125 results on '"Philip D. Jeffrey"'
Search Results
2. Structure-Function Studies of the Bacillus subtilis Ric Proteins Identify the Fe-S Cluster-Ligating Residues and Their Roles in Development and RNA Processing
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Felix Adusei-Danso, Faisal Tarique Khaja, Micaela DeSantis, Philip D. Jeffrey, Eugenie Dubnau, Borries Demeler, Matthew B. Neiditch, and David Dubnau
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Ric proteins ,iron sulfur cluster ,Bacillus subtilis ,RNA processing ,bacterial development ,Microbiology ,QR1-502 - Abstract
ABSTRACT In Bacillus subtilis, the RicA (YmcA), RicF (YlbF), and RicT (YaaT) proteins accelerate the phosphorylation of the transcription factor Spo0A, contributing to genetic competence, sporulation, and biofilm formation, and are also essential for the correct maturation of several protein-encoding and riboswitch RNAs. These proteins form a stable complex (RicAFT) that carries two [4Fe-4S]+2 clusters. We show here that the complex is a 1:1:1 heterotrimer, and we present the X-ray crystal structures of a RicAF heterotetramer and of a RicA dimer. We also demonstrate that one of the Fe-S clusters (cluster 1) is ligated by cysteine residues donated exclusively by RicT and can be retained when the RicT monomer is purified by itself. Cluster 2 is ligated by C167 from RicT, by C134 and C146 located near the C terminus of RicF, and by C141 at the C terminus of RicA. These findings imply the following novel arrangement: adjacent RicT residues C166 and 167 ligate clusters 1 and 2, respectively, while cluster 2 is ligated by cysteine residues from RicT, RicA, and RicF. Thus, the two clusters must lie close to one another and at the interface of the RicAFT protomers. We also show that the cluster-ligating cysteine residues, and therefore most likely both Fe-S clusters, are essential for cggR-gapA mRNA maturation, for the regulation of ricF transcript stability, and for several Ric-associated developmental phenotypes, including competence for transformation, biofilm formation, and sporulation. Finally, we present evidence that RicAFT, RicAF, and RicA and the RicT monomer may play distinct regulatory roles in vivo. IMPORTANCE The RicA, RicF, and RicT proteins are widely conserved among the firmicute bacteria and play multiple roles in Bacillus subtilis. Among the phenotypes associated with the inactivation of these proteins are the inability to be genetically transformed or to form biofilms, a decrease in sporulation frequency, and changes in the stability and maturation of multiple RNA species. Despite their importance, the molecular mechanisms of Ric protein activities have not been elucidated and the roles of the two iron-sulfur clusters on the complex of the three proteins are not understood. To unravel the mechanisms of Ric action, molecular characterization of the complex and of its constituent proteins is essential. This report represents a major step toward understanding the structures of the Ric proteins, the arrangement and roles of the Fe-S clusters, and the phenotypes associated with Ric mutations.
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- 2019
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3. The Sec1/Munc18 protein Vps45 holds the Qa-SNARE Tlg2 in an open conformation
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Travis J Eisemann, Frederick Allen, Kelly Lau, Gregory R Shimamura, Philip D Jeffrey, and Frederick M Hughson
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membrane fusion ,SNAREs ,sec1/munc18 (SM) proteins ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Fusion of intracellular trafficking vesicles is mediated by the assembly of SNARE proteins into membrane-bridging complexes. SNARE-mediated membrane fusion requires Sec1/Munc18-family (SM) proteins, SNARE chaperones that can function as templates to catalyze SNARE complex assembly. Paradoxically, the SM protein Munc18-1 traps the Qa-SNARE protein syntaxin-1 in an autoinhibited closed conformation. Here we present the structure of a second SM–Qa-SNARE complex, Vps45–Tlg2. Strikingly, Vps45 holds Tlg2 in an open conformation, with its SNARE motif disengaged from its Habc domain and its linker region unfolded. The domain 3a helical hairpin of Vps45 is unfurled, exposing the presumptive R-SNARE binding site to allow template complex formation. Although Tlg2 has a pronounced tendency to form homo-tetramers, Vps45 can rescue Tlg2 tetramers into stoichiometric Vps45–Tlg2 complexes. Our findings demonstrate that SM proteins can engage Qa-SNAREs using at least two different modes, one in which the SNARE is closed and one in which it is open.
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- 2020
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4. The PqsE Active Site as a Target for Small Molecule Antimicrobial Agents against Pseudomonas aeruginosa
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Isabelle R. Taylor, Philip D. Jeffrey, Dina A. Moustafa, Joanna B. Goldberg, and Bonnie L. Bassler
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Biochemistry - Published
- 2022
5. Structure of a Membrane Tethering Complex Incorporating Multiple SNAREs
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Kevin A. DAmico, Abigail E. Stanton, Jaden D. Shirkey, Sophie M. Travis, Philip D. Jeffrey, and Frederick M. Hughson
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Most membrane fusion reactions in eukaryotic cells are mediated by membrane tethering complexes (MTCs) and SNARE proteins. MTCs are much larger than SNAREs and are thought to mediate the initial attachment of two membranes. Complementary SNAREs then form membrane-bridging complexes whose assembly draws the membranes together for fusion. Here, we present a cryo-EM structure of the simplest known MTC, the 255-kDa Dsl1 complex, bound to the two SNAREs that anchor it to the endoplasmic reticulum. N-terminal domains of the SNAREs form an integral part of the structure, stabilizing a Dsl1 complex configuration with remarkable and unexpected similarities to the 850-kDa exocyst MTC. The structure of the SNARE-anchored Dsl1 complex and its comparison with exocyst reveal what are likely to be common principles underlying MTC function. Our structure also implies that tethers and SNAREs can work together as a single integrated machine.
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- 2023
6. Aerobic Partial Oxidation of Alkanes Using Photodriven Iron Catalysis
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Nathan Coutard, Jonathan M. Goldberg, Henry U. Valle, Yuan Cao, Xiaofan Jia, Philip D. Jeffrey, T. Brent Gunnoe, and John T. Groves
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Inorganic Chemistry ,Physical and Theoretical Chemistry - Abstract
Photodriven oxidations of alkanes in trifluoroacetic acid using commercial and synthesized Fe(III) sources as catalyst precursors and dioxygen (O
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- 2021
7. The human microbiome encodes resistance to the antidiabetic drug acarbose
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Liping Zhao, Alexei Korennykh, Guojun Wu, Abhishek Biswas, Mohamed S. Donia, Michael A. Estrella, Philip D. Jeffrey, and Jared N. Balaich
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Models, Molecular ,Biology ,Article ,Drug Resistance, Bacterial ,medicine ,Animals ,Humans ,Hypoglycemic Agents ,Microbiome ,Organism ,Acarbose ,chemistry.chemical_classification ,Mouth ,Multidisciplinary ,Human microbiome ,biology.organism_classification ,Gastrointestinal Microbiome ,Phosphotransferases (Alcohol Group Acceptor) ,Enzyme ,chemistry ,Biochemistry ,Metagenomics ,Amylases ,Inactivation, Metabolic ,Metagenome ,Oral Microbiome ,Bacteria ,medicine.drug - Abstract
The human microbiome encodes a large repertoire of biochemical enzymes and pathways, most of which remain uncharacterized. Here, using a metagenomics-based search strategy, we discovered that bacterial members of the human gut and oral microbiome encode enzymes that selectively phosphorylate a clinically used antidiabetic drug, acarbose1,2, resulting in its inactivation. Acarbose is an inhibitor of both human and bacterial α-glucosidases3, limiting the ability of the target organism to metabolize complex carbohydrates. Using biochemical assays, X-ray crystallography and metagenomic analyses, we show that microbiome-derived acarbose kinases are specific for acarbose, provide their harbouring organism with a protective advantage against the activity of acarbose, and are widespread in the microbiomes of western and non-western human populations. These results provide an example of widespread microbiome resistance to a non-antibiotic drug, and suggest that acarbose resistance has disseminated in the human microbiome as a defensive strategy against a potential endogenous producer of a closely related molecule. Bacteria in the human gut and oral microbiome encode enzymes that selectively phosphorylate the antidiabetic drug acarbose—an inhibitor of both human and bacterial α-glucosidases—resulting in its inactivation and limiting the drug's effects on the ability of the host to metabolize complex carbohydrates.
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- 2021
8. A NR2E1‐interacting peptide of LSD1 inhibits the proliferation of brain tumour initiating cells
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Rong Hu, Umar Farook Shahul Hameed, Xiang Sun, Balakrishnan Shenbaga Moorthy, Wen Zhang, Philip D. Jeffrey, Li Zhou, Xin Ma, Fangjin Chen, Jianfeng Pei, Pankaj K. Giri, Yonggao Mou, Kunchithapadam Swaminathan, and Ping Yuan
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Cell Biology ,General Medicine - Abstract
Elimination of brain tumour initiating cells (BTICs) is important for the good prognosis of malignant brain tumour treatment. To develop a novel strategy targeting BTICs, we studied NR2E1(TLX) involved self-renewal mechanism of BTICs and explored the intervention means.NR2E1 and its interacting protein-LSD1 in BTICs were studied by gene interference combined with cell growth, tumour sphere formation, co-immunoprecipitation and chromatin immunoprecipitation assays. NR2E1 interacting peptide of LSD1 was identified by Amide Hydrogen/Deuterium Exchange and Mass Spectrometry (HDX-MS) and analysed by in vitro functional assays. The in vivo function of the peptide was examined with intracranial mouse model by transplanting patient-derived BTICs.We found NR2E1 recruits LSD1, a lysine demethylase, to demethylate mono- and di-methylated histone 3 Lys4 (H3K4me/me2) at the Pten promoter and repress its expression, thereby promoting BTIC proliferation. Using Amide Hydrogen/Deuterium Exchange and Mass Spectrometry (HDX-MS) method, we identified four LSD1 peptides that may interact with NR2E1. One of the peptides, LSD1-197-211 that locates at the LSD1 SWIRM domain, strongly inhibited BTIC proliferation by promoting Pten expression through interfering NR2E1 and LSD1 function. Furthermore, overexpression of this peptide in human BTICs can inhibit intracranial tumour formation.Peptide LSD1-197-211 can repress BTICs by interfering the synergistic function of NR2E1 and LSD1 and may be a promising lead peptide for brain tumour therapy in future.
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- 2022
9. Structure of human NADK2 reveals atypical assembly and regulation of NAD kinases from animal mitochondria
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Jin Du, Michael Estrella, Kristina Solorio-Kirpichyan, Philip D. Jeffrey, and Alexei Korennykh
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Mitochondrial Proteins ,Phosphotransferases (Alcohol Group Acceptor) ,Multidisciplinary ,Animals ,Humans ,Protein Multimerization ,NAD ,NADP ,Mitochondria - Abstract
All kingdoms of life produce essential nicotinamide dinucleotide NADP(H) using NAD kinases (NADKs). A panel of published NADK structures from bacteria, eukaryotic cytosol, and yeast mitochondria revealed similar tetrameric enzymes. Here, we present the 2.8-Å structure of the human mitochondrial kinase NADK2 with a bound substrate, which is an exception to this uniformity, diverging both structurally and biochemically from NADKs. We show that NADK2 harbors a unique tetramer disruptor/dimerization e lement, which is conserved in m itochondrial k inases of a nimals (EMKA) and absent from other NADKs. EMKA stabilizes the NADK2 dimer but prevents further NADK2 oligomerization by blocking the tetramerization interface. This structural change bears functional consequences and alters the activation mechanism of the enzyme. Whereas tetrameric NADKs undergo cooperative activation via oligomerization, NADK2 is a constitutively active noncooperative dimer. Thus, our data point to a unique regulation of NADP(H) synthesis in animal mitochondria achieved via structural adaptation of the NADK2 kinase.
