy, Joshua M [0000-0002-8014-8552], Dunstan, Rhys A [0000-0002-7161-3993], Grinter, Rhys [0000-0002-8195-5348], Lithgow, Trevor [0000-0002-0102-7884], Coulibaly, Fasséli [0000-0003-3380-2117], Apollo - University of Cambridge Repository, Hardy, Joshua M. [0000-0002-8014-8552], and Dunstan, Rhys A. [0000-0002-7161-3993]
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0301 basic medicine ,145 ,Science ,Amino Acid Motifs ,Phage biology ,General Physics and Astronomy ,Flagellum ,Protein Structure, Secondary ,General Biochemistry, Genetics and Molecular Biology ,631/326/596/432 ,Bacteriophage ,82/80 ,03 medical and health sciences ,chemistry.chemical_compound ,Protein structure ,Viral genetics ,Dna genetics ,631/45/535/1258 ,Electron microscopy ,Bacteriophages ,lcsh:Science ,Herpesviridae ,Multidisciplinary ,030102 biochemistry & molecular biology ,biology ,82/16 ,Chemistry ,101/28 ,Virion ,article ,DNA ,General Chemistry ,Virus structures ,biology.organism_classification ,Dna translocation ,Vitrification ,030104 developmental biology ,Capsid ,Flagella ,631/326/596/2148 ,DNA, Viral ,Biophysics ,Capsid Proteins ,lcsh:Q ,Protein Multimerization - Abstract
Funder: Sir Henry Wellcome Fellow (106077/Z/14/Z), Funder: Australian Research Council Laureate Fellow (FL130100038), Flagellotropic bacteriophages engage flagella to reach the bacterial surface as an effective means to increase the capture radius for predation. Structural details of these viruses are of great interest given the substantial drag forces and torques they face when moving down the spinning flagellum. We show that the main capsid and auxiliary proteins form two nested chainmails that ensure the integrity of the bacteriophage head. Core stabilising structures are conserved in herpesviruses suggesting their ancestral origin. The structure of the tail also reveals a robust yet pliable assembly. Hexameric rings of the tail-tube protein are braced by the N-terminus and a β-hairpin loop, and interconnected along the tail by the splayed β-hairpins. By contrast, we show that the β-hairpin has an inhibitory role in the tail-tube precursor, preventing uncontrolled self-assembly. Dyads of acidic residues inside the tail-tube present regularly-spaced motifs well suited to DNA translocation into bacteria through the tail.
- Published
- 2021
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28. Routine sub-2.5 Å cryo-EM structure determination of GPCRs
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Xin Zhang, Denise Wootten, Yi Lynn Liang, Patrick M. Sexton, Fabian Eisenstein, Radostin Danev, and Matthew J. Belousoff
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Models, Molecular ,Computer science ,Cryo-electron microscopy ,viruses ,Science ,General Physics and Astronomy ,Image processing ,macromolecular substances ,environment and public health ,General Biochemistry, Genetics and Molecular Biology ,Article ,Receptors, G-Protein-Coupled ,03 medical and health sciences ,Phase plate ,0302 clinical medicine ,Receptor pharmacology ,Cryoelectron microscopy ,Image Processing, Computer-Assisted ,030304 developmental biology ,G protein-coupled receptor ,Structure (mathematical logic) ,0303 health sciences ,Multidisciplinary ,General Chemistry ,Grid ,diagnosis ,Multiple factors ,Gold ,Biological system ,Structural biology ,030217 neurology & neurosurgery ,Energy (signal processing) - Abstract
Cryo-electron microscopy (cryo-EM) of small membrane proteins, such as G protein-coupled receptors (GPCRs), remains challenging. Pushing the performance boundaries of the technique requires quantitative knowledge about the contribution of multiple factors. Here, we present an in-depth analysis and optimization of the main experimental parameters in cryo-EM. We combined actual structural studies with methods development to quantify the effects of the Volta phase plate, zero-loss energy filtering, objective lens aperture, defocus magnitude, total exposure, and grid type. By using this information to carefully maximize the experimental performance, it is now possible to routinely determine GPCR structures at resolutions better than 2.5 Å. The improved fidelity of such maps enables the building of better atomic models and will be crucial for the future expansion of cryo-EM into the structure-based drug design domain. The optimization guidelines given here are not limited to GPCRs and can be applied directly to other small proteins., There is a need to optimise cryo-EM data acquisition approaches to improve the resolution of GPCR cryo-EM structures to better than 2.5 Å, in order to use them for structure-based drug design purposes. Here, the authors present a systematic analysis of the main cryo-EM experimental parameters using three GPCRs as test cases, which is also of interest for the cryo-EM structure determination of other small membrane proteins.
