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Glutathione (GSH) and its oxidized dimer (GSSG) play an important role in living systems as an antioxidant, balancing the presence of reactive oxygen species (ROS). The central thiol (‐S‐S‐) bond in GSSG can undergo free rotation, providing multiple conformations with respect to the S‐S bridge. The six titratable sites of GSSG, which are influenced by pH variations, affect these conformations in solution, whereas in solids, additionally crystal packing effects come into play. In view of differing reports about the structure of GSSG in literature, we have here conducted an extensive reexamination of its conformations using NMR, and contrasting results have been obtained for solution and solid state. In solution, the existence of more than one antiparallel orientation of the monomer unit with different hydrogen bonding schemes has been indicated by NOE and amide temperature coefficient results. On the other hand, in the solid‐state, a 1H‐1H double‐quantum (DQ) to 13C single‐quantum (SQ) correlation study has confirmed a parallel orientation, consistent with the reported X‐ray crystal structure. Experimentally assigned solid‐state NMR resonances have been validated using GIPAW calculations incorporated in the Quantum ESPRESSO package. [ABSTRACT FROM AUTHOR]

Research on camelid-derived single-domain antibodies (sdAbs) has demonstrated their significant utility in diverse biotechnological applications, including therapy and diagnostic. This is largely due to their relative simplicity as monomeric proteins, ranging from 12 to 15 kDa, in contrast to immunoglobulin G (IgG) antibodies, which are glycosylated heterotetramers of 150–160 kDa. Single-domain antibodies exhibit high conformational stability and adopt the typical immunoglobulin domain fold, consisting of a two-layer sandwich of 7–9 antiparallel beta-strands. They contain three loops, known as complementary-determining regions (CDRs), which are assembled on the sdAb surface and are responsible for antigen recognition. The single-domain antibody examined in this study, sdAb-mrh-IgG, was engineered to recognize IgG from rats, mice, but it also weakly recognizes IgG from humans (Pleiner et al. 2018). A search of the Protein Data Bank revealed only one NMR structure of a single-domain antibody, which is unrelated to sdAb-mrh-IgG. The NMR chemical shift assignments of sdAb-mrh-IgG will be utilized to study its molecular dynamics and interactions with antigens in solution, which is fundamental for the rational design of novel single-domain antibodies. [ABSTRACT FROM AUTHOR]

Metallothioneins (MTs) are small proteins that coordinate d‐block metal ions in sulfur‐metal clusters to control metal ion concentrations within the cell. Here we study metal cluster formation in the MT of the periwinkle Littorina littorea (LlMT) by nuclear magnetic resonance (NMR). We demonstrate that the three Cd2+ ions in each domain are taken up highly cooperatively, that is, in an all‐or‐none fashion, with a four‐ to six‐fold higher affinity for the C‐terminal domain. During the transfer of metal ions from Cd2+‐loaded MT to apo MT, Cd2+ is most efficiently transferred from the metalated protein to the apo C‐terminal domain. This behavior might be connected to unique structural motifs in the C‐terminal domain, such as two double‐CXC motifs and an increased proportion of positively charged residues. In Cd2+/Zn2+ metal exchange experiments, the N‐terminal domain displayed the most efficient inter‐molecular metal exchange. Amide hydrogen exchange reveals fewer protected amides for the N‐terminal domain, suggesting the structure might more easily "open up" to facilitate metal exchange. Experiments with a physical separation of donor and acceptor species demonstrate that metal exchange and transfer require protein–protein contacts. These findings provide insights into the mechanism of metal uptake and metal transfer, which are important processes during metal detoxification in snail MTs. [ABSTRACT FROM AUTHOR]

Myotonic dystrophy type 2 (DM2) is a neurogenerative disease caused by caprylic/capric triglyceride (CCTG) tetranucleotide repeat expansions in intron 1 of the cellular nucleic acid‐binding protein (CNBP) gene. Non‐B DNA structures formed by CCTG repeats can promote genetic instability, whereas interrupting motifs of NCTG (N = A/T/G) within CCTG repeats help to maintain genomic stability. However, whether the interrupting motifs can affect DNA structures of CCTG repeats remains unclear. Here, we report that four CCTG repeats with an interrupting 3′‐A/T/G residue formed dumbbell structures, whereas a non‐interrupting 3′‐C residue resulted in a multi‐loop structure exhibiting conformational dynamics that may contribute to a higher tendency of escaping from DNA mismatch repair and causing repeat expansions. The results provide new structural insights into the genetic instability of CCTG repeats in DM2. [ABSTRACT FROM AUTHOR]