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- 2022
10. Inhibitor Mimetic Mutations in the Pseudomonas aeruginosa PqsE Enzyme Reveal a Protein–Protein Interaction with the Quorum-Sensing Receptor RhlR That Is Vital for Virulence Factor Production
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Brad R. Henke, Bonnie L. Bassler, Isabelle R. Taylor, Chari D. Smith, Philip D. Jeffrey, and Jon E. Paczkowski
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0301 basic medicine ,chemistry.chemical_classification ,010405 organic chemistry ,Chemistry ,Pseudomonas aeruginosa ,Drug discovery ,Tryptophan ,Human pathogen ,General Medicine ,medicine.disease_cause ,01 natural sciences ,Biochemistry ,Virulence factor ,0104 chemical sciences ,Microbiology ,03 medical and health sciences ,Quorum sensing ,030104 developmental biology ,Enzyme ,medicine ,Molecular Medicine ,Receptor - Abstract
Pseudomonas aeruginosa is an opportunistic human pathogen that causes fatal infections. There exists an urgent need for new antimicrobial agents to combat P. aeruginosa. We conducted a screen for molecules that bind the virulence-controlling protein PqsE and characterized hit compounds for inhibition of PqsE enzymatic activity. The binding conformations of two inhibitory molecules, BB391 and BB393, were identified by crystallography, and inhibitor binding was mimicked by the substitution of PqsE residues E182 and S285 with tryptophan. Comparison of the inhibitor-mimetic mutations to the catalytically inactive PqsE D73A protein demonstrated that catalysis is not responsible for the role PqsE plays in driving virulence factor production. Rather, the PqsE E182W protein fails to interact with the quorum-sensing receptor, RhlR, and our results suggest that it is this interaction that is responsible for promoting virulence factor production in P. aeruginosa. These findings provide a new route for drug discovery efforts targeting PqsE.
- Published
- 2021
11. Structure of trypanosome coat protein VSGsur and function in suramin resistance
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Natalie Wiedemar, F. Nina Papavasiliou, Johan Zeelen, Hamidreza Hashemi, Joseph Verdi, Silvan Hälg, C. Erec Stebbins, Kathryn Perez, Pascal Mäser, Alexander Hempelmann, Philip D. Jeffrey, and Monique van Straaten
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Trypanosoma brucei rhodesiense ,Microbiology (medical) ,Protein Conformation ,Suramin ,Immunology ,Drug Resistance ,Plasma protein binding ,Trypanosoma brucei ,Crystallography, X-Ray ,Applied Microbiology and Biotechnology ,Microbiology ,Article ,03 medical and health sciences ,Protein structure ,parasitic diseases ,polycyclic compounds ,Genetics ,medicine ,Antigenic variation ,Binding site ,Immune Evasion ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Binding Sites ,biology ,030306 microbiology ,Chemistry ,Cell Biology ,medicine.disease ,biology.organism_classification ,Antigenic Variation ,Trypanocidal Agents ,Endocytosis ,3. Good health ,Cell biology ,Trypanosomiasis, African ,Mutation ,Glycoprotein ,Trypanosomiasis ,Variant Surface Glycoproteins, Trypanosoma ,Protein Binding ,medicine.drug - Abstract
Suramin has been a primary early-stage treatment for African trypanosomiasis for nearly 100 yr. Recent studies revealed that trypanosome strains that express the variant surface glycoprotein (VSG) VSGsur possess heightened resistance to suramin. Here, we show that VSGsur binds tightly to suramin but other VSGs do not. By solving high-resolution crystal structures of VSGsur and VSG13, we also demonstrate that these VSGs define a structurally divergent subgroup of the coat proteins. The co-crystal structure of VSGsur with suramin reveals that the chemically symmetric drug binds within a large cavity in the VSG homodimer asymmetrically, primarily through contacts of its central benzene rings. Structure-based, loss-of-contact mutations in VSGsur significantly decrease the affinity to suramin and lead to a loss of the resistance phenotype. Altogether, these data show that the resistance phenotype is dependent on the binding of suramin to VSGsur, establishing that the VSG proteins can possess functionality beyond their role in antigenic variation. The co-crystal structure of VSGsur with the trypanocidal compound suramin directly links the binding of the drug to the resistance phenotype displayed by strains of Trypanosoma brucei expressing VSGsur. Therefore, VSGs can have a function beyond that of antigenic variation.
- Published
- 2021
12. The PqsE active site as a target for small molecule antimicrobial agents againstPseudomonas aeruginosa
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Isabelle R. Taylor, Philip D. Jeffrey, Dina A. Moustafa, Joanna B. Goldberg, and Bonnie L. Bassler
- Abstract
The opportunistic pathogenPseudomonas aeruginosacauses antibiotic resistant, nosocomial infections in immuno-compromised individuals, and is a high priority for antimicrobial development. Key to pathogenicity inP. aeruginosaare biofilm formation and virulence factor production. Both traits are controlled by the cell-to-cell communication process called quorum sensing (QS). QS involves the synthesis, release, and population-wide detection of signal molecules called autoinducers. We previously reported that activity of the RhlR QS transcription factor depends on a protein-protein interaction with the hydrolase, PqsE, and PqsE catalytic activity is dispensable for this interaction. Nonetheless, the PqsE-RhlR interaction could be disrupted by substitution of an active site glutamate residue with tryptophan (PqsE(E182W)). Here, we show that disruption of the PqsE-RhlR interaction via either the E182W change or alteration of PqsE surface residues that are essential for the interaction with RhlR, attenuatesP. aeruginosainfection in a murine host. We use crystallography to characterize the conformational changes induced by the PqsE(E182W) substitution to define the mechanism underlying disruption of the PqsE-RhlR interaction. A loop rearrangement that repositions the E280 residue in PqsE(E182W) is responsible for the loss of interaction. We verify the implications garnered from the PqsE(E182W) structure using mutagenic, biochemical, and additional structural analyses. We present the next generation of molecules targeting the PqsE active site, including a structure of the tightest binding of these compounds, BB584, in complex with PqsE. The findings presented here provide insight for drug discovery againstP. aeruginosawith PqsE as the target.Author SummaryThe human pathogenPseudomonas aeruginosais resistant to many currently used antibiotics, making it a burden of urgent clinical importance.P. aeruginosapathogenicity is controlled by the bacterial cell-to-cell communication process called quorum sensing (QS). The function of one protein that controlsP. aeruginosaQS-directed virulence, RhlR, requires a protein-protein interaction with an enzyme called PqsE. When PqsE is blocked from interacting with RhlR,P. aeruginosais avirulent and incapable of infecting an animal host. Here, we validate the PqsE-RhlR interaction as a target for antibiotic development, and we present a mechanism for how such antibiotics could disrupt the PqsE-RhlR interaction. Discovery of new antibiotics would fulfill an unmet healthcare need by providing treatments to combatP. aeruginosainfections.
- Published
- 2022
13. Structure, Regulation, and Inhibition of the Quorum-Sensing Signal Integrator LuxO.
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Hande Boyaci, Tayyab Shah, Amanda Hurley, Bashkim Kokona, Zhijie Li, Christian Ventocilla, Philip D Jeffrey, Martin F Semmelhack, Robert Fairman, Bonnie L Bassler, and Frederick M Hughson
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Biology (General) ,QH301-705.5 - Abstract
In a process called quorum sensing, bacteria communicate with chemical signal molecules called autoinducers to control collective behaviors. In pathogenic vibrios, including Vibrio cholerae, the accumulation of autoinducers triggers repression of genes responsible for virulence factor production and biofilm formation. The vibrio autoinducer molecules bind to transmembrane receptors of the two-component histidine sensor kinase family. Autoinducer binding inactivates the receptors' kinase activities, leading to dephosphorylation and inhibition of the downstream response regulator LuxO. Here, we report the X-ray structure of LuxO in its unphosphorylated, autoinhibited state. Our structure reveals that LuxO, a bacterial enhancer-binding protein of the AAA+ ATPase superfamily, is inhibited by an unprecedented mechanism in which a linker that connects the catalytic and regulatory receiver domains occupies the ATPase active site. The conformational change that accompanies receiver domain phosphorylation likely disrupts this interaction, providing a mechanistic rationale for LuxO activation. We also determined the crystal structure of the LuxO catalytic domain bound to a broad-spectrum inhibitor. The inhibitor binds in the ATPase active site and recapitulates elements of the natural regulatory mechanism. Remarkably, a single inhibitor molecule may be capable of inhibiting an entire LuxO oligomer.
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- 2016
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14. Controllable phycobilin modification: an alternative photoacclimation response in cryptophyte algae
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Leah Spangler, Philip D. Jeffrey, Mina Yu, and Gregory D. Scholes
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biology ,Chemistry ,General Chemical Engineering ,Phycobiliprotein ,General Chemistry ,Chromophore ,biology.organism_classification ,Absorbance ,Light-harvesting complex ,Light intensity ,chemistry.chemical_compound ,Algae ,Chlorophyll ,Biophysics ,Phycobilin - Abstract
Cryptophyte algae are well known for their ability to survive under low light conditions through the use of their auxiliary light harvesting antennas, phycobiliproteins. Mainly acting to absorb light where chlorophyll cannot (500-650 nm), phycobiliproteins also play an instrumental role in helping cryptophyte algae respond to changes in light intensity through the process of photoacclimation. Until recently, photoacclimation in cryptophyte algae was only observed as a change in the cellular concentration of phycobiliproteins; however, an additional photoacclimation response was recently discovered that causes shifts in the phycobiliprotein absorbance peaks following growth under red, blue, or green light. Here, we reproduce this newly identified photoacclimation response in two other species of cryptophyte algae, P. sulcata and H. pacifica, and elucidate the origin of the response on the protein level. We compare isolated native and photoacclimated phycobiliproteins for these two species using spectroscopy and mass spectrometry, and we report the x-ray structures of the PC577 light harvesting complex and corresponding photoacclimated complex. We find that neither the protein sequences, nor the protein structures are modified by photoacclimation. We conclude that cryptophyte algae change a chromophore in one site of their phycobiliprotein beta-subunits as part of the photoacclimation response to changes in the spectral quality of light. Ultrafast pump-probe spectroscopy shows that the energy transfer is weakly affected by the photoacclimation.
- Published
- 2021
15. Structural and Functional Analysis of Keratinicyclin Reveals Synergistic Antibiosis with Vancomycin Against Clostridium Difficile
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Vasiliki Chioti, Mohammad R. Seyedsayamdost, Philip D. Jeffrey, Kirklin L McWhorter, Fei Xu, and Katherine M. Davis
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biology ,medicine.drug_class ,Stereochemistry ,Chemistry ,Antibiotics ,Antibiosis ,Clostridium difficile ,biology.organism_classification ,Glycopeptide ,chemistry.chemical_compound ,medicine ,Moiety ,Vancomycin ,Peptidoglycan ,Bacteria ,medicine.drug - Abstract
Keratinicyclins and keratinimicins are recently discovered glycopeptide antibiotics (GPAs). The latter are canonical GPAs with broad-spectrum activity against Gram-positive bacteria, while keratinicyclins form a new chemotype by virtue of an unusual oxazolidinone moiety and exhibit specific antibiosis against Clostridium difficile. Here, we investigated the three-dimensional structures and functional consequences for both molecules. Equilibrium binding studies showed tight binding by keratinimicin A, but not keratinicyclin B, to the peptidoglycan terminus. Using protein crystallography methods, we solved the X-ray crystal structures of both GPAs, which, in conjunction with DFT calculations, indicate that the inability of keratinicyclin B to bind the peptidoglycan is governed by steric factors. Keratinicyclin B, therefore, interferes with an alternative target to inhibit C. difficile growth, a conclusion confirmed by checkerboard analysis that revealed synergistic activity with vancomycin. Our results set the stage for identifying the molecular target of keratinicyclins and for exploring their therapeutic utility in combination with vancomycin.