- Published
- 2021
29. Structures of the human cholecystokinin 1 (CCK1) receptor bound to Gs and Gq mimetic proteins provide insight into mechanisms of G protein selectivity
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Denise Wootten, Arthur Christopoulos, Radostin Danev, Sebastian G.B. Furness, Patrick M. Sexton, David M. Thal, Xiaomeng Xu, Hari Venugopal, Sarah J. Piper, Matthew J. Belousoff, Kaleeckal G. Harikumar, Laurence J. Miller, and Jesse I. Mobbs
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Proteomics ,Models, Molecular ,GTP-Binding Protein alpha Subunits ,Peptide Hormones ,Plasma protein binding ,Biochemistry ,Physical Chemistry ,Intracellular Receptors ,Database and Informatics Methods ,0302 clinical medicine ,Protein structure ,Heterotrimeric G protein ,Macromolecular Structure Analysis ,GTP-Binding Protein alpha Subunits, Gs ,Electron Microscopy ,Biology (General) ,0303 health sciences ,Microscopy ,Proteomic Databases ,General Neuroscience ,Lipids ,Chemistry ,Cholesterol ,Physical Sciences ,Signal transduction ,General Agricultural and Biological Sciences ,Cholecystokinin ,hormones, hormone substitutes, and hormone antagonists ,Signal Transduction ,Protein Binding ,Protein Structure ,Transmembrane Receptors ,G protein ,QH301-705.5 ,Discovery Report ,Biology ,Research and Analysis Methods ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Humans ,Molecular Biology ,030304 developmental biology ,G protein-coupled receptor ,General Immunology and Microbiology ,Chemical Bonding ,Biology and Life Sciences ,Proteins ,Electron Cryo-Microscopy ,Hydrogen Bonding ,Cell Biology ,Hormones ,Biological Databases ,HEK293 Cells ,Membrane protein ,Biophysics ,GTP-Binding Protein alpha Subunits, Gq-G11 ,Receptors, Cholecystokinin ,G Protein Coupled Receptors ,030217 neurology & neurosurgery - Abstract
G protein–coupled receptors (GPCRs) are critical regulators of cellular function acting via heterotrimeric G proteins as their primary transducers with individual GPCRs capable of pleiotropic coupling to multiple G proteins. Structural features governing G protein selectivity and promiscuity are currently unclear. Here, we used cryo-electron microscopy (cryo-EM) to determine structures of the cholecystokinin (CCK) type 1 receptor (CCK1R) bound to the CCK peptide agonist, CCK-8 and 2 distinct transducer proteins, its primary transducer Gq, and the more weakly coupled Gs. As seen with other Gq/11–GPCR complexes, the Gq–α5 helix (αH5) bound to a relatively narrow pocket in the CCK1R core. Surprisingly, the backbone of the CCK1R and volume of the G protein binding pocket were essentially equivalent when Gs was bound, with the Gs αH5 displaying a conformation that arises from “unwinding” of the far carboxyl-terminal residues, compared to canonically Gs coupled receptors. Thus, integrated changes in the conformations of both the receptor and G protein are likely to play critical roles in the promiscuous coupling of individual GPCRs., Cryo-EM structures of the G protein-coupled receptor CCK1R bound to the CCK peptide agonist CCK-8 and two distinct transducer proteins – its primary transducer Gq, and the more weakly coupled Gs – reveal unexpected modes of G protein interaction.
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- 2021
30. Structures of the human cholecystokinin 1 (CCK1) receptor bound to Gs and Gq mimetic proteins: insight into mechanisms of G protein selectivity
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Sarah J. Piper, Denise Wootten, Kaleeckal G. Harikumar, Laurence J. Miller, Jesse I. Mobbs, Arthur Christopoulos, Matthew J. Belousoff, Xiaomeng Xu, Hari Venugopal, Patrick M. Sexton, Sebastian G.B. Furness, David M. Thal, and Radostin Danev
- Subjects
Agonist ,chemistry.chemical_classification ,chemistry ,medicine.drug_class ,G protein ,Heterotrimeric G protein ,Helix ,medicine ,Biophysics ,Peptide ,Receptor ,Function (biology) ,G protein-coupled receptor - Abstract
G protein-coupled receptors (GPCRs) are critical regulators of cellular function acting via heterotrimeric G proteins as their primary transducers with individual GPCRs capable of pleiotropic coupling to multiple G proteins. Structural features governing G protein selectivity and promiscuity are currently unclear. Here we used cryo-electron microscopy to determine structures of the CCK1R bound to the CCK peptide agonist, CCK-8 and two distinct transducer proteins, its primary transducer Gq, and the more weakly coupled Gs. As seen with other Gq/11-GPCR complexes, the Gq-α5 helix bound to a relatively narrow pocket in the CCK1R core. Surprisingly, the backbone of the CCK1R and volume of the G protein binding pocket was essentially equivalent when Gs was bound, with the Gs α5 helix displaying a conformation that arises from “unwinding” of the far C-terminal residues, compared to canonically Gs coupled receptors. Thus, integrated changes in the conformations of both the receptor and G protein play critical roles in the promiscuous coupling of individual GPCRs.One-Sentence SummaryCryo-EM structures of the CCK-1R reveal key mechanisms for promiscuous G protein coupling.
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- 2021
31. cryoEM‐Guided Development of Antibiotics for Drug‐Resistant Bacteria
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Christopher J. Stubenrauch, Hari Venugopal, David W. Lupton, Alexander Wright, Matthew J. Belousoff, Samuel Seoner, Rebecca S. Bamert, Trevor Lithgow, and Isabella Stuart
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Methicillin-Resistant Staphylococcus aureus ,Models, Molecular ,Drug ,medicine.drug_class ,media_common.quotation_subject ,Antibiotics ,Microbial Sensitivity Tests ,Crystallography, X-Ray ,medicine.disease_cause ,01 natural sciences ,Biochemistry ,Vancomycin-Resistant Enterococci ,Microbiology ,Structure-Activity Relationship ,chemistry.chemical_compound ,Antibiotic resistance ,Acetamides ,Drug Discovery ,medicine ,heterocyclic compounds ,General Pharmacology, Toxicology and Pharmaceutics ,media_common ,Pharmacology ,Binding Sites ,Molecular Structure ,biology ,010405 organic chemistry ,Organic Chemistry ,Linezolid ,Staphylococcal Infections ,biochemical phenomena, metabolism, and nutrition ,Ribosomal RNA ,bacterial infections and mycoses ,biology.organism_classification ,Anti-Bacterial Agents ,0104 chemical sciences ,010404 medicinal & biomolecular chemistry ,chemistry ,Enterococcus ,Staphylococcus aureus ,Drug Design ,Molecular Medicine ,Ribosomes ,Acetamide - Abstract
While the ribosome is a common target for antibiotics, challenges with crystallography can impede the development of new bioactives using structure-based drug design approaches. In this study we exploit common structural features present in linezolid-resistant forms of both methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) to redesign the antibiotic. Enabled by rapid and facile cryoEM structures, this process has identified (S)-2,2-dichloro-N-((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (LZD-5) and (S)-2-chloro-N-((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl) acetamide (LZD-6), which inhibit the ribosomal function and growth of linezolid-resistant MRSA and VRE. The strategy discussed highlights the potential for cryoEM to facilitate the development of novel bioactive materials.