DNA aptamers have emerged as novel molecular tools in disease theranostics owing to their high binding affinity and specificity for protein targets, which rely on their ability to fold into distinctive three-dimensional (3D) structures. However, delicate atomic interactions that shape the 3D structures are often ignored when designing and modeling aptamers, leading to inefficient functional optimization. Challenges persist in determining high-resolution aptamer-protein complex structures. Moreover, the experimentally determined 3D structures of DNA molecules with exquisite functions remain scarce. These factors impede our comprehension and optimization of some important DNA aptamers. Here, we performed a streamlined solution NMR-based structural investigation on the 41-nt sgc8c, a prominent DNA aptamer used to target membrane protein tyrosine kinase 7, for cancer theranostics. We show that sgc8c prefolds into an intricate three-way junction (3WJ) structure stabilized by long-range tertiary interactions and extensive base-base stackings. Delineated by NMR chemical shift perturbations, site-directed mutagenesis, and 3D structural information, we identified essential nucleotides constituting the key functional elements of sgc8c that are centralized at the core of 3WJ. Leveraging the well-established structure-function relationship, we efficiently engineered two sgc8c variants by modifying the apical loop and introducing L-DNA base pairs to simultaneously enhance thermostability, biostability, and binding affinity for both protein and cell targets, a feat not previously attained despite extensive efforts. This work showcases a simplified NMR-based approach to comprehend and optimize sgc8c without acquiring the complex structure, and offers principles for the sophisticated structure-function organization of DNA molecules. [ABSTRACT FROM AUTHOR]

Photoconvertible fluorescent proteins (PCFP) are important cellular markers in advanced imaging modalities such as photoactivatable localization microscopy (PALM). However, their complex photophysical and photochemical behavior hampers applications such as quantitative and single‐particle‐tracking PALM. This work employs multidimensional NMR combined with ensemble fluorescence measurements to show that the popular mEos4b in its Green state populates two conformations (A and B), differing in side‐chain protonation of the conserved residues E212 and H62, altering the hydrogen‐bond network in the chromophore pocket. The interconversion (protonation/deprotonation) between these two states, which occurs on the minutes time scale in the dark, becomes strongly accelerated in the presence of UV light, leading to a population shift. This work shows that the reversible photoswitching and Green‐to‐Red photoconversion properties differ between the A and B states. The chromophore in the A‐state photoswitches more efficiently and is proposed to be more prone to photoconversion, while the B‐state shows a higher level of photobleaching. Altogether, this data highlights the central role of conformational heterogeneity in fluorescent protein photochemistry. [ABSTRACT FROM AUTHOR]

NMR studies can provide unique information about protein conformations in solution. In CASP14, three reference structures provided by solution NMR methods were available (T1027, T1029, and T1055), as well as a fourth data set of NMR-derived contacts for an integral membrane protein (T1088). For the three targets with NMR-based structures, the best prediction results ranged from very good (GDT_TS = 0.90, for T1055) to poor (GDT_TS = 0.47, for T1029). We explored the basis of these results by comparing all CASP14 prediction models against experimental NMR data. For T1027, NMR data reveal extensive internal dynamics, presenting a unique challenge for protein structure prediction methods. The analysis of T1029 motivated exploration of a novel method of "inverse structure determination," in which an AlphaFold2 model was used to guide NMR data analysis. NMR data provided to CASP predictor groups for target T1088, a 238-residue integral membrane porin, was also used to assess several NMR-assisted prediction methods. Most groups involved in this exercise generated similar beta-barrel models, with good agreement with the experimental data. However, as was also observed in CASP13, some pure prediction groups that did not use any NMR data generated models for T1088 that better fit the NMR data than the models generated using these experimental data. These results demonstrate the remarkable power of modern methods to predict structures of proteins with accuracies rivaling solution NMR structures, and that it is now possible to reliably use prediction models to guide and complement experimental NMR data analysis.

Abstract Photoconvertible fluorescent proteins (PCFP) are important cellular markers in advanced imaging modalities such as photoactivatable localization microscopy (PALM). However, their complex photophysical and photochemical behavior hampers applications such as quantitative and single‐particle‐tracking PALM. This work employs multidimensional NMR combined with ensemble fluorescence measurements to show that the popular mEos4b in its Green state populates two conformations (A and B), differing in side‐chain protonation of the conserved residues E212 and H62, altering the hydrogen‐bond network in the chromophore pocket. The interconversion (protonation/deprotonation) between these two states, which occurs on the minutes time scale in the dark, becomes strongly accelerated in the presence of UV light, leading to a population shift. This work shows that the reversible photoswitching and Green‐to‐Red photoconversion properties differ between the A and B states. The chromophore in the A‐state photoswitches more efficiently and is proposed to be more prone to photoconversion, while the B‐state shows a higher level of photobleaching. Altogether, this data highlights the central role of conformational heterogeneity in fluorescent protein photochemistry.

Staphylococcus aureus CzrA is a paradigmatic member of the ArsR family of transcriptional metalloregulators, which are critical for the bacterial response to stress. Zinc binding to CzrA, which induces DNA derepression, is entropically driven, as shown by calorimetry. A detailed equilibrium dynamics study of different allosteric states of CzrA revealed that zinc induces an entropy redistribution that controls for DNA binding regulation; however, this change in conformational entropy only accounts for a small net contribution to the total entropy. This difference between the change in conformational entropy vs. total entropy of zinc binding implies a significant contribution of solvent molecule rearrangements to this equilibrium. However, the absence of major structural changes suggests that solvent rearrangements occur mainly on the protein surface and/or from zinc desolvation, concomitant with a dynamical redistribution of conformational entropy. Previous results also suggest that zinc binding not only leads to a redistribution of protein internal dynamics, but also release of water molecules from the protein surface. In turn, these water molecules may make a significant contribution to the allosteric response that results in dissociation from the DNA.Quantifying the differential hydration of two conformational states that share very similar crystal structures and then correlating this with the protein's solvent entropy change constitutes an unresolved problem, even when thermodynamics suggest a significant contribution of solvent entropy. Here, we present different avenues to dissect hydration dynamics in a metal-binding transcriptional regulator that provide different insights into this complex problem. We explore primary solution NMR tools for probing protein–water interactions: the laboratory frame nuclear Overhauser effect (NOE) and its rotating frame counterpart (ROE) between long-lived water molecules and the protein residues. The wNOE/wROE ratio is a promising tool for the detection of hydration dynamics near the surface of a protein in a site-specific manner, minimizing contamination from bulk solvent. Molecular dynamics simulations and computational methods designed to provide a spatially resolved picture of solvent thermodynamics were also employed to provide a more complete panorama of solvent redistribution.

CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat and associated Cas9 protein) is a molecular tool with transformative genome editing capabilities. At the molecular level, an intricate allosteric signaling is critical for DNA cleavage, but its role in the specificity enhancement of the Cas9 endonuclease is poorly understood. Here, multi-microsecond molecular dynamics is combined with solution NMR and graph theory-derived models to probe the allosteric role of key specificity-enhancing mutations. We show that mutations responsible for increasing the specificity of Cas9 alter the allosteric structure of the catalytic HNH domain, impacting the signal transmission from the DNA recognition region to the catalytic sites for cleavage. Specifically, the K855A mutation strongly disrupts the allosteric connectivity of the HNH domain, exerting the highest perturbation on the signaling transfer, while K810A and K848A result in more moderate effects on the allosteric communication. This differential perturbation of the allosteric signal correlates to the order of specificity enhancement (K855A > K848A ~ K810A) observed in biochemical studies, with the mutation achieving the highest specificity most strongly perturbing the signaling transfer. These findings suggest that alterations of the allosteric communication from DNA recognition to cleavage are critical to increasing the specificity of Cas9 and that allosteric hotspots can be targeted through mutational studies for improving the system's function.

Expansions of short tandem repeats (STRs) are associated with approximately 50 human neurodegenerative diseases. These pathogenic STRs are prone to form non-B DNA structure, which has been considered as one of the causative factors for repeat expansions. Minidumbbell (MDB) is a relatively new type of non-B DNA structure formed by pyrimidine-rich STRs. An MDB is composed of two tetraloops or pentaloops, exhibiting a highly compact conformation with extensive loop-loop interactions. The MDB structures have been found to form in CCTG tetranucleotide repeats associated with myotonic dystrophy type 2, ATTCT pentanucleotide repeats associated with spinocerebellar ataxia type 10, and the recently discovered ATTTT/ATTTC repeats associated with spinocerebellar ataxia type 37 and familial adult myoclonic epilepsy. In this review, we first introduce the structures and conformational dynamics of MDBs with a focus on the high-resolution structural information determined by nuclear magnetic resonance spectroscopy. Then we discuss the effects of sequence context, chemical environment, and nucleobase modification on the structure and thermostability of MDBs. Finally, we provide perspectives on further explorations of sequence criteria and biological functions of MDBs.

Eight hundred and twenty-six human G protein-coupled receptors (GPCRs) mediate the actions of two-thirds of the human hormones and neurotransmitters and over one-third of clinically used drugs. Studying the structure and dynamics of human GPCRs in lipid bilayer environments resembling the native cell membrane milieu is of great interest as a basis for understanding structure–function relationships and thus benefits continued drug development. Here, we incorporate the human A2A adenosine receptor (A2AAR) into lipid nanodiscs, which represent a detergent-free environment for structural studies using nuclear magnetic resonance (NMR) in solution. The [15N,1H]-TROSY correlation spectra confirmed that the complex of [u-15N, ~70% 2H]-A2AAR with an inverse agonist adopts its global fold in lipid nanodiscs in solution at physiological temperature. The global assessment led to two observations of practical interest. First, A2AAR in nanodiscs can be stored for at least one month at 4 °C in an aqueous solvent. Second, LMNG/CHS micelles are a very close mimic of the environment of A2AAR in nanodiscs. The NMR signal of five individually assigned tryptophan indole 15N–1H moieties located in different regions of the receptor structure further enabled a detailed assessment of the impact of nanodiscs and LMNG/CHS micelles on the local structure and dynamics of A2AAR. As expected, the largest effects were observed near the lipid–water interface along the intra- and extracellular surfaces, indicating possible roles of tryptophan side chains in stabilizing GPCRs in lipid bilayer membranes. [ABSTRACT FROM AUTHOR]

G protein‐coupled receptors (GPCRs) transmit signals from drugs across cell membranes, leading to associated physiological effects. To study the structural basis of the transmembrane signalling, in‐membrane chemical modification (IMCM) has previously been introduced for 19F‐labelling of GPCRs expressed in Spodoptera frugiperda (Sf9) insect cells. Here, IMCM is used with the A2A adenosine receptor (A2AAR) expressed in Pichia pastoris; 19F‐NMR revealed nearly complete solvent protection of the A2AAR transmembrane domain in the membrane and in 2,2‐didecylpropane‐1,3‐bis‐β‐D‐maltopyranoside (LMNG)/cholesteryl hemisuccinate (CHS) micelles, and extensive solvent accessibility for A2AAR in n‐dodecyl β‐D‐maltoside (DDM)/CHS micelles. No Cys residue dominated non‐specific labelling with 2,2,2‐trifluoroethanethiol. These observations yield an improved protocol for IMCM 19F‐labelling of GPCRs and new insights into variable solvent accessibility for function‐related characterization of GPCRs. [ABSTRACT FROM AUTHOR]