- Published
- 2021
16. Structure and mechanism of pyrimidine–pyrimidone (6-4) photoproduct recognition by the Rad4/XPC nucleotide excision repair complex
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Jung Hyun Min, Ouathek Ouerfelli, Debamita Paul, Hong Zhao, Suse Broyde, Philip D. Jeffrey, and Hong Mu
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Saccharomyces cerevisiae Proteins ,DNA Repair ,Base pair ,Stereochemistry ,DNA damage ,DNA repair ,NAR Breakthrough Article ,Pyrimidine dimer ,Molecular Dynamics Simulation ,Biology ,03 medical and health sciences ,chemistry.chemical_compound ,Protein Domains ,Genetics ,Nucleotide ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,030302 biochemistry & molecular biology ,Nucleotide-excision repair complex ,DNA ,3. Good health ,DNA-Binding Proteins ,chemistry ,Pyrimidine Dimers ,Nucleic Acid Conformation ,Protein Binding ,Nucleotide excision repair - Abstract
Failure in repairing ultraviolet radiation-induced DNA damage can lead to mutations and cancer. Among UV-lesions, the pyrimidine–pyrimidone (6-4) photoproduct (6-4PP) is removed from the genome much faster than the cyclobutane pyrimidine dimer (CPD), owing to the more efficient recognition of 6-4PP by XPC-RAD23B, a key initiator of global-genome nucleotide excision repair (NER). Here, we report a crystal structure of a Rad4–Rad23 (yeast XPC-Rad23B ortholog) bound to 6-4PP-containing DNA and 4-μs molecular dynamics (MD) simulations examining the initial binding of Rad4 to 6-4PP or CPD. This first structure of Rad4/XPC bound to a physiological substrate with matched DNA sequence shows that Rad4 flips out both 6-4PP-containing nucleotide pairs, forming an ‘open’ conformation. The MD trajectories detail how Rad4/XPC initiates ‘opening’ 6-4PP: Rad4 initially engages BHD2 to bend/untwist DNA from the minor groove, leading to unstacking and extrusion of the 6-4PP:AA nucleotide pairs towards the major groove. The 5′ partner adenine first flips out and is captured by a BHD2/3 groove, while the 3′ adenine extrudes episodically, facilitating ensuing insertion of the BHD3 β-hairpin to open DNA as in the crystal structure. However, CPD resists such Rad4-induced structural distortions. Untwisting/bending from the minor groove may be a common way to interrogate DNA in NER.
- Published
- 2019
17. ParST is a widespread toxin–antitoxin module that targets nucleotide metabolism
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Philip D. Jeffrey, Frank J. Piscotta, and A.J. Link
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medicine.disease_cause ,03 medical and health sciences ,chemistry.chemical_compound ,Escherichia coli ,Ribose-Phosphate Pyrophosphokinase ,RefSeq ,medicine ,Amino Acid Sequence ,030304 developmental biology ,ADP Ribose Transferases ,chemistry.chemical_classification ,0303 health sciences ,Crystallography ,Multidisciplinary ,biology ,Nucleotides ,030306 microbiology ,Toxin ,Phosphoribosyl pyrophosphate ,Toxin-Antitoxin Systems ,biology.organism_classification ,Toxin-antitoxin system ,Sphingomonadaceae ,Enzyme ,PNAS Plus ,chemistry ,Biochemistry ,ADP-ribosylation ,Antitoxin ,Bacteria - Abstract
Toxin–antitoxin (TA) systems interfere with essential cellular processes and are implicated in bacterial lifestyle adaptations such as persistence and the biofilm formation. Here, we present structural, biochemical, and functional data on an uncharacterized TA system, the COG5654–COG5642 pair. Bioinformatic analysis showed that this TA pair is found in 2,942 of the 16,286 distinct bacterial species in the RefSeq database. We solved a structure of the toxin bound to a fragment of the antitoxin to 1.50 Å. This structure suggested that the toxin is a mono-ADP-ribosyltransferase (mART). The toxin specifically modifies phosphoribosyl pyrophosphate synthetase (Prs), an essential enzyme in nucleotide biosynthesis conserved in all organisms. We propose renaming the toxin ParT for Prs ADP-ribosylating toxin and ParS for the cognate antitoxin. ParT is a unique example of an intracellular protein mART in bacteria and is the smallest known mART. This work demonstrates that TA systems can induce bacteriostasis through interference with nucleotide biosynthesis.
- Published
- 2018
18. Inhibitor Mimetic Mutations in the
- Author
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Isabelle R, Taylor, Jon E, Paczkowski, Philip D, Jeffrey, Brad R, Henke, Chari D, Smith, and Bonnie L, Bassler
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Virulence Factors ,Molecular Mimicry ,Mutation ,Pseudomonas aeruginosa ,Quorum Sensing ,Articles - Abstract
Pseudomonas aeruginosa is an opportunistic human pathogen that causes fatal infections. There exists an urgent need for new antimicrobial agents to combat P. aeruginosa. We conducted a screen for molecules that bind the virulence-controlling protein PqsE and characterized hit compounds for inhibition of PqsE enzymatic activity. The binding conformations of two inhibitory molecules, BB391 and BB393, were identified by crystallography, and inhibitor binding was mimicked by the substitution of PqsE residues E182 and S285 with tryptophan. Comparison of the inhibitor-mimetic mutations to the catalytically inactive PqsE D73A protein demonstrated that catalysis is not responsible for the role PqsE plays in driving virulence factor production. Rather, the PqsE E182W protein fails to interact with the quorum-sensing receptor, RhlR, and our results suggest that it is this interaction that is responsible for promoting virulence factor production in P. aeruginosa. These findings provide a new route for drug discovery efforts targeting PqsE.
- Published
- 2021
19. A NR2E1-Interacting Peptide of LSD1 Inhibits the Proliferation of Brain Tumor Initiating Cells
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Umar F. Shahul Hameed, Pankaj Kumar Giri, Xiang Sun, Ping Yuan, Balakrishnan Shenbaga Moorthy, Fangjin Chen, Philip D. Jeffrey, Wen Zhang, Yonggao Mou, Jianfeng Pei, Xin Ma, Rong Hu, Kunchithapadam Swaminathan, and Li Zhou
- Subjects
Gene knockdown ,animal structures ,Cell ,Brain tumor ,Biology ,medicine.disease ,In vitro ,medicine.anatomical_structure ,medicine ,Cancer research ,biology.protein ,H3K4me3 ,PTEN ,Epigenetics ,Chromatin immunoprecipitation - Abstract
Background: Brain tumor initiating cells (BTICs) play a key role in the progression and relapse of the dreadful glioblastoma. Targeting the TLX involved self-renewal mechanism of BITCs may enable us to develop novel therapeutic strategy for glioblastoma. Methods: The function of NR2E (TLX) and its interacting protein-LSD1 in BTICs were characterized by gene knockdown combined with tumor sphere formation assay, cell-growth assay, co-immunoprecipitation and chromatin immunoprecipitation assays. NR2E interacting peptide of LSD1 was identified and confirmed by Amide Hydrogen/Deuterium Exchange and Mass Spectrometry (HDX-MS) and in vitro functional assay with LSD1 fragment deletion mutants. The in vivo function of the peptide was further examined with patient-derived glioblastoma initiating cell xenograft mouse model. Findings: NR2E1 recruits LSD1 to remove the active epigenetic marker H3K4me3 at Pten promoter and repress its expression, thereby promoting BTIC proliferation. LSD1 peptide (L197-211) that locates at the LSD1 SWIRM domain is critical for the interaction between NR2E1 and LSD1. Overexpression of this peptide inhibits BTIC proliferation and brain tumor growth. Interpretation: Our studies show that NR2E1 partners with LSD1 to promote the self-renewal of BTICs. Interference of NR2E1 and LSD1 interaction by peptide L197-211 may be explored as a novel therapeutic strategy for malignant brain tumors. Funding Information: This work was supported by funds from National Natural Science Foundation of China (NSFC) (Grant No. 81773156 to PY and Grant No. 8187232 to YM) and the support of the Ministry of Education of Singapore Academic Research Fund to the Singapore researchers to KS. Declaration of Interests: The authors declare that no competing interest exists. Ethics Approval Statement: The animal experiments were performed under the approval from Animal Experimentation Ethics Committee (AEEC) in the Sixth Affiliated Hospital of Sun Yatsen University.
- Published
- 2021
20. Conformational change-induced repeat domain expansion regulates Rap phosphatase quorum-sensing signal receptors.
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Vijay Parashar, Philip D Jeffrey, and Matthew B Neiditch
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Biology (General) ,QH301-705.5 - Abstract
The large family of Gram-positive quorum-sensing receptors known as the RNPP proteins consists of receptors homologous to the Rap, NprR, PlcR, and PrgX proteins that are regulated by imported oligopeptide autoinducers. Rap proteins are phosphatases and transcriptional anti-activators, and NprR, PlcR, and PrgX proteins are DNA binding transcription factors. Despite their obvious importance, the mechanistic basis of oligopeptide receptor regulation is largely unknown. Here, we report the X-ray crystal structure of the Bacillus subtilis quorum-sensing receptor RapJ in complex with the centrally important oligopeptide autoinducer competence and sporulation factor (CSF, also termed PhrC), a member of the Phr family of quorum-sensing signals. Furthermore, we present the crystal structure of RapI. Comparison of the RapJ-PhrC, RapI, RapH-Spo0F, and RapF-ComA(C) crystal structures reveals the mechanistic basis of Phr activity. More specifically, when complexed with target proteins, Rap proteins consist of a C-terminal tetratricopeptide repeat (TPR) domain connected by a flexible helix-containing linker to an N-terminal 3-helix bundle. In the absence of a target protein or regulatory peptide, the Rap protein 3-helix bundle adopts different conformations. However, in the peptide-bound conformation, the Rap protein N-terminal 3-helix bundle and linker undergo a radical conformational change, form TPR-like folds, and merge with the existing C-terminal TPR domain. To our knowledge, this is the first example of conformational change-induced repeat domain expansion. Furthermore, upon Phr binding, the entire Rap protein is compressed along the TPR superhelical axis, generating new intramolecular contacts that lock the Rap protein in an inactive state. The fact that Rap proteins are conformationally flexible is surprising considering that it is accepted dogma that TPR proteins do not undergo large conformational changes. Repeat proteins are widely used as scaffolds for the development of designed affinity reagents, and we propose that Rap proteins could be used as scaffolds for engineering novel ligand-switchable affinity reagents.
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- 2013
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21. The Sec1/Munc18 protein Vps45 holds the Qa-SNARE Tlg2 in an open conformation
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Frederick M. Hughson, Travis J Eisemann, Philip D. Jeffrey, Frederick Allen, Kelly Lau, and Gregory R. Shimamura
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SNAREs ,QH301-705.5 ,Protein Conformation ,Science ,Structural Biology and Molecular Biophysics ,membrane fusion ,Chaetomium ,General Biochemistry, Genetics and Molecular Biology ,Fungal Proteins ,Munc18 Proteins ,None ,Biology (General) ,Binding site ,SNARE complex assembly ,General Immunology and Microbiology ,Qa-SNARE Proteins ,Chemistry ,General Neuroscience ,Vesicle ,Molecular biophysics ,Lipid bilayer fusion ,Cell Biology ,General Medicine ,Syntaxin 1 ,Fusion protein ,Recombinant Proteins ,Structural biology ,Biophysics ,Medicine ,sec1/munc18 (SM) proteins ,biological phenomena, cell phenomena, and immunity ,Linker ,VPS45 ,Protein Binding ,Research Article - Abstract
Fusion of intracellular trafficking vesicles is mediated by the assembly of soluble N-ethylmaleimide-sensitive fusion protein receptors (SNAREs) to form membrane-bridging complexes. Also required for SNARE-mediated membrane fusion are Sec1/Munc18-family (SM) proteins, SNARE chaperones that can function as templates to catalyze SNARE complex assembly. In the paradigmatic structure of an SM–SNARE complex, Munc18-1 bound to the Qa-SNARE syntaxin 1, the SNARE protein is trapped in an autoinhibited closed conformation that prevents it from entering into SNARE complexes. Here, we present the structure of a second SM–Qa-SNARE complex, Vps45–Tlg2. Strikingly, Vps45 holds Tlg2 in an open conformation, with its SNARE motif disengaged from its three-helical Habc domain and its linker region unfolded. The domain 3a helical hairpin of Vps45 is unfurled, exposing the presumptive R-SNARE binding site to allow template complex formation. Tlg2 has a pronounced tendency to self-associate via its SNARE motif, and we demonstrate that Vps45 can rescue Tlg2 oligomers into stoichiometric Vps45–Tlg2 complexes. Our findings demonstrate that SM proteins can engage Qa-SNAREs using at least two different modes, one in which the SNARE is closed and one in which it is open.