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- 2019
32. Structural and Functional Diversity among Agonist-Bound States of the GLP-1 Receptor
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Patrick M. Sexton, Peishen Zhao, Brian P. Cary, Samuel H. Gellman, Tin T. Truong, Sarah J. Piper, Radostin Danev, Matthew J. Belousoff, and Denise Wootten
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chemistry.chemical_classification ,Agonist ,Gs alpha subunit ,chemistry ,medicine.drug_class ,Biophysics ,medicine ,Peptide ,Receptor ,Conformational isomerism ,Function (biology) ,Glucagon-like peptide 1 receptor ,G protein-coupled receptor - Abstract
Recent advances in G protein-coupled receptor (GPCR) structural elucidation have strengthened previous hypotheses that multi-dimensional signal propagation mediated by these receptors is, in part, dependent on their conformational mobility. However, the relationship between receptor function and static structures determined via crystallography or cryo-electron microscopy is not always clear. This study examines the contribution of peptide agonist conformational plasticity to activation of the glucagon-like peptide-1 receptor (GLP-1R), an important clinical target. We employ variants of the peptides GLP-1 and exendin-4 to explore the interplay between helical propensity near the agonist N-terminus and the ability to bind to and activate the receptor. Cryo-EM analysis of a complex involving an exendin-4 analogue, the GLP-1R and Gs protein revealed two receptor conformers with distinct modes of peptide-receptor engagement. Our functional and structural data suggest that receptor conformational dynamics associated with flexibility of the peptide N-terminal activation domain may be a key determinant of agonist efficacy.
- Published
- 2021
33. Molecular basis for control of antibiotic production by a bacterial hormone
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Max J. Cryle, Christophe Corre, Vilmos Fülöp, Peter J. Harrison, Hussain Bhukya, Dean Rea, Paulina K. Sydor, Shanshan Zhou, Kathryn M Styles, Nicolas Malet, Lona M. Alkhalaf, Matthew J. Belousoff, Gregory L. Challis, Hariprasad Venugopal, and Lijiang Song
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0301 basic medicine ,Multidisciplinary ,biology ,Chemistry ,030106 microbiology ,Streptomyces coelicolor ,Repressor ,biology.organism_classification ,Methylenomycin ,QR ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Biochemistry ,Hormone receptor ,Gene cluster ,TetR ,Hormone metabolism ,DNA - Abstract
Actinobacteria produce numerous antibiotics and other specialized metabolites that have important applications in medicine and agriculture1. Diffusible hormones frequently control the production of such metabolites by binding TetR family transcriptional repressors (TFTRs), but the molecular basis for this remains unclear2. The production of methylenomycin antibiotics in Streptomyces coelicolor A3(2) is initiated by the binding of 2-alkyl-4-hydroxymethylfuran-3-carboxylic acid (AHFCA) hormones to the TFTR MmfR3. Here we report the X-ray crystal structure of an MmfR–AHFCA complex, establishing the structural basis for hormone recognition. We also elucidate the mechanism for DNA release upon hormone binding through the single-particle cryo-electron microscopy structure of an MmfR–operator complex. DNA binding and release assays with MmfR mutants and synthetic AHFCA analogues define the role of individual amino acid residues and hormone functional groups in ligand recognition and DNA release. These findings will facilitate the exploitation of actinobacterial hormones and their associated TFTRs in synthetic biology and in the discovery of new antibiotics. X-ray crystallography and cryo-electron microscopy structures of the transcriptional repressor of the methylomycin gene cluster, MmfR, reveal the molecular basis for regulation of antibiotic biosynthesis by AHFCA hormones in Actinobacteria.
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- 2021
34. Structure and dynamics of semaglutide and taspoglutide bound GLP-1R-Gs complexes
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Patrick M. Sexton, Yi Lynn Liang, Radostin Danev, Denise Wootten, Matthew J. Belousoff, and Xin Zhang
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chemistry.chemical_classification ,endocrine system ,Semaglutide ,media_common.quotation_subject ,digestive, oral, and skin physiology ,Taspoglutide ,Peptide ,Peptide binding ,Appetite ,Carbohydrate metabolism ,Pharmacology ,chemistry ,High incidence ,Receptor ,hormones, hormone substitutes, and hormone antagonists ,media_common - Abstract
SUMMARYThe glucagon-like peptide-1 receptor (GLP-1R) regulates insulin secretion, carbohydrate metabolism and appetite, and is an important target for treatment of type II diabetes and obesity. Multiple GLP-1R agonists have entered into clinical trials, such as semaglutide, progressing to approval. Others, including taspoglutide, failed through high incidence of side-effects or insufficient efficacy. GLP-1R agonists have a broad spectrum of signalling profiles. However, molecular understanding is limited by a lack of structural information on how different GLP-1R agonists engage with the GLP-1R. In this study, we determined cryo-electron microscopy (cryo-EM) structures of GLP-1R-Gs protein complexes bound with semaglutide and taspoglutide. These revealed similar peptide binding modes to that previously observed for GLP-1. However, 3D variability analysis of the cryo-EM micrographs revealed different motions within the bound peptides and the receptor relative to when GLP-1 is bound. This work provides novel insights into the molecular determinants of peptide engagement with the GLP-1R.
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- 2021
35. Evolving cryo-EM structural approaches for GPCR drug discovery
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Ieva Drulyte, Abhay Kotecha, Matthew J. Belousoff, Lingbo Yu, Radostin Danev, Patrick M. Sexton, Xin Zhang, Rachel M. Johnson, and Denise Wootten
- Subjects
Cryo-electron microscopy ,Binding pocket ,Computational biology ,CHO Cells ,Spodoptera ,Receptors, G-Protein-Coupled ,Small Molecule Libraries ,03 medical and health sciences ,0302 clinical medicine ,Cricetulus ,Structural Biology ,Cricetinae ,Drug Discovery ,Sf9 Cells ,Image acquisition ,Animals ,Humans ,Molecular Biology ,030304 developmental biology ,G protein-coupled receptor ,0303 health sciences ,Chemistry ,Drug discovery ,030302 biochemistry & molecular biology ,Cryoelectron Microscopy ,Single-Domain Antibodies ,Method development ,Bound drug ,030217 neurology & neurosurgery ,Protein Binding - Abstract
G protein-coupled receptors (GPCRs) are the largest class of cell surface drug targets. Advances in biochemical approaches for the stabilisation of GPCR:transducer complexes together with improvements in the technology and application of cryo-EM has recently opened up new possibilities for structure-assisted drug design of GPCR agonists. Nonetheless, limitations in the commercial application of some of these approaches, including the use of nanobody 35 (Nb35) for stabilisation of GPCR:Gs complexes, and the high cost of 300kV imaging have restricted broad application of cryo-EM in drug discovery. Here, using the PF 06882961-bound GLP-1R as exemplar, we validated formation of stable complexes with a modified Gs protein in the absence of Nb35 that had equivalent resolution in the drug binding pocket to complexes solved in the presence of Nb35, while the G protein displayed increased conformational dynamics. In parallel, we assessed the performance of 200kV versus 300kV image acquisition using a Falcon 4 or K3 direct electron detector. We show that with 300kV Krios, both bottom mounted Falcon 4 and energy filtered (25eV slit) Bio-Quantum K3 produced similar resolution. Moreover, the 200kV Glacios with bottom mounted Falcon 4 yielded a 3.2 Å map with clear density for bound drug and multiple structurally ordered waters. Our work paves the way for broader commercial application of cryo-EM for GPCR drug discovery.