Staphylococcus epidermidis is the leading causative agent for hospital-acquired infections, especially device-related infections, due to its ability to form biofilms. The accumulation-associated protein (Aap) of S. epidermidis is primarily responsible for biofilm formation and consists of two domains, A and B. It was found that the A domain is responsible for the attachment to the abiotic/biotic surface, whereas the B domain is responsible for the accumulation of bacteria during biofilm formation. One of the parts of the A domain is the Aap lectin, which is a carbohydrate-binding domain having 222 amino acids in its structure. Here we report the near complete backbone chemical shift assignments for the lectin domain, as well as its predicted secondary structure. This data will provide a platform for future NMR studies to explore the role of lectin in biofilm formation. [ABSTRACT FROM AUTHOR]

Selective isotopic labeling of methyl side chain groups in proteins and other biomolecules, combined with advances in perdeuteration, new pulse sequences, and high field spectrometers with cryogenic probes, has revolutionized the field of solution nuclear magnetic resonance (NMR) spectroscopy by enabling characterization of macromolecular systems with molecular weights above 1 MDa in their native aqueous environment. This tutorial provides a basic overview for how modern NMR spectroscopists can utilize methyl side chain probes to study their system of interest. The advantages and limitations of methyl side chain probes are discussed. In addition, the preparation of selectively 13C-methyl labeled recombinant protein samples, strategies for manual and automated methyl NMR resonance assignment, and the application of methyl probes for characterization of dynamics and conformational exchange are discussed. A sneak preview for ways in which methyl probes are expected to continue to advance the field of biomolecular NMR towards new horizons in solution studies of large supramolecular complexes is also presented.

To completely treat and ultimately prevent dementia, it is essential to elucidate its pathogenic mechanisms in detail. There are two major hypotheses for the pathogenesis of Alzheimer’s dementia: the β-amyloid (Aβ) hypothesis and the tau hypothesis. The modified amyloid hypothesis, which proposes that toxic oligomers rather than amyloid fibrils are the essential cause, has recently emerged. Aβ peptides [Aβ(1–40) and Aβ(1–42)] form highly insoluble aggregates in vivo and in vitro. These Aβ aggregates contain many polymorphisms, whereas Aβ peptides are intrinsically disordered in physiological aqueous solutions without any compact conformers. Over the last three decades, solid-state nuclear magnetic resonance (NMR) has greatly contributed to elucidating the structure of each polymorph, while solution NMR has revealed the dynamic nature of the transient conformations of the monomer. Moreover, several methods to investigate the aggregation process based on the observation of magnetization saturation transfer have also been developed. The complementary use of NMR methods with cryo-electron microscopy, which has rapidly matured, is expected to clarify the relationship between the amyloid and molecular pathology of Alzheimer’s dementia in the near future. This review article is an extended version of the Japanese article, Insights into the Mechanisms of Oligomerization/Fibrilization of Amyloid β Peptide from Nuclear Magnetic Resonance, published in SEIBUTSU BUTSURI Vol. 62, p. 39–42 (2022).

Metallothioneins (MTs) are small proteins that coordinate d-block metal ions in sulfur-metal clusters to control metal ion concentrations within the cell. Here we study metal cluster formation in the MT of the periwinkle Littorina littorea (LlMT) by nuclear magnetic resonance (NMR). We demonstrate that the three Cd 2+ ions in each domain are taken up highly cooperatively, that is, in an all-or-none fashion, with a four- to six-fold higher affinity for the C-terminal domain. During the transfer of metal ions from Cd 2+ -loaded MT to apo MT, Cd 2+ is most efficiently transferred from the metalated protein to the apo C-terminal domain. This behavior might be connected to unique structural motifs in the C-terminal domain, such as two double-CXC motifs and an increased proportion of positively charged residues. In Cd 2+ /Zn 2+ metal exchange experiments, the N-terminal domain displayed the most efficient inter-molecular metal exchange. Amide hydrogen exchange reveals fewer protected amides for the N-terminal domain, suggesting the structure might more easily "open up" to facilitate metal exchange. Experiments with a physical separation of donor and acceptor species demonstrate that metal exchange and transfer require protein-protein contacts. These findings provide insights into the mechanism of metal uptake and metal transfer, which are important processes during metal detoxification in snail MTs., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Nuclear magnetic resonance (NMR) is a major method used to study protein structure at atomic resolution. Besides presenting the high-resolution structure, NMR facilitates studies on protein dynamics near physiological conditions that are intimately related to the biological mechanism of the proteins. Unnatural amino acids (UAA) labeling could significantly reduce the complexity of protein NMR spectra. In this review, we briefly summarize the widely used UAA labeling strategies for proteins, including chemical peptide synthesis, residue-specific peptide modification, 19F labeled aromatic amino acids incorporation and genetic code expansion based UAA incorporation. The recent applicants of UAA in characterizing protein structure and dynamics, the limitations and prospects of UAA are also highlighted.