- Published
- 2020
22. The Structural Basis of Rubisco Phase Separation in the Pyrenoid
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Weronika Patena, Tobias Wunder, Hui-Ting Chou, Sarah A. Port, Nicky Atkinson, Philip D. Jeffrey, Vivian K. Chen, Moritz T. Meyer, Doreen Matthies, Alistair J. McCormick, Oliver Mueller-Cajar, Zhiheng Yu, Shan He, Benjamin D. Engel, Guanhua He, Martin C. Jonikas, Frederick M. Hughson, and Antonio Martinez-Sanchez
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0106 biological sciences ,0303 health sciences ,biology ,Chemistry ,fungi ,RuBisCO ,Carbon fixation ,food and beverages ,Chlamydomonas reinhardtii ,Sequence (biology) ,biology.organism_classification ,01 natural sciences ,Pyrenoid ,03 medical and health sciences ,Organelle ,biology.protein ,Biophysics ,Binding site ,Linker ,030304 developmental biology ,010606 plant biology & botany - Abstract
Approximately one-third of global CO2 fixation occurs in a phase separated algal organelle called the pyrenoid. Existing data suggest that the pyrenoid forms by the phase-separation of the CO2-fixing enzyme Rubisco with a linker protein; however, the molecular interactions underlying this phase-separation remain unknown. Here we present the structural basis of the interactions between Rubisco and its intrinsically disordered linker protein EPYC1 (Essential Pyrenoid Component 1) in the model alga Chlamydomonas reinhardtii. We find that EPYC1 consists of five evenly-spaced Rubisco-binding regions that share sequence similarity. Single-particle cryo-electron microscopy of one of these regions in complex with Rubisco indicates that each Rubisco holoenzyme has eight binding sites for EPYC1, one on each Rubisco small subunit. Interface mutations disrupt binding, phase separation, and pyrenoid formation. Cryo-electron tomography supports a model where EPYC1 and Rubisco form a co-dependent multivalent network of specific low-affinity bonds, giving the matrix liquid-like properties. Our results advance the structural and functional understanding of the phase separation underlying the pyrenoid, an organelle that plays a fundamental role in the global carbon cycle.
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- 2020
23. Author response: The Sec1/Munc18 protein Vps45 holds the Qa-SNARE Tlg2 in an open conformation
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Travis J Eisemann, Frederick M. Hughson, Philip D. Jeffrey, Kelly Lau, Frederick Allen, and Gregory R. Shimamura
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Physics ,Biophysics ,VPS45 - Published
- 2020
24. A Parasite Coat Protein Binds Suramin to Confer Drug Resistance
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Joseph Verdi, Natalie Wiedemar, Monique van Straaten, Philip D. Jeffrey, Pascal Maeser, Johan Zeelen, F. Nina Papavasiliou, Alexander Hempelmann, Hamidreza Hashemi, Kathryn Perez, Silvan Haelg, and C. Erec Stebbins
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chemistry.chemical_classification ,Suramin ,Drug resistance ,Coat protein ,medicine.disease ,Phenotype ,Cell biology ,chemistry ,parasitic diseases ,polycyclic compounds ,medicine ,Antigenic variation ,Parasite hosting ,African trypanosomiasis ,Glycoprotein ,medicine.drug - Abstract
Suramin has been a primary early-stage treatment for African trypanosomiasis for nearly one hundred years. Recent studies revealed that trypanosome strains that express the Variant Surface Glycoprotein VSGsur possess heightened resistance to suramin. We show here that VSGsur binds tightly to suramin, other VSGs do not, and that together with VSG13 it defines a structurally divergent subgroup of these coat proteins. The co-crystal structure of VSGsur with suramin reveals that the chemically symmetric drug binds within a large cavity in the VSG homodimer asymmetrically, primarily through contacts of its central benzene rings. Structure-based, loss-of-contact mutations in VSGsur significantly decrease the affinity to suramin and lead to a loss of the resistance phenotype. Altogether, these data show that the resistance phenotype is dependent on the binding of suramin to VSGsur, establishing that the VSG proteins can possess functionality beyond their role in antigenic variation.
- Published
- 2020
25. Structural basis for the binding of SNAREs to the multisubunit tethering complex Dsl1
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Safraz Hamid, I-Mei Yu, Sophie M. Travis, Philip D. Jeffrey, Kevin DAmico, Frederick M. Hughson, and Gabriel Ramirez-Arellano
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0303 health sciences ,Tethering ,Chemistry ,Vesicle ,Endoplasmic reticulum ,USE1 ,Lipid bilayer fusion ,COPI ,Golgi apparatus ,03 medical and health sciences ,symbols.namesake ,0302 clinical medicine ,symbols ,Biophysics ,SNARE complex ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
Multisubunit tethering complexes (MTCs) are large (250 to >750 kDa), conserved macromolecular machines that are essential for SNARE-mediated membrane fusion in all eukaryotes. MTCs are thought to function as organizers of membrane trafficking, mediating the initial, long-range interaction between a vesicle and its target membrane and promoting the formation of membrane-bridging SNARE complexes. Previously, we reported the structure of the Dsl1 complex, the simplest known MTC, which is essential for COPI-mediated transport from the Golgi to the endoplasmic reticulum (ER). This structure suggested how the Dsl1 complex might function to tether a vesicle to its target membrane by binding at one end to the COPI coat and at the other end to ER SNAREs. Here, we use x-ray crystallography to investigate these Dsl1-SNARE interactions in greater detail. The Dsl1 complex comprises three subunits that together form a two-legged structure with a central hinge. Our results show that distal regions of each leg bind N-terminal Habc domains of the ER SNAREs Sec20 (a Qb-SNARE) and Use1 (a Qc-SNARE). The observed binding modes appear to anchor the Dsl1 complex to the ER target membrane while simultaneously ensuring that both SNAREs are in open conformations with their SNARE motifs available for assembly. The proximity of the two SNARE motifs, and therefore their ability to enter the same SNARE complex, depends on the relative orientation of the two Dsl1 legs.
- Published
- 2020
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26. Structural basis for the binding of SNAREs to the multisubunit tethering complex Dsl1
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Sophie M. Travis, Safraz Hamid, Gabriel Ramirez-Arellano, Conor McMahon, Philip D. Jeffrey, Frederick M. Hughson, I-Mei Yu, and Kevin DAmico
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0301 basic medicine ,Models, Molecular ,Saccharomyces cerevisiae Proteins ,Vesicle docking ,Saccharomyces cerevisiae ,Crystallography, X-Ray ,Biochemistry ,03 medical and health sciences ,symbols.namesake ,Protein Structure, Quaternary ,Molecular Biology ,030102 biochemistry & molecular biology ,Chemistry ,Vesicle ,Endoplasmic reticulum ,USE1 ,Lipid bilayer fusion ,Cell Biology ,COPI ,Golgi apparatus ,030104 developmental biology ,Biophysics ,symbols ,Editors' Picks Highlights ,biological phenomena, cell phenomena, and immunity ,SNARE complex ,SNARE Proteins - Abstract
Multisubunit-tethering complexes (MTCs) are large (250 to >750 kDa), conserved macromolecular machines that are essential for soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE)–mediated membrane fusion in all eukaryotes. MTCs are thought to organize membrane trafficking by mediating the initial long-range interaction between a vesicle and its target membrane and promoting the formation of membrane-bridging SNARE complexes. Previously, we reported the structure of the yeast Dsl1 complex, the simplest known MTC, which is essential for coat protein I (COPI) mediated transport from the Golgi to the endoplasmic reticulum (ER). This structure suggests how the Dsl1 complex might tether a vesicle to its target membrane by binding at one end to the COPI coat and at the other to ER-associated SNAREs. Here, we used X-ray crystallography to investigate these Dsl1–SNARE interactions in greater detail. The Dsl1 complex comprises three subunits that together form a two-legged structure with a central hinge. We found that distal regions of each leg bind N-terminal Habc domains of the ER SNAREs Sec20 (a Qb-SNARE) and Use1 (a Qc-SNARE). The observed binding modes appear to anchor the Dsl1 complex to the ER target membrane while simultaneously ensuring that both SNAREs are in open conformations, with their SNARE motifs available for assembly. The proximity of the two SNARE motifs, and therefore their ability to enter the same SNARE complex, will depend on the relative orientation of the two Dsl1 legs. These results underscore the critical roles of SNARE N-terminal domains in mediating interactions with other elements of the vesicle docking and fusion machinery.
- Published
- 2020
27. Structural basis of competitive recognition of p53 and MDM2 by HAUSP/USP7: implications for the regulation of the p53-MDM2 pathway.
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Min Hu, Lichuan Gu, Muyang Li, Philip D Jeffrey, Wei Gu, and Yigong Shi
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Biology (General) ,QH301-705.5 - Abstract
Herpesvirus-associated ubiquitin-specific protease (HAUSP, also known as USP7), a deubiquitylating enzyme of the ubiquitin-specific processing protease family, specifically deubiquitylates both p53 and MDM2, hence playing an important yet enigmatic role in the p53-MDM2 pathway. Here we demonstrate that both p53 and MDM2 specifically recognize the N-terminal tumor necrosis factor-receptor associated factor (TRAF)-like domain of HAUSP in a mutually exclusive manner. HAUSP preferentially forms a stable HAUSP-MDM2 complex even in the presence of excess p53. The HAUSP-binding elements were mapped to a peptide fragment in the carboxy-terminus of p53 and to a short-peptide region preceding the acidic domain of MDM2. The crystal structures of the HAUSP TRAF-like domain in complex with p53 and MDM2 peptides, determined at 2.3-A and 1.7-A resolutions, respectively, reveal that the MDM2 peptide recognizes the same surface groove in HAUSP as that recognized by p53 but mediates more extensive interactions. Structural comparison led to the identification of a consensus peptide-recognition sequence by HAUSP. These results, together with the structure of a combined substrate-binding-and-deubiquitylation domain of HAUSP, provide important insights into regulation of the p53-MDM2 pathway by HAUSP.
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- 2006
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28. Structure-Function Studies of the Bacillus subtilis Ric Proteins Identify the Fe-S Cluster-Ligating Residues and Their Roles in Development and RNA Processing
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Matthew B. Neiditch, David Dubnau, Micaela DeSantis, Faisal Tarique Khaja, Borries Demeler, Eugenie Dubnau, Philip D. Jeffrey, and Felix Adusei-Danso
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Riboswitch ,Molecular Biology and Physiology ,Iron–sulfur cluster ,Bacillus subtilis ,Ric proteins ,Microbiology ,03 medical and health sciences ,chemistry.chemical_compound ,bacterial development ,hemic and lymphatic diseases ,Virology ,Transcription factor ,030304 developmental biology ,0303 health sciences ,Molecular Reproduction, Development & Genetics ,biology ,030306 microbiology ,C-terminus ,RNA ,biology.organism_classification ,Heterotetramer ,QR1-502 ,chemistry ,Biochemistry ,RNA processing ,iron sulfur cluster ,Research Article ,Cysteine - Abstract
The RicA, RicF, and RicT proteins are widely conserved among the firmicute bacteria and play multiple roles in Bacillus subtilis. Among the phenotypes associated with the inactivation of these proteins are the inability to be genetically transformed or to form biofilms, a decrease in sporulation frequency, and changes in the stability and maturation of multiple RNA species. Despite their importance, the molecular mechanisms of Ric protein activities have not been elucidated and the roles of the two iron-sulfur clusters on the complex of the three proteins are not understood. To unravel the mechanisms of Ric action, molecular characterization of the complex and of its constituent proteins is essential. This report represents a major step toward understanding the structures of the Ric proteins, the arrangement and roles of the Fe-S clusters, and the phenotypes associated with Ric mutations., In Bacillus subtilis, the RicA (YmcA), RicF (YlbF), and RicT (YaaT) proteins accelerate the phosphorylation of the transcription factor Spo0A, contributing to genetic competence, sporulation, and biofilm formation, and are also essential for the correct maturation of several protein-encoding and riboswitch RNAs. These proteins form a stable complex (RicAFT) that carries two [4Fe-4S]+2 clusters. We show here that the complex is a 1:1:1 heterotrimer, and we present the X-ray crystal structures of a RicAF heterotetramer and of a RicA dimer. We also demonstrate that one of the Fe-S clusters (cluster 1) is ligated by cysteine residues donated exclusively by RicT and can be retained when the RicT monomer is purified by itself. Cluster 2 is ligated by C167 from RicT, by C134 and C146 located near the C terminus of RicF, and by C141 at the C terminus of RicA. These findings imply the following novel arrangement: adjacent RicT residues C166 and 167 ligate clusters 1 and 2, respectively, while cluster 2 is ligated by cysteine residues from RicT, RicA, and RicF. Thus, the two clusters must lie close to one another and at the interface of the RicAFT protomers. We also show that the cluster-ligating cysteine residues, and therefore most likely both Fe-S clusters, are essential for cggR-gapA mRNA maturation, for the regulation of ricF transcript stability, and for several Ric-associated developmental phenotypes, including competence for transformation, biofilm formation, and sporulation. Finally, we present evidence that RicAFT, RicAF, and RicA and the RicT monomer may play distinct regulatory roles in vivo.