- Published
- 2021
36. Structure and dynamics of the CGRP receptor in apo and peptide-bound forms
- Author
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Patrick M. Sexton, Sarah J. Piper, Denise Wootten, Karen J. Gregory, Arthur Christopoulos, Daniel J. Garama, Yi Lynn Liang, Debbie L. Hay, Matthew J. Belousoff, Jianjun Cao, Tracy M. Josephs, Katie Leach, and Radostin Danev
- Subjects
Models, Molecular ,G protein ,Protein Conformation ,Calcitonin Gene-Related Peptide ,Peptide ,Hydrogen Deuterium Exchange-Mass Spectrometry ,Plasma protein binding ,Calcitonin gene-related peptide ,Moths ,Ligands ,Protein Structure, Secondary ,Cell Line ,Receptor Activity-Modifying Protein 1 ,03 medical and health sciences ,0302 clinical medicine ,Protein structure ,GTP-Binding Protein alpha Subunits, Gs ,Animals ,Humans ,Protein Interaction Domains and Motifs ,Receptor ,CGRP receptor complex ,030304 developmental biology ,G protein-coupled receptor ,chemistry.chemical_classification ,0303 health sciences ,Multidisciplinary ,Chemistry ,Calcitonin Receptor-Like Protein ,Cell Membrane ,Cryoelectron Microscopy ,3. Good health ,Biophysics ,Apoproteins ,030217 neurology & neurosurgery ,Protein Binding ,Receptors, Calcitonin Gene-Related Peptide - Abstract
Dynamic activation of a GPCR G protein–coupled receptors (GPCRs) coordinate a complex information flow between the outside and inside of a cell. An increasing number of GPCR structures provide insight into function. However, the dynamics that link extracellular sensing to intracellular signaling are not completely understood, because GPCRs used in structure determination are generally modified to constrain their dynamics. Josephs et al. succeeded in determining the structures of an unmodified calcitonin gene–related peptide receptor, which is implicated in migraines, both alone and bound to its neuropeptide ligand. Based on the structures and data from complementary biophysical techniques, they show that initial binding of the peptide causes only minor conformational changes of the GPCR, but dynamically causes changes at the intracellular side that facilitate G protein binding and activation. Science , this issue p. eabf7258
- Published
- 2020
37. Structure and dynamics of semaglutide- and taspoglutide-bound GLP-1R-Gs complexes
- Author
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Xin Zhang, Patrick M. Sexton, Yi Lynn Liang, Denise Wootten, Radostin Danev, and Matthew J. Belousoff
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Models, Molecular ,endocrine system ,QH301-705.5 ,media_common.quotation_subject ,cryoelectron microscopy ,Glucagon-Like Peptides ,Taspoglutide ,Peptide ,Type 2 diabetes ,Carbohydrate metabolism ,Pharmacology ,General Biochemistry, Genetics and Molecular Biology ,GPCRs ,Glucagon-Like Peptide-1 Receptor ,Cell Line ,Structure-Activity Relationship ,Protein Domains ,GTP-Binding Proteins ,medicine ,Humans ,Biology (General) ,Receptor ,G protein-coupled receptor ,media_common ,GLP-1R ,chemistry.chemical_classification ,semaglutide ,Chemistry ,Semaglutide ,digestive, oral, and skin physiology ,Appetite ,medicine.disease ,Multiprotein Complexes ,Peptides ,GPCR dynamics ,hormones, hormone substitutes, and hormone antagonists ,Protein Binding - Abstract
Summary: The glucagon-like peptide-1 receptor (GLP-1R) regulates insulin secretion, carbohydrate metabolism, and appetite and is an important target for treatment of type 2 diabetes and obesity. Multiple GLP-1R agonists have entered into clinical trials, with some, such as semaglutide, progressing to approval. Others, including taspoglutide, failed due to the high incidence of side effects or insufficient efficacy. GLP-1R agonists have a broad spectrum of signaling profiles, but molecular understanding is limited by a lack of structural information on how different agonists engage with the GLP-1R. Here, we report cryoelectron microscopy (cryo-EM) structures and cryo-EM 3D variability analysis of semaglutide- and taspoglutide-bound GLP-1R-Gs protein complexes. These reveal similar peptide interactions to GLP-1 but different motions within the receptor and bound peptides, providing insights into the molecular determinants of GLP-1R peptide engagement.
- Published
- 2020
38. Metal Dependent Dynamic Equilibrium: A Regulatory Mechanism for M17 Aminopeptidases fromPlasmodium falciparumandPlasmodium vivax
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Sheena McGowan, Natalie A. Borg, Sarah C. Atkinson, Matthew J Belousoff, Hariprasad Venugopal, Tess R. Malcolm, and Nyssa Drinkwater
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biology ,Stereochemistry ,Chemistry ,Metal ions in aqueous solution ,Active site ,Plasmodium falciparum ,Random hexamer ,Exopeptidase ,biology.organism_classification ,Metal ,Tetramer ,visual_art ,biology.protein ,visual_art.visual_art_medium ,Dynamic equilibrium - Abstract
M17 leucyl aminopeptidases are metal-dependent exopeptidases that rely on oligomerization to diversify their functional roles. The M17 aminopeptidases fromPlasmodium falciparum(PfA-M17) andPlasmodium vivax(Pv-M17) function as catalytically active hexamers to acquire free amino acids from human hemoglobin and are drug targets for the design of novel anti-malarial agents. In this study, we found that the active site metal ions essential for catalytic activity have a secondary structural role mediating the formation of active hexamers. We found thatPfA-M17 andPv-M17 exist in a metal-dependent dynamic equilibrium between active hexameric species and smaller inactive species, that can be controlled by manipulating the identity and concentration of metal ions available. Mutation of residues involved in metal ion binding impaired catalytic activity and the formation of active hexamers. Structural resolution of thePv-M17 hexameric species revealed thatPfA-M17 andPv-M17 bind metal ions and substrates in a conserved fashion, althoughPv-M17 forms the active hexamer more readily and processes substrates faster thanPfA-M17. On the basis of solution studies and structures determined by cryo-electron microscopy, we propose a dynamic equilibrium between monomer dimer tetramer hexamer, which becomes directional towards the large oligomeric states with the addition of metal ions. M17 aminopeptidases can exploit this sophisticated metal-dependent dynamic equilibrium to regulate formation of the catalytically active hexamer and therefore regulate catalysis.