Understanding the effects of a fertilization regime on the long-term accumulation and transformation of soil phosphorus (P) is essential for promoting the development of sustainable management of soil P. Based on a 29-year field experiment in Mollisol, the compositions and changes of P forms using a modified Hedley sequential extraction method, solution 31P-NMR and P K-edge XANES and soil properties were investigated under continuous mono maize with and without manure (NPKM and NPK). Results showed a stronger positive related coefficient between soil total P and labile P, and mid-labile P fraction was found in NPKM than in NPK treatment. It indicated NPKM could improve the availability of soil accumulated P and reduce its transformation to stable P. Accumulated inorganic P (Pi) was dominated by aluminum phosphate (Al-P) and monobasic calcium phosphate monohydrate (MCP) for NPK treatment, Al-P, MCP, and tricalcium phosphate for NPKM treatment with XANES analysis, which contributed to the P availability in Mollisol. Moreover, the proportion of IHP with XANES and ratio of orthophosphate diesters to monoesters in NPK compared to NPKM indicated the higher Po lability with NPK treatment. Pi, especially NaHCO3-Pi and NaOH-Pi, were the potential sources of resin-Pi. Soil organic matter (SOM), organic-bound iron, and alumina oxide (Fep + Alp) showed significant influence on the transformation of P forms. Our research suggested that due to the rise in SOM and Fep + Alp, the fertilization regime significantly increased most highly active soil P fractions, especially in NPKM treatment. This work gives new insight into sustainable P management, which benefits the reduction in soil P accumulation. [ABSTRACT FROM AUTHOR]

The dysbindin domain-containing protein 1 (DBNDD1) is a conserved protein among higher eukaryotes whose structure and function are poorly investigated so far. Here, we present the backbone and side chain nuclear magnetic resonance assignments for the human DBNDD1 protein. Our chemical-shift based secondary structure analysis reveals the human DBNDD1 as an intrinsically disordered protein. [ABSTRACT FROM AUTHOR]

Eight hundred and twenty-six human G protein-coupled receptors (GPCRs) mediate the actions of two-thirds of the human hormones and neurotransmitters and over one-third of clinically used drugs. Studying the structure and dynamics of human GPCRs in lipid bilayer environments resembling the native cell membrane milieu is of great interest as a basis for understanding structure–function relationships and thus benefits continued drug development. Here, we incorporate the human A2A adenosine receptor (A2AAR) into lipid nanodiscs, which represent a detergent-free environment for structural studies using nuclear magnetic resonance (NMR) in solution. The [15N,1H]-TROSY correlation spectra confirmed that the complex of [u-15N, ~70% 2H]-A2AAR with an inverse agonist adopts its global fold in lipid nanodiscs in solution at physiological temperature. The global assessment led to two observations of practical interest. First, A2AAR in nanodiscs can be stored for at least one month at 4 °C in an aqueous solvent. Second, LMNG/CHS micelles are a very close mimic of the environment of A2AAR in nanodiscs. The NMR signal of five individually assigned tryptophan indole 15N–1H moieties located in different regions of the receptor structure further enabled a detailed assessment of the impact of nanodiscs and LMNG/CHS micelles on the local structure and dynamics of A2AAR. As expected, the largest effects were observed near the lipid–water interface along the intra- and extracellular surfaces, indicating possible roles of tryptophan side chains in stabilizing GPCRs in lipid bilayer membranes.

Multidrug and toxin extrusion (MATE) family transporters excrete toxic compounds coupled to Na+/H+ influx. Although structures of MATE transporters are available, the mechanism by which substrate export is coupled to ion influx remains unknown. To address this issue, we conducted a structural analysis of Pyrococcus furiosus MATE (PfMATE) using solution nuclear magnetic resonance (NMR). The NMR analysis, along with thorough substitutions of all non-exposed acidic residues, confirmed that PfMATE is under an equilibrium between inward-facing (IF) and outward-facing (OF) conformations, dictated by the Glu163 protonation. Importantly, we found that only the IF conformation exhibits a mid-μM affinity for substrate recognition. In contrast, the OF conformation exhibited only weak mM substrate affinity, suitable for releasing substrate to the extracellular side. These results indicate that PfMATE is an affinity-directed H+ antiporter where substrates selectively bind to the protonated IF conformation in the equilibrium, and subsequent proton release mechanistically ensures H+-coupled substrate excretion by the transporter. [Display omitted] • The substrate/H+-antiport mechanism of PfMATE was structurally analyzed by solution NMR • The equilibrium between the IF/OF conformations is dictated by Glu163 protonation • Only the IF conformation has a sufficient affinity for substrate binding • The affinity-directed transport mechanism ensures H+-coupled substrate excretion Takeuchi et al. structurally characterized the substrate/H+-antiport mechanism of the multidrug transporter PfMATE. PfMATE is an affinity-directed H+ antiporter and substrates selectively bind to its protonated IF conformation. Subsequent proton release is coupled to a structural rearrangement to the OF conformation, which mechanistically ensures H+-coupled substrate excretion. [ABSTRACT FROM AUTHOR]