- Published
- 2019
29. A metagenomic strategy for harnessing the chemical repertoire of the human microbiome
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Philip D. Jeffrey, Yuki Sugimoto, Francine R. Camacho, Arman Odabas, Pranatchareeya Chankhamjon, Abhishek Biswas, Shuo Wang, and Mohamed S. Donia
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0303 health sciences ,Multidisciplinary ,Host Microbial Interactions ,030306 microbiology ,Drug discovery ,Microbiota ,Human microbiome ,Bacterial genome size ,Computational biology ,Biology ,03 medical and health sciences ,Synthetic biology ,Polyketide ,Metagenomics ,Multigene Family ,Polyketides ,Humans ,Metagenome ,Oral Microbiome ,Microbiome ,030304 developmental biology - Abstract
INTRODUCTION The human microbiome has been correlated with several health and disease conditions, but the molecular mechanisms underlying these correlations remain largely unexplored. Biologically active small molecules that are produced by the human microbiome offer an important route for exploring these mechanisms because they often mediate important microbe-microbe and microbe-host interactions. In bacterial genomes, small-molecule biosynthetic genes are usually encoded in distinct clusters known as biosynthetic gene clusters (BGCs), which enables scientists to use computational tools for recognizing them and predicting their products. Here, we present a hybrid strategy that uses computational and synthetic biology tools for discovering microbiome-encoded small molecules. RATIONALE Previous efforts to discover small-molecule BGCs from the human microbiome relied mainly on analyzing genomic data of sequenced bacterial isolates. Although this approach has revealed the enormous and largely untapped diversity of microbiome-encoded BGCs, it fails to report on the biosynthetic potential of members of the human microbiome that have not yet been cultured or isolated: the majority of species in metagenomic sequencing data. Therefore, we sought to develop a computational algorithm that discovers small-molecule BGCs directly in complex metagenomic sequencing data of the human microbiome: metagenomic identifier of biosynthetic gene clusters (MetaBGC). First, high-performance probabilistic models for identifying homologs of a biosynthetic enzyme of interest are built specifically for use with complex metagenomic datasets(MetaBGC-Build). Next, these models are used to identify biosynthetic genes in thousands of metagenomic datasets of the human microbiome at the single-read level (MetaBGC-Identify). Finally, identified biosynthetic reads are quantified in the entire cohort of samples (MetaBGC-Quantify) and clustered into biosynthetic read bins on the basis of their abundance profiles across samples (MetaBGC-Cluster). To evaluate the utility of this approach, we used it to discover BGCs for type II polyketides, a clinically relevant class of small molecules, directly from metagenomic sequencing data of the human microbiome. RESULTS We applied MetaBGC to 3203 metagenomic samples of the human microbiome originating from Western (subjects from the United States, Spain, and Denmark) and non-Western (subjects from China and Fiji) populations and from every major human body site (gut, mouth, skin, and vagina). Overall, we discovered 13 complete BGCs that potentially encode type II polyketides; eight of these were encoded by diverse bacterial isolates of the human microbiome in a strain-specific manner and five could not be assigned to any sequenced species. Type II polyketide BGCs are found in three major human body sites, gut, mouth, and skin, and at least six of them are transcribed under host-colonization conditions and widely distributed in different human populations (e.g., 46% of healthy subjects from the United States encode at least one BGC in their gut, oral, or skin microbiome). Next, we selected two of the identified BGCs for experimental characterization, one from the oral microbiome and another from the gut microbiome. We used a synthetic biology strategy in which metagenomically discovered BGCs are genetically engineered and expressed in various heterologous hosts without the need for cultivation of the native producer. Using this strategy, we successfully purified and solved the structures of five new type II polyketide molecules as the products of the two characterized BGCs. Finally, we show that two of the discovered molecules exert strong antibacterial activities against members of the human microbiome that occupy the same niche as their producer, implying a possible role in microbe-microbe competition. CONCLUSION We developed a hybrid strategy that combines computational and experimental techniques for discovering and characterizing small-molecule BGCs directly from complex datasets of the human microbiome. Using this strategy, we discovered that a clinically relevant class of molecules, type II polyketides, are widely encoded in the human microbiome and that human microbiome–derived polyketides resemble in structure and biological activity clinically used ones. Our approach is generally applicable to other classes of small molecules and can be used to systematically unveil the chemical potential of the human microbiome—a goal that is useful for both mechanistic microbiome explorations and drug discovery.
- Published
- 2019
30. An autoinducer analog reveals an alternative mode of ligand binding for the LasR quorum-sensing receptor
- Author
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Jian Ping Cong, Chari D. Smith, Philip D. Jeffrey, Brad R. Henke, Amelia R. McCready, Jon E. Paczkowski, Bonnie L. Bassler, Zhijie Li, and Frederick M. Hughson
- Subjects
0301 basic medicine ,Agonist ,Stereochemistry ,medicine.drug_class ,Homoserine ,Ligands ,01 natural sciences ,Biochemistry ,Chemical synthesis ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Structure-Activity Relationship ,4-Butyrolactone ,Bacterial Proteins ,medicine ,Escherichia coli ,Molecule ,Amino Acids ,Receptor ,biology ,Molecular Structure ,010405 organic chemistry ,Chemistry ,Quorum Sensing ,General Medicine ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,0104 chemical sciences ,Quorum sensing ,030104 developmental biology ,Mutation ,Pseudomonas aeruginosa ,Trans-Activators ,Molecular Medicine ,Autoinducer ,Bacteria ,Protein Binding ,Signal Transduction - Abstract
Bacteria use a cell-cell communication process called quorum sensing to coordinate collective behaviors. Quorum sensing relies on production and group-wide detection of extracellular signal molecules called autoinducers. Here, we probe the activity of the Pseudomonas aeruginosa LasR quorum-sensing receptor using synthetic agonists based on the structure of the native homoserine lactone autoinducer. The synthetic compounds range from low to high potency, and agonist activity tracks with the ability of the agonist to stabilize the LasR protein. Structural analyses of the LasR ligand binding domain complexed with representative synthetic agonists reveal two modes of ligand binding, one mimicking the canonical autoinducer binding arrangement, and the other with the lactone head group rotated approximately 150°. Iterative mutagenesis combined with chemical synthesis reveals the amino acid residues and the chemical moieties, respectively, that are key to enabling each mode of binding. Simultaneous alteration of LasR residues Thr75, Tyr93, and Ala127 converts low-potency compounds into high-potency compounds and converts ligands that are nearly inactive into low-potency compounds. These results show that the LasR binding pocket displays significant flexibility in accommodating different ligands. The ability of LasR to bind ligands in different conformations, and in so doing, alter their potency as agonists, could explain the difficulties that have been encountered in the development of competitive LasR inhibitors.
- Published
- 2019
31. Generation of a Selective Small Molecule Inhibitor of the CBP/p300 Bromodomain for Leukemia Therapy
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Julia Meier, Katharina Leonards, Jürg Schwaller, Marjeta Urh, Danette L. Daniels, Christopher Wells, C. Bountra, Martin Philpott, Nigel J. Parr, Andrew J. Bannister, Clarence Yapp, Simon Taylor, Dave Lugo, Samuel Robson, Matteo Vecellio, Sharlene Velichko, Umesh Kumar, Tony Kouzarides, Panagis Filippakopoulos, Paul Brennan, Alison O'Mahony, Heidi Olzscha, Stefan Knapp, Oleg Fedorov, Sarah Picaud, Kevin Lee, Katherine Louise Jones, Sarah Martin, Duncan Hay, Rab K. Prinjha, Ka Hing Che, Angeliki Thanasopoulou, Philip D. Jeffrey, Nicholas B. La Thangue, Octovia P. Monteiro, Anthony Tumber, and Susanne Müller
- Subjects
Models, Molecular ,Cancer Research ,Cellular differentiation ,Molecular Sequence Data ,P300-CBP Transcription Factors ,Biology ,Article ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Cell Line, Tumor ,Antineoplastic Combined Chemotherapy Protocols ,medicine ,Animals ,Humans ,Cytotoxic T cell ,p300-CBP Transcription Factors ,Doxorubicin ,Amino Acid Sequence ,Enzyme Inhibitors ,030304 developmental biology ,0303 health sciences ,Cell growth ,Myeloid leukemia ,Drug Synergism ,medicine.disease ,Xenograft Model Antitumor Assays ,Molecular biology ,Protein Structure, Tertiary ,3. Good health ,Bromodomain ,Leukemia, Myeloid, Acute ,Oxazepines ,Leukemia ,Oncology ,030220 oncology & carcinogenesis ,Cancer research ,medicine.drug - Abstract
The histone acetyltransferases CBP/p300 are involved in recurrent leukemia-associated chromosomal translocations and are key regulators of cell growth. Therefore, efforts to generate inhibitors of CBP/p300 are of clinical value. We developed a specific and potent acetyl-lysine competitive protein–protein interaction inhibitor, I-CBP112, that targets the CBP/p300 bromodomains. Exposure of human and mouse leukemic cell lines to I-CBP112 resulted in substantially impaired colony formation and induced cellular differentiation without significant cytotoxicity. I-CBP112 significantly reduced the leukemia-initiating potential of MLL-AF9+ acute myeloid leukemia cells in a dose-dependent manner in vitro and in vivo. Interestingly, I-CBP112 increased the cytotoxic activity of BET bromodomain inhibitor JQ1 as well as doxorubicin. Collectively, we report the development and preclinical evaluation of a novel, potent inhibitor targeting CBP/p300 bromodomains that impairs aberrant self-renewal of leukemic cells. The synergistic effects of I-CBP112 and current standard therapy (doxorubicin) as well as emerging treatment strategies (BET inhibition) provide new opportunities for combinatorial treatment of leukemia and potentially other cancers. Cancer Res; 75(23); 5106–19. ©2015 AACR.
- Published
- 2015
32. Unraveling the sequence of cytosolic reactions in the export of GspB adhesin from Streptococcus gordonii
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Wei Mi, Paul M. Sullam, Barbara A. Bensing, Philip D. Jeffrey, Parastoo Azadi, Roberto Sonon, Tom A. Rapoport, Asif Shajahan, Maofu Liao, Yu Chen, and Ravin Seepersaud
- Subjects
0301 basic medicine ,Biochemistry & Molecular Biology ,Glycosylation ,bacterial pathogenesis ,030106 microbiology ,Plasma protein binding ,Crystallography, X-Ray ,Biochemistry ,glycosyltransferase ,Medical and Health Sciences ,Acetylglucosamine ,03 medical and health sciences ,chemistry.chemical_compound ,Cytosol ,Bacterial Proteins ,adhesin ,Glycosyltransferase ,protein secretion ,Editors' Picks ,Adhesins, Bacterial ,crystallography ,Molecular Biology ,biology ,Membrane transport protein ,Streptococcus gordonii ,Bacterial ,Glycosyltransferases ,Membrane Transport Proteins ,Cell Biology ,Biological Sciences ,biology.organism_classification ,Adhesins ,protein export ,Transport protein ,Bacterial adhesin ,Protein Transport ,Secretory protein ,Infectious Diseases ,chemistry ,Chemical Sciences ,biology.protein ,Editors' Picks Highlights ,X-Ray ,Generic health relevance ,Protein Processing, Post-Translational ,Protein Binding - Abstract
Many pathogenic bacteria, including Streptococcus gordonii, possess a pathway for the cellular export of a single serine-rich-repeat protein that mediates the adhesion of bacteria to host cells and the extracellular matrix. This adhesin protein is O-glycosylated by several cytosolic glycosyltransferases and requires three accessory Sec proteins (Asp1–3) for export, but how the adhesin protein is processed for export is not well understood. Here, we report that the S. gordonii adhesin GspB is sequentially O-glycosylated by three enzymes (GtfA/B, Nss, and Gly) that attach N-acetylglucosamine and glucose to Ser/Thr residues. We also found that modified GspB is transferred from the last glycosyltransferase to the Asp1/2/3 complex. Crystal structures revealed that both Asp1 and Asp3 are related to carbohydrate-binding proteins, suggesting that they interact with carbohydrates and bind glycosylated adhesin, a notion that was supported by further analyses. We further observed that Asp1 also has an affinity for phospholipids, which is attenuated by Asp2. In summary, our findings support a model in which the GspB adhesin is sequentially glycosylated by GtfA/B, Nss, and Gly and then transferred to the Asp1/2/3 complex in which Asp1 mediates the interaction of the Asp1/2/3 complex with the lipid bilayer for targeting of matured GspB to the export machinery.