- Published
- 2020
39. BonA from Acinetobacter baumannii forms a divisome-localized decamer that supports outer envelope function
- Author
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Faye C. Morris, Pok Man Leung, Trevor Lithgow, Matthew J. Belousoff, Anton Y. Peleg, Simone Beckham, Jian Li, Eva Heinz, Chris Greening, Sachith D. Gunasinghe, Rhys Grinter, and Rhys A. Dunstan
- Subjects
chemistry.chemical_compound ,Strain (chemistry) ,biology ,Cell division ,Phylogenetic tree ,Chemistry ,Peptidoglycan ,biology.organism_classification ,Bacterial outer membrane ,Bacteria ,Function (biology) ,Acinetobacter baumannii ,Cell biology - Abstract
Acinetobacter baumannii is a high-risk pathogen due to the rapid global spread of multi-drug resistant lineages. Its phylogenetic divergence from other ESKAPE pathogens means that determinants of its antimicrobial resistance can be difficult to extrapolate from other widely studied bacteria. A recent study showed that A. baumannii upregulates production of an outer-membrane lipoprotein, which we designate BonA, in response to challenge with polymyxins. Here we show that BonA has limited sequence similarity and distinct structural features compared to lipoproteins from other bacterial species. Analyses through X-ray crystallography, small-angle X-ray scattering, electron microscopy, and multiangle light scattering demonstrate that BonA has a dual BON-domain architecture and forms a decamer via an unusual oligomerization mechanism. This analysis also indicates this decamer is transient, suggesting dynamic oligomerization plays a role in BonA function. Antisera recognizing BonA shows it is an outer membrane protein localized to the divisome. Loss of BonA modulates the density of the outer membrane, consistent with a change in its structure or link to the peptidoglycan, and prevents motility in a clinical strain (ATCC 17978). Consistent with these findings, the dimensions of the BonA decamer are sufficient to permeate the peptidoglycan layer, with the potential to form a membrane-spanning complex during cell division.
- Published
- 2020
40. Routine sub-2.5 Å cryo-EM structure determination of B-family G protein-coupled receptors
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Patrick M. Sexton, Xin Zhang, Yi Lynn Liang, Matthew J. Belousoff, Radostin Danev, and Denise Wootten
- Subjects
Flexibility (engineering) ,Software ,Membrane protein ,Computer science ,Cryo-electron microscopy ,business.industry ,Microscopy ,Biological system ,Grid ,business ,Energy (signal processing) ,Nanodisc ,G protein-coupled receptor - Abstract
Cryo-electron microscopy (cryo-EM) experienced game-changing hardware and software advances about a decade ago. Since then, there have been gradual and steady improvements in experimental and data analysis methods. Nonetheless, structural analysis of nonsymmetric membrane proteins, such as G protein-coupled receptors (GPCRs), remains challenging. Their relatively low molecular weight and obstruction by the micelle/nanodisc result in marginal signal levels, which combined with the intrinsic flexibility of such complexes creates difficult structural study scenarios. Pushing the performance limits of cryo-EM requires careful optimization of all experimental aspects. To this end, it is necessary to build quantitative knowledge of the effect each parameter has on the outcome. Here, we present in-depth analysis of the influence of the main cryo-EM experimental factors on the performance for GPCR structure determination. We used a tandem experiment approach that combined real-world structural studies with parameter testing. We quantified the effects of using a Volta phase plate, zero-loss energy filtering, objective lens aperture, defocus magnitude, total exposure, and grid type. Through such systematic optimization of the experimental conditions, it has been possible to routinely determine class B1 GPCR structures at resolutions better than 2.5 Å. The improved fidelity of such maps helps to build higher confidence atomic models and will be crucial for the future expansion of cryo-EM into the structure-based drug design domain. The optimization guidelines drafted here are not limited to GPCRs and can be applied directly for the study of other challenging membrane protein targets.