Bacterial tyrosine kinases (BY-kinases) comprise a family of protein tyrosine kinases that are structurally distinct from their functional counterparts in eukaryotes and are highly conserved across the bacterial kingdom. BY-kinases act in concert with their counteracting phosphatases to regulate a variety of cellular processes, most notably the synthesis and export of polysaccharides involved in biofilm and capsule biogenesis. Biochemical data suggest that BY-kinase function involves the cyclic assembly and disassembly of oligomeric states coupled to the overall phosphorylation levels of a C-terminal tyrosine cluster. This process is driven by the opposing effects of intermolecular autophosphorylation, and dephosphorylation catalyzed by tyrosine phosphatases. In the absence of structural insight into the interactions between a BY-kinase and its phosphatase partner in atomic detail, the precise mechanism of this regulatory process has remained poorly defined. To address this gap in knowledge, we have determined the structure of the transiently assembled complex between the catalytic core of the Escherichia coli (K-12) BY-kinase Wzc and its counteracting low-molecular weight protein tyrosine phosphatase (LMW-PTP) Wzb using solution NMR techniques. Unambiguous distance restraints from paramagnetic relaxation effects were supplemented with ambiguous interaction restraints from static spectral perturbations and transient chemical shift changes inferred from relaxation dispersion measurements and used in a computational docking protocol for structure determination. This structurepresents an atomic picture of the mode of interaction between an LMW-PTP and its BY-kinase substrate, and provides mechanistic insight into the phosphorylationcoupled assembly/disassembly process proposed to drive BY-kinase function. [ABSTRACT FROM AUTHOR]

Allosteric modulators of G-protein-coupled receptors (GPCRs) enhance signaling by binding to GPCRs concurrently with their orthosteric ligands, offering a novel approach to overcome the efficacy limitations of conventional orthosteric ligands. However, the structural mechanism by which allosteric modulators mediate GPCR signaling remains largely unknown. Here, to elucidate the mechanism of µ-opioid receptor (MOR) activation by allosteric modulators, we conducted solution NMR analyses of MOR by monitoring the signals from methionine methyl groups. We found that the intracellular side of MOR exists in an equilibrium between three conformations with different activities. Interestingly, the populations in the equilibrium determine the apparent signaling activity of MOR. Our analyses also revealed that the equilibrium is not fully shifted to the conformation with the highest activity even in the full agonistbound state, where the intracellular half of TM6 is outward-shifted. Surprisingly, an allosteric modulator for MOR, BMS-986122, shifted the equilibrium toward the conformation with the highest activity, leading to the increased activity of MOR in the full agonist-bound state. We also determined that BMS-986122 binds to a cleft in the transmembrane region around T162 on TM3. Together, these results suggest that BMS-986122 binding to TM3 increases the activity of MOR by rearranging the direct interactions of TM3 and TM6, thus stabilizing TM6 in the outward-shifted position which is favorable for G-protein binding. These findings shed light on the rational developments of novel allosteric modulators that activate GPCRs further than orthosteric ligands alone and pave the way for next-generation GPCR-targeting therapeutics. [ABSTRACT FROM AUTHOR]

ATP binding cassette (ABC) proteins are present in all phyla of life and form one of the largest protein families. The Bacillus subtilis ABC transporter BmrA is a functional homodimer that can extrude many different harmful compounds out of the cell. Each BmrA monomer is composed of a transmembrane domain (TMD) and a nucleotide binding domain (NBD). While the TMDs of ABC transporters are sequentially diverse, the highly conserved NBDs harbor distinctive conserved motifs that enable nucleotide binding and hydrolysis, interdomain communication and that mark a protein as a member of the ABC superfamily. In the catalytic cycle of an ABC transporter, the NBDs function as the molecular motor that fuels substrate translocation across the membrane via the TMDs and are thus pivotal for the entire transport process. For a better understanding of the structural and dynamic consequences of nucleotide interactions within the NBD at atomic resolution, we determined the 1H, 13C and 15N backbone chemical shift assignments of the 259 amino acid wildtype BmrA-NBD in its post-hydrolytic, ADP-bound state. [ABSTRACT FROM AUTHOR]

Minidumbbell (MDB) is a noncanonical DNA structure found to form in several pyrimidine-rich short tandem repeats associated with neurodegenerative diseases. The most recently reported MDB contains two pentaloops formed by ATTCT repeats. Here, we studied the effects of a purine residue and a backbone discontinuous site on the structure and thermal stability of MDBs containing two pentaloops. It was found that a purine as the fourth loop residue improved the thermal stability of MDBs containing two regular pentaloops, while a backbone discontinuous site between the third and fourth, or between the fourth and fifth loop residues enhanced the thermal stability of MDBs containing a regular and a quasi pentaloops. The results of this study provide new insights into the sequence criteria and structural basis of MDBs. [ABSTRACT FROM AUTHOR]