- Published
- 2018
33. Structure and function of accessory Sec proteins involved in the adhesin export pathway of Streptococcus gordonii
- Author
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Tom A. Rapoport, Ravin Seepersaud, Maofu Liao, Asif Shajahan, Parastoo Azadi, Roberto Sonon, Yu Chen, Wei Mi, Philip D. Jeffrey, Barbara A. Bensing, and Paul M. Sullam
- Subjects
chemistry.chemical_classification ,0303 health sciences ,biology ,030306 microbiology ,Streptococcus gordonii ,Pathogenic bacteria ,medicine.disease_cause ,biology.organism_classification ,Bacterial adhesin ,03 medical and health sciences ,Cytosol ,Enzyme ,chemistry ,Biochemistry ,Glycosyltransferase ,medicine ,biology.protein ,Secretion ,Bacteria ,030304 developmental biology - Abstract
Many pathogenic bacteria, includingStreptococcus gordonii, possess a pathway for the export of a single serine-rich-repeat protein that mediates the adhesion of bacteria to host cells and the extracellular matrix. These adhesins areO-glycosylated by several cytosolic glycosyltransferases and require three accessory Sec proteins (Asp1-3) for export, but how the adhesins are processed for secretion is not well defined. Here, we show thatO-glycosylation ofS. gordoniiadhesin GspB occurs in a sequential manner by three enzymes (GtfA/B, Nss, Gly) that attach N-acetylglucosamine and glucose to Ser/Thr residues. The modified substrate is subsequently transferred from the last glycosyltransferase to the Asp1/2/3 complex. Crystal structures show that both Asp1 and Asp3 are related to carbohydrate binding proteins. Asp1 also has an affinity for phospholipids, which is attenuated by Asp2. These results suggest a mechanism for the modification of adhesin in the cytosol and its subsequent targeting to the export machinery.
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- 2017
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34. A direct role for the Sec1/Munc18-family protein Vps33 as a template for SNARE assembly
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Ben P. Phillips, Philip D. Jeffrey, Richard W. Baker, Michael Zick, Frederick M. Hughson, and William Wickner
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Munc18 Proteins ,Saccharomyces cerevisiae Proteins ,Synaptosomal-Associated Protein 25 ,Protein subunit ,Vesicular Transport Proteins ,Nanotechnology ,Saccharomyces cerevisiae ,Biology ,Crystallography, X-Ray ,Article ,Protein Structure, Secondary ,R-SNARE Proteins ,Protein structure ,SNARE complex assembly ,Multidisciplinary ,Qa-SNARE Proteins ,Membrane Proteins ,Protein Structure, Tertiary ,Cell biology ,Transport protein ,Membrane protein ,biological phenomena, cell phenomena, and immunity ,Protein Binding - Abstract
Unravelling the SM-SNARE conundrum So-called SNARE proteins mediate and lend specificity to the fusion between different intracellular membranes. The SM proteins are universally required for intracellular vesicle fusion, yet their mechanism of action has long been enigmatic. Baker et al. have solved a piece of the puzzle by “capturing” SNAREs in the process of assembling into fusogenic complexes on the surface of an SM protein. The findings suggest exactly how and why SM proteins help vesicular fusion during intracellular membrane trafficking. Science , this issue p. 1111
- Published
- 2015
35. Cog5–Cog7 crystal structure reveals interactions essential for the function of a multisubunit tethering complex
- Author
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Tetyana Kudlyk, Vladimir Lupashin, Irina D. Pokrovskaya, Leslie K. Climer, Frederick M. Hughson, Gregory R. Shimamura, Jun Yong Ha, Philip D. Jeffrey, and Томский государственный университет Институт биологии, экологии, почвоведения, сельского и лесного хозяйства (Биологический институт) Научные подразделения БИ
- Subjects
Glycosylation ,Vesicle fusion ,мультисубъединичные комплексы ,Protein subunit ,Vesicle docking ,Exocyst ,Biology ,Crystallography, X-Ray ,Protein Structure, Secondary ,Гольджи комплекс ,chemistry.chemical_compound ,symbols.namesake ,Cog ,Humans ,Protein Structure, Quaternary ,Multidisciplinary ,Biological Sciences ,Golgi apparatus ,Cell biology ,Adaptor Proteins, Vesicular Transport ,chemistry ,Multiprotein Complexes ,Chaperone (protein) ,symbols ,biology.protein ,кристаллическая структура - Abstract
The conserved oligomeric Golgi (COG) complex is required, along with SNARE and Sec1/Munc18 (SM) proteins, for vesicle docking and fusion at the Golgi. COG, like other multisubunit tethering complexes (MTCs), is thought to function as a scaffold and/or chaperone to direct the assembly of productive SNARE complexes at the sites of membrane fusion. Reflecting this essential role, mutations in the COG complex can cause congenital disorders of glycosylation. A deeper understanding of COG function and dysfunction will likely depend on elucidating its molecular structure. Despite some progress toward this goal, including EM studies of COG lobe A (subunits 1-4) and higher-resolution structures of portions of Cog2 and Cog4, the structures of COG's eight subunits and the principles governing their assembly are mostly unknown. Here, we report the crystal structure of a complex between two lobe B subunits, Cog5 and Cog7. The structure reveals that Cog5 is a member of the complexes associated with tethering containing helical rods (CATCHR) fold family, with homology to subunits of other MTCs including the Dsl1, exocyst, and Golgi-associated retrograde protein (GARP) complexes. The Cog5-Cog7 interaction is analyzed in relation to the Dsl1 complex, the only other CATCHR-family MTC for which subunit interactions have been characterized in detail. Biochemical and functional studies validate the physiological relevance of the observed Cog5-Cog7 interface, indicate that it is conserved from yeast to humans, and demonstrate that its disruption in human cells causes defects in trafficking and glycosylation.
- Published
- 2014
36. Structure of the Ca2+-dependent PP2A heterotrimer and insights into Cdc6 dephosphorylation
- Author
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Yongna Xing, Vitali Stanevich, Nathan Wlodarchak, Feng Guo, Kenneth A. Satyshur, Li Jiang, Philip D. Jeffrey, Marc C. Mumby, and Tingwan Sun
- Subjects
calcium-binding motif ,Protein subunit ,Phosphatase ,Cell Cycle Proteins ,Biology ,Crystallography, X-Ray ,Cdc6 ,Substrate Specificity ,Dephosphorylation ,03 medical and health sciences ,0302 clinical medicine ,Holoenzymes ,Animals ,Humans ,Protein Phosphatase 2 ,Phosphorylation ,Cell Cycle Protein ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,protein phosphatase 2A ,DNA replication ,Cell Biology ,Protein phosphatase 2 ,regulatory subunit ,3. Good health ,Cell biology ,Protein Subunits ,030220 oncology & carcinogenesis ,Calcium ,Original Article ,cell cycle - Abstract
The B″/PR72 family of protein phosphatase 2A (PP2A) is an important PP2A family involved in diverse cellular processes, and uniquely regulated by calcium binding to the regulatory subunit. The PR70 subunit in this family interacts with cell division control 6 (Cdc6), a cell cycle regulator important for control of DNA replication. Here, we report crystal structures of the isolated PR72 and the trimeric PR70 holoenzyme at a resolution of 2.1 and 2.4 Å, respectively, and in vitro characterization of Cdc6 dephosphorylation. The holoenzyme structure reveals that one of the PR70 calcium-binding motifs directly contacts the scaffold subunit, resulting in the most compact scaffold subunit conformation among all PP2A holoenzymes. PR70 also binds distinctively to the catalytic subunit near the active site, which is required for PR70 to enhance phosphatase activity toward Cdc6. Our studies provide a structural basis for unique regulation of B″/PR72 holoenzymes by calcium ions, and suggest the mechanisms for precise control of substrate specificity among PP2A holoenzymes.
- Published
- 2013
37. Optimized End-Stacking Provides Specificity of N-Methyl Mesoporphyrin IX for Human Telomeric G-Quadruplex DNA
- Author
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Stephen T. Miller, Thomas J. Lawton, John M. Nicoludis, Philip D. Jeffrey, Steven P. Barrett, Liliya A. Yatsunyk, and Paul R. Rablen
- Subjects
Models, Molecular ,Circular dichroism ,Dna duplex ,Stereochemistry ,Stacking ,Crystallography, X-Ray ,G-quadruplex ,Biochemistry ,Catalysis ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Fluorescence Resonance Energy Transfer ,Humans ,heterocyclic compounds ,Principal Component Analysis ,Chemistry ,Circular Dichroism ,General Chemistry ,Telomere ,G-Quadruplexes ,Förster resonance energy transfer ,Mesoporphyrins ,Mesoporphyrin IX ,Spectrophotometry, Ultraviolet ,DNA - Abstract
N-methyl mesoporphyrin IX (NMM) is exceptionally selective for G-quadruplexes (GQ) relative to duplex DNA and, as such, has found a wide range of applications in biology and chemistry. In addition, NMM is selective for parallel versus antiparallel GQ folds, as was recently demonstrated in our laboratory. Here, we present the X-ray crystal structure of a complex between NMM and human telomeric DNA dAGGG(TTAGGG)(3), Tel22, determined in two space groups, P2(1)2(1)2 and P6, at 1.65 and 2.15 Å resolution, respectively. The former is the highest resolution structure of the human telomeric GQ DNA reported to date. The biological unit contains a Tel22 dimer of 5'-5' stacked parallel-stranded quadruplexes capped on both ends with NMM, supporting the spectroscopically determined 1:1 stoichiometry. NMM is capable of adjusting its macrocycle geometry to closely match that of the terminal G-tetrad required for efficient π-π stacking. The out-of-plane N-methyl group of NMM fits perfectly into the center of the parallel GQ core where it aligns with potassium ions. In contrast, the interaction of the N-methyl group with duplex DNA or antiparallel GQ would lead to steric clashes that prevent NMM from binding to these structures, thus explaining its unique selectivity. On the basis of the biochemical data, binding of NMM to Tel22 does not rely on relatively nonspecific electrostatic interactions, which characterize most canonical GQ ligands, but rather it is hydrophobic in nature. The structural features observed in the NMM-Tel22 complex described here will serve as guidelines for developing new quadruplex ligands that have excellent affinity and precisely defined selectivity.