- Published
- 2020
41. Structure and dynamics of the active Gs-coupled human secretin receptor
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Arthur Christopoulos, Sebastian G.B. Furness, Yi Lynn Liang, Giuseppe Deganutti, Matthew J. Belousoff, Maoqing Dong, Christopher A. Reynolds, Alisa Glukhova, Sarah J. Piper, Radostin Danev, Patrick M. Sexton, Maryam Khoshouei, Denise Wootten, Kaleeckal G. Harikumar, and Laurence J. Miller
- Subjects
Models, Molecular ,0301 basic medicine ,Insecta ,General Physics and Astronomy ,Peptide ,02 engineering and technology ,Plasma protein binding ,Crystallography, X-Ray ,Protein Structure, Secondary ,Receptors, G-Protein-Coupled ,Secretin ,Protein structure ,Cricetinae ,GTP-Binding Protein alpha Subunits, Gs ,lcsh:Science ,Peptide sequence ,chemistry.chemical_classification ,Multidisciplinary ,Chemistry ,Gastroenterology ,021001 nanoscience & nanotechnology ,Structural biology ,0210 nano-technology ,Hydrophobic and Hydrophilic Interactions ,hormones, hormone substitutes, and hormone antagonists ,Protein Binding ,Science ,Protein domain ,Molecular Dynamics Simulation ,digestive system ,Article ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Receptors, Gastrointestinal Hormone ,03 medical and health sciences ,Protein Domains ,Animals ,Humans ,Amino Acid Sequence ,Cysteine ,G protein-coupled receptor ,Binding Sites ,Cryoelectron Microscopy ,Hydrogen Bonding ,General Chemistry ,030104 developmental biology ,Biophysics ,lcsh:Q - Abstract
The class B secretin GPCR (SecR) has broad physiological effects, with target potential for treatment of metabolic and cardiovascular disease. Molecular understanding of SecR binding and activation is important for its therapeutic exploitation. We combined cryo-electron microscopy, molecular dynamics, and biochemical cross-linking to determine a 2.3 Å structure, and interrogate dynamics, of secretin bound to the SecR:Gs complex. SecR exhibited a unique organization of its extracellular domain (ECD) relative to its 7-transmembrane (TM) core, forming more extended interactions than other family members. Numerous polar interactions formed between secretin and the receptor extracellular loops (ECLs) and TM helices. Cysteine-cross-linking, cryo-electron microscopy multivariate analysis and molecular dynamics simulations revealed that interactions between peptide and receptor were dynamic, and suggested a model for initial peptide engagement where early interactions between the far N-terminus of the peptide and SecR ECL2 likely occur following initial binding of the peptide C-terminus to the ECD., The class B secretin GPCR (SecR) has broad physiological effects, with target potential for treatment of metabolic and cardiovascular disease. Here, authors present a cryo-EM structure and biochemical studies of secretin binding to the SecR:Gs complex which show that interactions between peptide and receptor were dynamic.
- Published
- 2020
42. Characterization of the Core Ribosomal Binding Region for the Oxazolidone Family of Antibiotics Using Cryo-EM
- Author
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Hari Venugopal, David W. Lupton, Kieran Deane-Alder, Trevor Lithgow, Edward Marschall, Rebecca S. Bamert, Matthew J. Belousoff, and Alexander Wright
- Subjects
Pharmacology ,Peptidyl transferase ,biology ,Stereochemistry ,Oxazolidone ,Ribosomal RNA ,Ribosome ,chemistry.chemical_compound ,chemistry ,Linezolid ,biology.protein ,Pharmacology (medical) ,Tedizolid ,Structural motif ,50S - Abstract
[Image: see text] Linezolid and tedizolid are oxazolidinones with established clinical utility for the treatment of Gram-positive pathogens. Over time it has become apparent that even modest structural changes to the core phenyl oxazolidinone leads to drastic changes in biological activity. Consequently, the structure–activity relationship around the core oxazolidinone is constantly evolving, often reflected with new structural motifs present in nascent oxazolidinones. Herein we describe the use of cryo-electron microscopy to examine the differences in binding of several functionally different oxazolidinones in the hopes of enhanced understanding of their SAR. Tedizolid, radezolid, T145, and contezolid have been examined within the peptidyl transferase center (PTC) of the 50S ribosomal subunit from methicillin resistant Staphylococcus aureus. The ribosome–antibiotic complexes were resolved to a resolution of around 3 Å enabling unambiguous assignment of how each antibiotic interacts with the PTC.
- Published
- 2020
43. Cryo-electron microscopy structure of the glucagon receptor with a dual-agonist peptide
- Author
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Denise Wootten, Guangyao Lin, Qiuxiang Tan, Li Zhong, Ming-Wei Wang, Patrick M. Sexton, Rulue Chang, Antao Dai, Radostin Danev, Matthew J. Belousoff, Shuo Han, Yi Lynn Liang, Dehua Yang, Xin Zhang, Anna Qiao, Lijun Shao, Beili Wu, and Limin Ma
- Subjects
0301 basic medicine ,Peptide ,Biochemistry ,Glucagon-Like Peptide-1 Receptor ,03 medical and health sciences ,Protein Domains ,Functional selectivity ,Receptors, Glucagon ,Animals ,Humans ,Receptor ,Protein Structure, Quaternary ,Molecular Biology ,Glucagon-like peptide 1 receptor ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,C-terminus ,Cryoelectron Microscopy ,Cell Biology ,Transmembrane domain ,030104 developmental biology ,chemistry ,Structural biology ,Protein Structure and Folding ,Biophysics ,Peptides ,Glucagon receptor - Abstract
Unimolecular dual agonists of the glucagon (GCG) receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R) are a new class of drugs that are potentially superior to GLP-1R–specific agonists for the management of metabolic disease. The dual-agonist, peptide 15 (P15), is a glutamic acid 16 analog of GCG with GLP-1 peptide substitutions between amino acids 17 and 24 that has potency equivalent to those of the cognate peptide agonists at the GCGR and GLP-1R. Here, we have used cryo-EM to solve the structure of an active P15-GCGR-G(s) complex and compared this structure to our recently published structure of the GCGR-G(s) complex bound to GCG. This comparison revealed that P15 has a reduced interaction with the first extracellular loop (ECL1) and the top of transmembrane segment 1 (TM1) such that there is increased mobility of the GCGR extracellular domain and at the C terminus of the peptide compared with the GCG-bound receptor. We also observed a distinct conformation of ECL3 and could infer increased mobility of the far N-terminal His-1 residue in the P15-bound structure. These regions of conformational variance in the two peptide-bound GCGR structures were also regions that were distinct between GCGR structures and previously published peptide-bound structures of the GLP-1R, suggesting that greater conformational dynamics may contribute to the increased efficacy of P15 in activation of the GLP-1R compared with GCG. The variable domains in this receptor have previously been implicated in biased agonism at the GLP-1R and could result in altered signaling of P15 at the GCGR compared with GCG.