Glycolysis plays a fundamental role in energy production and metabolic homeostasis. The intracellular [adenosine triphosphate]/[adenosine diphosphate] ([ATP]/[ADP]) ratio controls glycolytic flux; however, the regulatory mechanism underlying reactions catalyzed by individual glycolytic enzymes enabling flux adaptation remains incompletely understood. Phosphoglycerate kinase (PGK) catalyzes the reversible phosphotransfer reaction, which directly produces ATP in a near-equilibrium step of glycolysis. Despite extensive studies on the transcriptional regulation of PGK expression, the mechanism in response to changes in the [ATP]/[ADP] ratio remains obscure. Here, we report a protein-level regulation of human PGK (hPGK) by utilizing the switching ligand-binding cooperativities between adenine nucleotides and 3-phosphoglycerate (3PG). This was revealed by nuclear magnetic resonance (NMR) spectroscopy at physiological salt concentrations. MgADP and 3PG bind to hPGK with negative cooperativity, whereas MgAMPPNP (a nonhydrolyzable ATP analog) and 3PG bind to hPGK with positive cooperativity. These opposite cooperativities enable a shift between different ligand-bound states depending on the intracellular [ATP]/[ADP] ratio. Based on these findings, we present an atomic-scale description of the reaction scheme for hPGK under physiological conditions. Our results indicate that hPGK intrinsically modulates its function via ligand-binding cooperativities that are finely tuned to respond to changes in the [ATP]/[ADP] ratio. The alteration of ligand-binding cooperativities could be one of the self-regulatory mechanisms for enzymes in bidirectional pathways, which enables rapid adaptation to changes in the intracellular environment. [ABSTRACT FROM AUTHOR]

CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat and associated Cas9 protein) is a molecular tool with transformative genome editing capabilities. At the molecular level, an intricate allosteric signaling is critical for DNA cleavage, but its role in the specificity enhancement of the Cas9 endonuclease is poorly understood. Here, multi-microsecond molecular dynamics is combined with solution NMR and graph theory-derived models to probe the allosteric role of key specificity-enhancing mutations. We show that mutations responsible for increasing the specificity of Cas9 alter the allosteric structure of the catalytic HNH domain, impacting the signal transmission from the DNA recognition region to the catalytic sites for cleavage. Specifically, the K855A mutation strongly disrupts the allosteric connectivity of the HNH domain, exerting the highest perturbation on the signaling transfer, while K810A and K848A result in more moderate effects on the allosteric communication. This differential perturbation of the allosteric signal correlates to the order of specificity enhancement (K855A > K848A ~ K810A) observed in biochemical studies, with the mutation achieving the highest specificity most strongly perturbing the signaling transfer. These findings suggest that alterations of the allosteric communication from DNA recognition to cleavage are critical to increasing the specificity of Cas9 and that allosteric hotspots can be targeted through mutational studies for improving the system’s function.

Ribonuclease P (RNase P) is a universal RNA-protein endonuclease that catalyzes 5’ precursor-tRNA (ptRNA) processing. The RNase P RNA plays the catalytic role in ptRNA processing; however, the RNase P protein is required for catalysis in vivo and interacts with the 5’ leader sequence. A single P RNA and a P protein form the functional RNase P holoenzyme yet dimeric forms of bacterial RNase P can interact with non-tRNA substrates and influence bacterial cell growth. Oligomeric forms of the P protein can also occur in vitro and occlude the 5’ leader ptRNA binding interface, presenting a challenge in accurately defining the substrate recognition properties. To overcome this, concentration and temperature dependent NMR studies were performed on a thermostable RNase P protein from Thermatoga maritima. NMR relaxation (R1, R2), heteronuclear NOE, and diffusion ordered spectroscopy (DOSY) experiments were analyzed, identifying a monomeric species through the determination of the diffusion coefficients (D) and rotational correlation times (τc). Experimental diffusion coefficients and τc values for the predominant monomer (2.17 ± 0.36 * 10−10 m2/s, τc = 5.3 ns) or dimer (1.87 ± 0.40* 10−10 m2/s, τc = 9.7 ns) protein assemblies at 45°C correlate well with calculated diffusion coefficients derived from the crystallographic P protein structure (PDB 1NZ0). The identification of a monomeric P protein conformer from relaxation data and chemical shift information enabled us to gain novel insight into the structure of the P protein, highlighting a lack of structural convergence of the N-terminus (residues 1–14) in solution. We propose that the N-terminus of the bacterial P protein is partially disordered and adopts a stable conformation in the presence of RNA. In addition, we have determined the location of the 5’ leader RNA in solution and measured the affinity of the 5’ leader RNA–P protein interaction. We show that the monomer P protein interacts with RNA at the 5’ leader binding cleft that was previously identified using X-ray crystallography. Data support a model where N-terminal protein flexibility is stabilized by holoenzyme formation and helps to accommodate the 5’ leader region of ptRNA. Taken together, local structural changes of the P protein and the 5’ leader RNA provide a means to obtain optimal substrate alignment and activation of the RNase P holoenzyme.