- Published
- 2012
38. The Structure of Sec12 Implicates Potassium Ion Coordination in Sar1 Activation
- Author
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Chaevia Clendinen, Conor McMahon, Philip D. Jeffrey, Frederick M. Hughson, Geoffrey P. Dann, and Sean Studer
- Subjects
animal structures ,Saccharomyces cerevisiae Proteins ,Vesicular Transport Proteins ,Small G Protein ,Saccharomyces cerevisiae ,Biology ,Crystallography, X-Ray ,environment and public health ,Biochemistry ,Protein Structure, Secondary ,Structure-Activity Relationship ,symbols.namesake ,Protein structure ,Guanine Nucleotide Exchange Factors ,Molecular Biology ,COPII ,Monomeric GTP-Binding Proteins ,Membrane Glycoproteins ,Endoplasmic reticulum ,fungi ,Cell Biology ,COPI ,Cations, Monovalent ,Golgi apparatus ,COP-Coated Vesicles ,enzymes and coenzymes (carbohydrates) ,Potassium ,Biophysics ,symbols ,Guanine nucleotide exchange factor ,biological phenomena, cell phenomena, and immunity - Abstract
Coat protein II (COPII)-coated vesicles transport proteins and lipids from the endoplasmic reticulum to the Golgi. Crucial for the initiation of COPII coat assembly is Sec12, a guanine nucleotide exchange factor responsible for activating the small G protein Sar1. Once activated, Sar1/GTP binds to endoplasmic reticulum membranes and recruits COPII coat components (Sec23/24 and Sec13/31). Here, we report the 1.36 Å resolution crystal structure of the catalytically active, 38-kDa cytoplasmic portion of Saccharomyces cerevisiae Sec12. Sec12 adopts a β propeller fold. Conserved residues cluster around a loop we term the "K loop," which extends from the N-terminal propeller blade. Structure-guided site-directed mutagenesis, in conjunction with in vitro and in vivo functional studies, reveals that this region of Sec12 is catalytically essential, presumably because it makes direct contact with Sar1. Strikingly, the crystal structure also reveals that a single potassium ion stabilizes the K loop; bound potassium is, moreover, essential for optimum guanine nucleotide exchange activity in vitro. Thus, our results reveal a novel role for a potassium-stabilized loop in catalyzing guanine nucleotide exchange.
- Published
- 2012
39. Crystal structure of the mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) paracaspase region
- Author
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Philip D. Jeffrey, Yigong Shi, Jun Yu, Jun Yong Ha, and Xiaolu Yang
- Subjects
Models, Molecular ,chemistry.chemical_classification ,Multidisciplinary ,Protease ,biology ,Protein Conformation ,medicine.medical_treatment ,Chromosomal translocation ,Biological Sciences ,Paracaspase ,BCL10 ,Neoplasm Proteins ,Amino acid ,MALT1 ,Protein structure ,Biochemistry ,chemistry ,Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein ,Caspases ,medicine ,biology.protein ,Humans ,Crystallization ,Dimerization ,Caspase - Abstract
The mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) paracaspase, a key component of the Carma1/Bcl10/MALT1 signalosome, is critical for NF-κB signaling in multiple contexts. MALT1 is thought to function as a scaffold and protease to promote signaling; however, the biochemical and structural basis of paracaspase action remains largely unknown. Here we report the 1.75-Å resolution crystal structure of the MALT1 paracaspase region, which contains the paracaspase domain and an ensuing Ig-like domain. The paracaspase and the Ig domains appear as a single folding unit and interact with each other through extensive van der Waals contacts and hydrogen bonds. The paracaspase domain adopts a fold that is nearly identical to that of classic caspases and homodimerizes similarly to form an active protease. Unlike caspases, the active and mature form of the paracaspase domain remains a single uncleaved polypeptide and specifically recognizes the bound peptide inhibitor Val-Arg-Pro-Arg. In particular, the carboxyl-terminal amino acid Arg of the inhibitor is coordinated by three highly conserved acidic residues. This structure serves as an important framework for deciphering the function and mechanism of paracaspases exemplified by MALT1.
- Published
- 2011
40. A Strategy for Antagonizing Quorum Sensing
- Author
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Frederick M. Hughson, Devin L. Stauff, Danielle L. Swem, Lee R. Swem, Colleen T. O'Loughlin, Philip D. Jeffrey, Guozhou Chen, and Bonnie L. Bassler
- Subjects
Models, Molecular ,Protein Conformation ,Homoserine ,Virulence ,Biology ,Crystallography, X-Ray ,Ligands ,Article ,Virulence factor ,Lactones ,Structure-Activity Relationship ,chemistry.chemical_compound ,4-Butyrolactone ,Chromobacterium ,Binding site ,Molecular Biology ,Binding Sites ,Dose-Response Relationship, Drug ,Molecular Structure ,Quorum Sensing ,DNA ,Cell Biology ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,Anti-Bacterial Agents ,Repressor Proteins ,Quorum sensing ,Biochemistry ,chemistry ,Biofilms ,Mutation ,Trans-Activators ,Autoinducer - Abstract
Quorum-sensing bacteria communicate via small molecules called autoinducers to coordinate collective behaviors. Because quorum sensing controls virulence factor expression in many clinically relevant pathogens, membrane-permeable quorum sensing antagonists that prevent population-wide expression of virulence genes offer a potential route to novel antibacterial therapeutics. Here, we report a strategy for inhibiting quorum-sensing receptors of the widespread LuxR family. Structure-function studies with natural and synthetic ligands demonstrate that the dimeric LuxR-type transcription factor CviR from Chromobacterium violaceum is potently antagonized by molecules that bind in place of the native acylated homoserine lactone autoinducer, provided that they stabilize a closed conformation. In such conformations, each of the two DNA-binding domains interacts with the ligand-binding domain of the opposing monomer. Consequently, the DNA-binding helices are held apart by ~60 Å, twice the ~30 Å separation required for operator binding. This approach may represent a general strategy for the inhibition of multi-domain proteins.
- Published
- 2011
41. A cis-Proline in α-Hemoglobin Stabilizing Protein Directs the Structural Reorganization of α-Hemoglobin
- Author
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Yigong Shi, Suiping Zhou, Joel P. Mackay, David A. Gell, Mitchell J. Weiss, Katerina Bendak, Andrew J. Gow, Philip D. Jeffrey, and Liang Feng
- Subjects
Proline ,Biology ,Crystallography, X-Ray ,medicine.disease_cause ,Biochemistry ,Protein Structure, Secondary ,Structure-Activity Relationship ,chemistry.chemical_compound ,Protein structure ,medicine ,Humans ,Structure–activity relationship ,Protein Structure, Quaternary ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,Heme ,Histidine ,Mutation ,Protein Stability ,Mutagenesis ,Oxygen transport ,Hemoglobin A ,Blood Proteins ,Cell Biology ,chemistry ,Protein Structure and Folding ,Helix ,Biophysics ,Molecular Chaperones - Abstract
alpha-Hemoglobin (alphaHb) stabilizing protein (AHSP) is expressed in erythropoietic tissues as an accessory factor in hemoglobin synthesis. AHSP forms a specific complex with alphaHb and suppresses the heme-catalyzed evolution of reactive oxygen species by converting alphaHb to a conformation in which the heme is coordinated at both axial positions by histidine side chains (bis-histidyl coordination). Currently, the detailed mechanism by which AHSP induces structural changes in alphaHb has not been determined. Here, we present x-ray crystallography, NMR spectroscopy, and mutagenesis data that identify, for the first time, the importance of an evolutionarily conserved proline, Pro(30), in loop 1 of AHSP. Mutation of Pro(30) to a variety of residue types results in reduced ability to convert alphaHb. In complex with alphaHb, AHSP Pro(30) adopts a cis-peptidyl conformation and makes contact with the N terminus of helix G in alphaHb. Mutations that stabilize the cis-peptidyl conformation of free AHSP, also enhance the alphaHb conversion activity. These findings suggest that AHSP loop 1 can transmit structural changes to the heme pocket of alphaHb, and, more generally, highlight the importance of cis-peptidyl prolyl residues in defining the conformation of regulatory protein loops.
- Published
- 2009
42. Mechanism of procaspase-8 activation by c-FLIP L
- Author
-
Yigong Shi, Jun Yu, and Philip D. Jeffrey
- Subjects
Caspase 8 ,Enzyme Precursors ,Programmed cell death ,Multidisciplinary ,biology ,Protein Conformation ,CASP8 and FADD-Like Apoptosis Regulating Protein ,Regulator ,Active site ,Apoptosis ,Biological Sciences ,Crystallography, X-Ray ,Cleavage (embryo) ,Cell biology ,Enzyme Activation ,Enzyme activator ,Protein structure ,Zymogen ,biology.protein ,Animals ,Humans ,Protein Multimerization - Abstract
Cellular FLICE-inhibitory protein (c-FLIP L ) is a key regulator of the extrinsic cell death pathway. Although widely regarded as an inhibitor of initiator caspase activation and cell death, c-FLIP L is also capable of enhancing procaspase-8 activation through heterodimerization of their respective protease domains. However, the underlying mechanism of this activation process remains enigmatic. Here, we demonstrate that cleavage of the intersubunit linker of c-FLIP L by procaspase-8 potentiates the activation process by enhancing heterodimerization between the two proteins and vastly improving the proteolytic activity of unprocessed caspase-(C)8. The crystal structures of the protease-like domain of c-FLIP L alone and in complex with zymogen C8 identify the unique determinants that favor heterodimerization over procaspase-8 homodimerization, and induce the latent active site of zymogen C8 into a productive conformation. Together, these findings provide molecular insights into a key aspect of c-FLIP L function that modulates procaspase-8 activation to elicit diverse responses in different cellular contexts.
- Published
- 2009
43. Modifying specificity of antidigoxin antibodies using insertional mutagenesis
- Author
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Rustem Krykbaev, Philip D. Jeffrey, Panayota Tsantili, and Michael N. Margolies
- Subjects
Digoxin ,Digitoxin ,Molecular Sequence Data ,Mutant ,Biology ,Biochemistry ,Article ,Insertional mutagenesis ,Mice ,chemistry.chemical_compound ,Antibody Specificity ,polycyclic compounds ,medicine ,Consensus sequence ,Animals ,Digoxigenin ,Amino Acid Sequence ,Molecular Biology ,Peptide sequence ,Cardiac glycoside ,Base Sequence ,Antibodies, Monoclonal ,Molecular biology ,carbohydrates (lipids) ,Mutagenesis, Insertional ,chemistry ,Mutagenesis, Site-Directed ,Binding Sites, Antibody ,medicine.drug - Abstract
Certain antibodies (Abs) elicited using the cardiac glycoside digoxin (digoxigenin tridigitoxoside) bind preferentially to analogs that differ from digoxin by substitutions on the cardenolide rings, the lactone, or by the presence or absence of attached sugars. Antibody 26-10 binds equally well to digoxin and digitoxin, which differ only by the presence in the former and the absence in the latter of an hydroxyl group at C12. Other antidigoxin Abs, however, can distinguish between these ligands by three orders of magnitude in binding. Inspection of the structure of Fab 26-10 complexed with digoxin shows a gap in complementarity in the region between the digoxin O12 and LCDR3. We proposed that insertions in LCDR3 might result in Abs that bind digitoxin preferentially. We produced libraries of mutants displayed on bacteriophage which were randomized at LCDR3 and contained LCDR3 insertions. Mutants were selected by panning against digoxin and analogs. The mutants bound digitoxin preferentially up to 47-fold greater than digoxin. The mutants that bound well to digitoxin demonstrated a consensus sequence including the substitution of Trp at position L:94. Using site-directed mutagenesis, the binding to digitoxin was shown to be maximized by the combination of an insertion and L:Trp94 mutation, moving the L 94 side chain closer to digoxin. We also selected mutants that bound preferentially to gitoxin, which, like digitoxin, lacks the 12-hydroxyl, increasing relative binding to gitoxin up to 600-fold compared to the unmutated Ab 26-10.
- Published
- 2009
44. Structural characterization of Tip20p and Dsl1p, subunits of the Dsl1p vesicle tethering complex
- Author
-
Yi Ren, Frederick M. Hughson, Arati Tripathi, and Philip D. Jeffrey
- Subjects
Models, Molecular ,Saccharomyces cerevisiae Proteins ,Vesicular Transport Proteins ,Golgi Apparatus ,Exocyst ,Saccharomyces cerevisiae ,Biology ,Crystallography, X-Ray ,Endoplasmic Reticulum ,Article ,Vesicle tethering ,symbols.namesake ,Structural Biology ,Animals ,Molecular Biology ,Glycoproteins ,Tethering ,Endoplasmic reticulum ,Vesicle ,Membrane Proteins ,Golgi apparatus ,Transport protein ,Cell biology ,Membrane ,symbols ,Carrier Proteins ,SNARE Proteins ,Protein Binding - Abstract
Multisubunit tethering complexes are essential for intracellular trafficking and have been proposed to mediate the initial interaction between vesicles and the membranes with which they fuse. Here we report initial structural characterization of the Dsl1p complex, whose three subunits are essential for trafficking from the Golgi apparatus to the endoplasmic reticulum (ER). Crystal structures reveal that two of the three subunits, Tip20p and Dsl1p, resemble known subunits of the exocyst complex, establishing a structural connection among several multisubunit tethering complexes and implying that many of their subunits are derived from a common progenitor. We show, moreover, that Tip20p and Dsl1p interact directly via N-terminal alpha-helices. Finally, we establish that different Dsl1p complex subunits bind independently to different ER SNARE proteins. Our results map out two alternative protein-interaction networks capable of tethering COPI-coated vesicles, via the Dsl1p complex, to ER membranes.