- Published
- 2020
44. Structural basis for control of antibiotic production by bacterial hormones
- Author
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Shanshan Zhou, Hussain Bhukya, Nicolas Malet, Peter J. Harrison, Dean Rea, Matthew J. Belousoff, Hariprasad Venugopal, Paulina K. Sydor, Kathryn M. Styles, Lijiang Song, Max J. Cryle, Lona M. Alkhalaf, Vilmos Fülöp, Gregory L. Challis, and Christophe Corre
- Subjects
2. Zero hunger ,0303 health sciences ,03 medical and health sciences ,010304 chemical physics ,0103 physical sciences ,01 natural sciences ,030304 developmental biology ,3. Good health - Abstract
SummaryActinobacteria produce numerous antibiotics and other specialised metabolites with important applications in medicine and agriculture. Diffusible hormones frequently control the production of such metabolites by binding TetR family transcriptional repressors (TFTRs), but the molecular basis for this remains unclear. The production of methylenomycin antibiotics in Streptomyces coelicolor A3(2) is initiated by binding of 2-alkyl-4-hydroxymethylfuran-3-carboxylic acid (AHFCA) hormones to the TFTR MmfR. Here, we report the X-ray crystal structure of an MmfR-AHFCA complex, establishing the structural basis for hormone recognition. We also elucidate the mechanism for DNA release upon hormone binding by single particle cryo-electron microscopy of an MmfR-operator complex. Electrophoretic mobility shift assays with MmfR mutants and synthetic AHFCA analogues illuminate the role played by individual amino acid residues and hormone functional groups in ligand recognition and DNA release. These findings will facilitate the exploitation of Actinobacterial hormones and their associated TFTRs in synthetic biology and novel antibiotic discovery.
- Published
- 2020
- Full Text
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45. Differential GLP-1R Binding and Activation by Peptide and Non-peptide Agonists
- Author
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Thomas Egebjerg, Steffen Reedtz-Runge, Christopher J. Langmead, Hari Venugopal, Peishen Zhao, Sheng Y. Ang, Radostin Danev, Patrick M. Sexton, Gregory D. Stewart, David E. Gloriam, Alisa Glukhova, Tin T. Truong, Sebastian G.B. Furness, Christina Rye Underwood, Xin Zhang, Albert J. Kooistra, Laurence J. Miller, Arthur Christopoulos, Petr Šenel, Yi Lynn Liang, Matthew J. Belousoff, and Denise Wootten
- Subjects
Agonist ,medicine.drug_class ,High resolution ,Peptide ,Biology ,Non peptide ,Glucagon-Like Peptide-1 Receptor ,Receptors, G-Protein-Coupled ,Structure-Activity Relationship ,03 medical and health sciences ,0302 clinical medicine ,Glucagon-Like Peptide 1 ,Functional selectivity ,Extracellular ,medicine ,Animals ,Humans ,Structure–activity relationship ,Binding site ,Receptor ,Molecular Biology ,030304 developmental biology ,G protein-coupled receptor ,chemistry.chemical_classification ,0303 health sciences ,Binding Sites ,Chemistry ,Cryoelectron Microscopy ,Cell Biology ,Cell biology ,Peptides ,030217 neurology & neurosurgery - Abstract
Peptide drugs targeting class B1 G-protein-coupled receptors (GPCRs) can treat multiple diseases; however, there remains substantial interest in the development of orally delivered non-peptide drugs. Here, we reveal unexpected overlap between signaling and regulation of the glucagon-like peptide-1 (GLP-1) receptor by the non-peptide agonist PF 06882961 and GLP-1 that was not observed for another compound, CHU-128. Compounds from these patent series, including PF 06882961, are currently in clinical trials for treatment of type 2 diabetes. High-resolution cryoelectron microscopy (cryo-EM) structures reveal that the binding sites for PF 06882961 and GLP-1 substantially overlap, whereas CHU-128 adopts a unique binding mode with a more open receptor conformation at the extracellular face. Structural differences involving extensive water-mediated hydrogen bond networks could be correlated to functional data to understand how PF 06882961, but not CHU-128, can closely mimic the pharmacological properties of GLP-1. These findings will facilitate rational structure-based discovery of non-peptide agonists targeting class B GPCRs.
- Published
- 2020
46. Delivery of femtolitre droplets using surface acoustic wave based atomisation for cryo-EM grid preparation
- Author
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Johnson Mak, Hari Venugopal, Alex de Marco, Dariush Ashtiani, Adrian Neild, Matthew J. Belousoff, and Bradley A. Spicer
- Subjects
0301 basic medicine ,Materials science ,Cryo-electron microscopy ,Sample (material) ,Acoustics ,Cryoelectron Microscopy ,Microfluidics ,Surface acoustic wave ,Detector ,Acoustic wave ,Grid ,Vitrification ,Specimen Handling ,03 medical and health sciences ,030104 developmental biology ,Femtolitre ,Structural Biology ,Freezing - Abstract
Cryo-Electron Microscopy (cryo-EM) has become an invaluable tool for structural biology. Over the past decade, the advent of direct electron detectors and automated data acquisition has established cryo-EM as a central method in structural biology. However, challenges remain in the reliable and efficient preparation of samples in a manner which is compatible with high time resolution. The delivery of sample onto the grid is recognized as a critical step in the workflow as it is a source of variability and loss of material due to the blotting which is usually required. Here, we present a method for sample delivery and plunge freezing based on the use of Surface Acoustic Waves to deploy 6–8 µm droplets to the EM grid. This method minimises the sample dead volume and ensures vitrification within 52.6 ms from the moment the sample leaves the microfluidics chip. We demonstrate a working protocol to minimize the atomised volume and apply it to plunge freeze three different samples and provide proof that no damage occurs due to the interaction between the sample and the acoustic waves.
- Published
- 2018
47. Structure of Pseudomonas aeruginosa ribosomes from an aminoglycoside-resistant clinical isolate
- Author
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Søren Molin, Ruggero La Rosa, Y. Halfon, Matthew J. Belousoff, Ada Yonath, Rocio Espinosa Portero, Donna Matzov, Z. Eyal, Anat Bashan, Helle Krogh Johansen, Alicia Jiménez-Fernández, and Ella Zimmerman
- Subjects
0303 health sciences ,Modern medicine ,Multidisciplinary ,Aminoglycoside ,030306 microbiology ,Chemistry ,Resistance ,Antibiotic ,Translation (biology) ,Ribosomal RNA ,Genetic code ,Ribosome ,Cystic fibrosis ,3. Good health ,03 medical and health sciences ,Biochemistry ,Ribosomal protein ,Protein biosynthesis ,030304 developmental biology - Abstract
Resistance to antibiotics has become a major threat to modern medicine. The ribosome plays a fundamental role in cell vitality by the translation of the genetic code into proteins; hence, it is a major target for clinically useful antibiotics. We report here the cryo-electron microscopy structures of the ribosome of a pathogenic aminoglycoside (AG)-resistant Pseudomonas aeruginosa strain, as well as of a nonresistance strain isolated from a cystic fibrosis patient. The structural studies disclosed defective ribosome complex formation due to a conformational change of rRNA helix H69, an essential intersubunit bridge, and a secondary binding site of the AGs. In addition, a stable conformation of nucleotides A1486 and A1487, pointing into helix h44, is created compared to a non-AG-bound ribosome. We suggest that altering the conformations of ribosomal protein uL6 and rRNA helix H69, which interact with initiation-factor IF2, interferes with proper protein synthesis initiation.