- Published
- 2009
45. Analysis of errors in the structure determination of MsbA
- Author
-
Philip D. Jeffrey
- Subjects
Bacterial protein ,Biochemistry ,Bacterial Proteins ,Structural Biology ,protein crystallography ,ATP-binding cassette transporter ,methodology ,ATP-Binding Cassette Transporters ,General Medicine ,Biology ,Crystallography, X-Ray ,Research Papers - Abstract
An analysis is presented of the methodological errors that led to the incorrect structure of MsbA., The determination of incorrect structures for the ABC transporter MsbA gave rise to questions of how this could have occurred. Methodological aspects of the MsbA structure determination are explored in light of this error.
- Published
- 2009
46. Structural Mechanism of Demethylation and Inactivation of Protein Phosphatase 2A
- Author
-
Zhu Li, Yu Chen, Yongna Xing, Yigong Shi, Philip D. Jeffrey, and Jeffry B. Stock
- Subjects
Models, Molecular ,PROTEINS ,Protein subunit ,Molecular Sequence Data ,Phosphatase ,DUSP6 ,macromolecular substances ,Crystallography, X-Ray ,Methylation ,environment and public health ,General Biochemistry, Genetics and Molecular Biology ,Serine ,03 medical and health sciences ,0302 clinical medicine ,Catalytic triad ,Animals ,Humans ,Amino Acid Sequence ,Protein Phosphatase 2 ,030304 developmental biology ,Demethylation ,0303 health sciences ,biology ,Biochemistry, Genetics and Molecular Biology(all) ,Protein phosphatase 2 ,enzymes and coenzymes (carbohydrates) ,Biochemistry ,SIGNALING ,embryonic structures ,biology.protein ,Carboxylic Ester Hydrolases ,Sequence Alignment ,030217 neurology & neurosurgery - Abstract
SummaryProtein phosphatase 2A (PP2A) is an important serine/threonine phosphatase that plays a role in many biological processes. Reversible carboxyl methylation of the PP2A catalytic subunit is an essential regulatory mechanism for its function. Demethylation and negative regulation of PP2A is mediated by a PP2A-specific methylesterase PME-1, which is conserved from yeast to humans. However, the underlying mechanism of PME-1 function remains enigmatic. Here we report the crystal structures of PME-1 by itself and in complex with a PP2A heterodimeric core enzyme. The structures reveal that PME-1 directly binds to the active site of PP2A and that this interaction results in the activation of PME-1 by rearranging the catalytic triad into an active conformation. Strikingly, these interactions also lead to inactivation of PP2A by evicting the manganese ions that are required for the phosphatase activity of PP2A. These observations identify a dual role of PME-1 that regulates PP2A activation, methylation, and holoenzyme assembly in cells.
- Published
- 2008
47. Structure of a Site-2 Protease Family Intramembrane Metalloprotease
- Author
-
Yigong Shi, Hanchi Yan, Nieng Yan, Liang Feng, Zhe Wang, Philip D. Jeffrey, and Zhuoru Wu
- Subjects
Protein Conformation ,Stereochemistry ,Intramembrane protease ,Archaeal Proteins ,Methanococcus ,medicine.medical_treatment ,Molecular Sequence Data ,Crystallography, X-Ray ,Regulated Intramembrane Proteolysis ,Catalysis ,Protein Structure, Secondary ,Protein structure ,Bacterial Proteins ,Catalytic Domain ,medicine ,Amino Acid Sequence ,Integral membrane protein ,Binding Sites ,Multidisciplinary ,Protease ,biology ,Chemistry ,Membrane Proteins ,Metalloendopeptidases ,Water ,Active site ,Transmembrane protein ,Protein Structure, Tertiary ,Zinc ,Transmembrane domain ,biology.protein ,Crystallization ,Dimerization - Abstract
Regulated intramembrane proteolysis by members of the site-2 protease (S2P) family is an important signaling mechanism conserved from bacteria to humans. Here we report the crystal structure of the transmembrane core domain of an S2P metalloprotease from Methanocaldococcus jannaschii . The protease consists of six transmembrane segments, with the catalytic zinc atom coordinated by two histidine residues and one aspartate residue ∼14 angstroms into the lipid membrane surface. The protease exhibits two distinct conformations in the crystals. In the closed conformation, the active site is surrounded by transmembrane helices and is impermeable to substrate peptide; water molecules gain access to zinc through a polar, central channel that opens to the cytosolic side. In the open conformation, transmembrane helices α1 and α6 separate from each other by 10 to 12 angstroms, exposing the active site to substrate entry. The structure reveals how zinc embedded in an integral membrane protein can catalyze peptide cleavage.
- Published
- 2007
48. Structural and biochemical insights into the regulation of protein phosphatase 2A by small t antigen of SV40
- Author
-
Yanhui Xu, Qing Bao, Zhu Li, Zheng Lin, Yigong Shi, Yongna Xing, Philip D. Jeffrey, Jeffry B. Stock, and Yu Chen
- Subjects
Models, Molecular ,Protein subunit ,Molecular Sequence Data ,Phosphatase ,Simian virus 40 ,Biology ,environment and public health ,chemistry.chemical_compound ,Holoenzymes ,Antigen ,Structural Biology ,Humans ,Amino Acid Sequence ,Protein Phosphatase 2 ,Binding site ,Antigens, Viral, Tumor ,Molecular Biology ,chemistry.chemical_classification ,Protein phosphatase 2 ,Cell Transformation, Viral ,Protein Structure, Tertiary ,Protein Subunits ,enzymes and coenzymes (carbohydrates) ,Enzyme ,Biochemistry ,chemistry ,Electrophoresis, Polyacrylamide Gel ,Crystallization ,DNA - Abstract
The small t antigen (ST) of DNA tumor virus SV40 facilitates cellular transformation by disrupting the functions of protein phosphatase 2A (PP2A) through a poorly defined mechanism. The crystal structure of the core domain of SV40 ST bound to the scaffolding subunit of human PP2A reveals that the ST core domain has a novel zinc-binding fold and interacts with the conserved ridge of HEAT repeats 3-6, which overlaps with the binding site for the B' (also called PR61 or B56) regulatory subunit. ST has a lower binding affinity than B' for the PP2A core enzyme. Consequently, ST does not efficiently displace B' from PP2A holoenzymes in vitro. Notably, ST inhibits PP2A phosphatase activity through its N-terminal J domain. These findings suggest that ST may function mainly by inhibiting the phosphatase activity of the PP2A core enzyme, and to a lesser extent by modulating assembly of the PP2A holoenzymes.
- Published
- 2007
49. Crystal Structure of the Bcl-XL-Beclin 1 Peptide Complex
- Author
-
Yigong Shi, Adam Oberstein, and Philip D. Jeffrey
- Subjects
Autophagy ,Bcl-xL ,Cell Biology ,BH3-only protein ,Peptide complex ,Biology ,Biochemistry ,Cell biology ,law.invention ,Protein structure ,Apoptosis ,law ,biology.protein ,Suppressor ,biological phenomena, cell phenomena, and immunity ,Molecular Biology ,Function (biology) - Abstract
Bcl-2 family proteins are key regulators of apoptosis and have recently been shown to modulate autophagy. The tumor suppressor Beclin 1 has been proposed to coordinate both apoptosis and autophagy through direct interaction with anti-apoptotic family members Bcl-2 and/or Bcl-XL. However, the molecular basis for this interaction remains enigmatic. Here we report that Beclin 1 contains a conserved BH3 domain, which is both necessary and sufficient for its interaction with Bcl-XL. We also report the crystal structure of a Beclin BH3 peptide in complex with Bcl-XL at 2.5A resolution. Reminiscent of previously determined Bcl-XL-BH3 structures, the amphipathic BH3 helix of Beclin 1 bound to a conserved hydrophobic groove of Bcl-XL. These results define Beclin 1 as a novel BH3-only protein, implying that Beclin 1 may have a direct role in initiating apoptotic signaling. We propose that this putative apoptotic function may be linked to the ability of Beclin 1 to suppress tumor formation in mammals.
- Published
- 2007
50. BET inhibitor resistance emerges from leukaemia stem cells
- Author
-
Mark A. Dawson, Dave Lugo, Richard Gregory, Paul Yeh, Alan F. Rubin, Omer Gilan, Steven W. Lane, Devbarna Sinha, Rab K. Prinjha, Sarah Ftouni, Jessica Kate Morison, Dean Tyler, Kym Stanley, Chun Yew Fong, Christopher L. Carpenter, Sarah-Jane Dawson, Anthony T. Papenfuss, Brian J. P. Huntly, Robert G. Ramsay, Stanley Chun-Wei Lee, Ricky W. Johnstone, Philip D. Jeffrey, Omar Abdel-Wahab, George Giotopoulos, Enid Y.N. Lam, and Tony Kouzarides
- Subjects
BRD4 ,Transcription, Genetic ,Genes, myc ,Cell Cycle Proteins ,chemical and pharmacologic phenomena ,Biology ,Article ,Epigenesis, Genetic ,BET inhibitor ,Benzodiazepines ,Mice ,03 medical and health sciences ,0302 clinical medicine ,In vivo ,Cell Line, Tumor ,Animals ,Humans ,Molecular Targeted Therapy ,Progenitor cell ,Wnt Signaling Pathway ,Cells, Cultured ,beta Catenin ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,Wnt signaling pathway ,Nuclear Proteins ,hemic and immune systems ,Azepines ,Triazoles ,Hematopoietic Stem Cells ,Molecular biology ,Chromatin ,Clone Cells ,3. Good health ,Bromodomain ,Gene Expression Regulation, Neoplastic ,Leukemia, Myeloid, Acute ,Haematopoiesis ,Drug Resistance, Neoplasm ,030220 oncology & carcinogenesis ,Neoplastic Stem Cells ,Cancer research ,Stem cell ,Transcription Factors - Abstract
Bromodomain and extra terminal protein (BET) inhibitors are first-in-class targeted therapies that deliver a new therapeutic opportunity by directly targeting bromodomain proteins that bind acetylated chromatin marks1,2. Early clinical trials have shown promise, especially in acute myeloid leukaemia3, and therefore the evaluation of resistance mechanisms is crucial to optimize the clinical efficacy of these drugs. Here we use primary mouse haematopoietic stem and progenitor cells immortalized with the fusion protein MLL-AF9 to generate several single-cell clones that demonstrate resistance, in vitro and in vivo, to the prototypical BET inhibitor, I-BET. Resistance to I-BET confers cross-resistance to chemically distinct BET inhibitors such as JQ1, as well as resistance to genetic knockdown of BET proteins. Resistance is not mediated through increased drug efflux or metabolism, but is shown to emerge from leukaemia stem cells both ex vivo and in vivo. Chromatin-bound BRD4 is globally reduced in resistant cells, whereas the expression of key target genes such as Myc remains unaltered, highlighting the existence of alternative mechanisms to regulate transcription. We demonstrate that resistance to BET inhibitors, in human and mouse leukaemia cells, is in part a consequence of increased Wnt/β-catenin signalling, and negative regulation of this pathway results in restoration of sensitivity to I-BET in vitro and in vivo. Together, these findings provide new insights into the biology of acute myeloid leukaemia, highlight potential therapeutic limitations of BET inhibitors, and identify strategies that may enhance the clinical utility of these unique targeted therapies.
- Published
- 2015
- Full Text
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