- Published
- 2019
48. Molecular basis for control of antibiotic production by a bacterial hormone
- Author
-
Shanshan, Zhou, Hussain, Bhukya, Nicolas, Malet, Peter J, Harrison, Dean, Rea, Matthew J, Belousoff, Hariprasad, Venugopal, Paulina K, Sydor, Kathryn M, Styles, Lijiang, Song, Max J, Cryle, Lona M, Alkhalaf, Vilmos, Fülöp, Gregory L, Challis, and Christophe, Corre
- Subjects
Models, Molecular ,Cryoelectron Microscopy ,Streptomyces coelicolor ,DNA ,Crystallography, X-Ray ,Ligands ,Hormones ,Anti-Bacterial Agents ,Repressor Proteins ,Structure-Activity Relationship ,Bacterial Proteins ,Apoproteins ,Furans ,Peptides ,Signal Transduction - Abstract
Actinobacteria produce numerous antibiotics and other specialized metabolites that have important applications in medicine and agriculture
- Published
- 2019
49. Structure and Dynamics of Adrenomedullin Receptors AM
- Author
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Yi-Lynn, Liang, Matthew J, Belousoff, Madeleine M, Fletcher, Xin, Zhang, Maryam, Khoshouei, Giuseppe, Deganutti, Cassandra, Koole, Sebastian G B, Furness, Laurence J, Miller, Debbie L, Hay, Arthur, Christopoulos, Christopher A, Reynolds, Radostin, Danev, Denise, Wootten, and Patrick M, Sexton
- Abstract
[Image: see text] Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) receptors are critically important for metabolism, vascular tone, and inflammatory response. AM receptors are also required for normal lymphatic and blood vascular development and angiogenesis. They play a pivotal role in embryo implantation and fertility and can provide protection against hypoxic and oxidative stress. CGRP and AM receptors are heterodimers of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) (CGRPR), as well as RAMP2 or RAMP3 (AM(1)R and AM(2)R, respectively). However, the mechanistic basis for RAMP modulation of CLR phenotype is unclear. In this study, we report the cryo-EM structure of the AM(1)R in complex with AM and Gs at a global resolution of 3.0 Å, and structures of the AM(2)R in complex with either AM or intermedin/adrenomedullin 2 (AM2) and Gs at 2.4 and 2.3 Å, respectively. The structures reveal distinctions in the primary orientation of the extracellular domains (ECDs) relative to the receptor core and distinct positioning of extracellular loop 3 (ECL3) that are receptor-dependent. Analysis of dynamic data present in the cryo-EM micrographs revealed additional distinctions in the extent of mobility of the ECDs. Chimeric exchange of the linker region of the RAMPs connecting the TM helix and the ECD supports a role for this segment in controlling receptor phenotype. Moreover, a subset of the motions of the ECD appeared coordinated with motions of the G protein relative to the receptor core, suggesting that receptor ECD dynamics could influence G protein interactions. This work provides fundamental advances in our understanding of GPCR function and how this can be allosterically modulated by accessory proteins.
- Published
- 2019
50. Activation of the GLP-1 receptor by a non-peptidic agonist
- Author
-
Arthur Christopoulos, Patrick M. Sexton, Lachlan Clydesdale, Asuka Inoue, Yi Lynn Liang, Giuseppe Deganutti, Tin T. Truong, Matthew J. Belousoff, Michael E. Christe, Sebastian G.B. Furness, Michael Gregory Bell, Christopher A. Reynolds, Denise Wootten, Kyle W. Sloop, Ming-Wei Wang, Francis S. Willard, Peishen Zhao, Laurence J. Miller, Madeleine M. Fletcher, and Radostin Danev
- Subjects
0301 basic medicine ,Agonist ,Models, Molecular ,medicine.drug_class ,Pyridines ,Phenylalanine ,CHO Cells ,Glucagon-Like Peptide-1 Receptor ,03 medical and health sciences ,0302 clinical medicine ,Protein structure ,Cricetulus ,Cricetinae ,medicine ,Functional selectivity ,Extracellular ,Animals ,Humans ,Receptor ,Protein Structure, Quaternary ,Glucagon-like peptide 1 receptor ,Multidisciplinary ,Chemistry ,Ligand (biochemistry) ,Isoquinolines ,Protein Structure, Tertiary ,Transmembrane domain ,Kinetics ,030104 developmental biology ,Structural Homology, Protein ,Biophysics ,030217 neurology & neurosurgery - Abstract
Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, including diabetes and obesity1. Structures of active receptors reveal peptide agonists engage deep within the receptor core, leading to an outward movement of extracellular loop 3 and the tops of transmembrane helices 6 and 7, an inward movement of transmembrane helix 1, reorganization of extracellular loop 2 and outward movement of the intracellular side of transmembrane helix 6, resulting in G-protein interaction and activation2,3,4,5,6. Here we solved the structure of a non-peptide agonist, TT-OAD2, bound to the glucagon-like peptide-1 (GLP-1) receptor. Our structure identified an unpredicted non-peptide agonist-binding pocket in which reorganization of extracellular loop 3 and transmembrane helices 6 and 7 manifests independently of direct ligand interaction within the deep transmembrane domain pocket. TT-OAD2 exhibits biased agonism, and kinetics of G-protein activation and signalling that are distinct from peptide agonists. Within the structure, TT-OAD2 protrudes beyond the receptor core to interact with the lipid or detergent, providing an explanation for the distinct activation kinetics that may contribute to the clinical efficacy of this compound series. This work alters our understanding of the events that drive the activation of class B receptors.
- Published
- 2019
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