Crystallin aggregation is characterized by light scattering of large molecular aggregates due to their phase separation in the lens. Low‐resolution biophysical studies using multiple techniques have characterized the folding, stability, binding, and aggregation of crystallins in the past but with limited access to their structure, dynamics, and interactions. In this Viewpoint, three schools of experimental structural biology, that is, X‐ray crystallography, solution and solid‐state NMR spectroscopy, and cryo‐electron microscopy, combine to provide atomic resolution details of native crystallins, soluble oligomers, and insoluble amyloid fibrils and amorphous aggregates. Computational structural biology provides additional details on crystallin dynamics and the crucial intercrystallin interactions in these events. Our current understanding of the diverse structural biology of crystallins is consistent with multiple pathways of protein aggregation for different structural intermediates. This Viewpoint combines our efforts with those of others to elucidate the recent progress in these high‐resolution studies and proposes an integrated structural biology approach to resolve the complex lens interactome. Overall, I discuss the outstanding questions and evaluate the experimental and theoretical caveats in the field. [ABSTRACT FROM AUTHOR]

As part of an International consortium aiming at the characterization by NMR of the proteins of the SARS-CoV-2 virus, we have obtained the virtually complete assignment of the backbone atoms of the non-structural protein nsp9. This small (12 kDa) protein is encoded by ORF1a, binds to RNA and seems to be essential for viral RNA synthesis. The crystal structures of the SARS-CoV-2 protein and other homologues suggest that the protein is dimeric as also confirmed by analytical ultracentrifugation and dynamic light scattering. Our data constitute the prerequisite for further NMR-based characterization, and provide the starting point for the identification of small molecule lead compounds that could interfere with RNA binding and prevent viral replication. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum transmembrane protein vesicle‐associated membrane protein‐associated protein (VAP) plays a central role in the formation and function of membrane contact sites (MCS) through its interactions with proteins. The major sperm protein (MSP) domain of VAP binds to a variety of sequences which are referred to as FFAT‐like motifs. In this study, we investigated the interactions of eight peptides containing FFAT‐like motifs with the VAP‐A MSP domain (VAP‐AMSP) by solution NMR. Six of eight peptides are specifically bound to VAP‐A. Furthermore, we found that the RNA‐dependent RNA polymerase of severe acute respiratory syndrome coronavirus 2 has an FFAT‐like motif which specifically binds to VAP‐AMSP as well as other FFAT‐like motifs. Our results will contribute to the discovery of new VAP interactors. [ABSTRACT FROM AUTHOR]

It is well known that poly(2-methoxyethyl acrylate) (PMEA) has good blood compatibility and its performance is attributed to its water structure. Recently, we applied solution nuclear magnetic resonance spectroscopy (solution-NMR) for analyzing the water structure in PMEA at ambient temperature and concluded that this method is useful because of the clear observation of the resonance peaks at low and high magnetic field (downfield and upfield, respectively) areas indicating the existence of more than two types of water. The present study was performed to compare the water structure of poly(tetrahydrofurfuryl acrylate) (PTHFA) and poly(2-hydroxyethyl methacrylate) (PHEMA) using solution 2H-NMR and deuterium oxide as water at the temperature range 15–45 °C. It was found that PTHFA has a different water structure from that of PHEMA. Water in PTHFA clearly showed two resonance peaks at downfield and upfield areas, with different spin-lattice relaxation times, T12H (high and low values, respectively). These observations are similar to those of PMEA. In contrast, PHEMA showed only one broad resonance peak (at downfield) with a low T12H value. Based on these observations, this study discusses the effect of water structures on the blood compatibility of these polymers. [ABSTRACT FROM AUTHOR]

In solution NMR, chemical shift perturbation (CSP) experiments are widely employed to study intermolecular interactions. However, excluding the nonsignificant peak shift is difficult because little is known about errors in CSP. Here, to address this issue, we introduce a method for estimating errors in CSP based on the noise level. First, we developed a technique that involves line shape fitting to estimate errors in peak position via Monte Carlo simulations. Second, this technique was applied to estimate errors in CSP. In intermolecular interaction analysis of VAP-A with SNX2, error estimation of CSP enabled the evaluation of small but significant changes in peak position and yielded detailed insights that are unattainable with conventional CSP analysis. Third, this technique was successfully applied to estimate errors in residual dipolar couplings. In conclusion, our error estimation method improves CSP analysis by excluding the nonsignificant peak shift. [Display omitted] • A method to estimate errors in NMR peak positions was developed. • A method to estimate errors in chemical shift perturbations was developed. • Small changes in peak position were evaluated through estimating the error of CSP. [ABSTRACT FROM AUTHOR]

[Display omitted] • Milli-to-micro-second conformational exchange data contained in 1HN NMR R 2 protein relaxation data is reinvestigated. • The 1HN relaxation terms for a fully proteated protein are theoretically analyzed. • Multi-spin cross correlations are quantitated and exploited. • TOCSY coherence transfer is quantified and eliminated. • ROESY cross peaks are eliminated. It is advantageous to investigate milli-to-micro-second conformational exchange data contained in the solution NMR protein relaxation data other than 15N nuclei. Not only does one search under another lamp post, one also looks at dynamics at other time scales. The HSQC-ROESY 1HN relaxation dispersion experiment for amide protons as introduced by Ishima, et al (1998). J. Am. Soc. 120, 10534 – 10542, is such an experiment, but has by the authors been advised to only be used for perdeuterated proteins to avoid complication with the 1H–1H multiple-spin effects. This is regretful, since not all proteins can be perdeuterated. Here we analyze in detail the 1HN relaxation terms for this experiment for a fully proteated protein. Indeed, the 1HN relaxation theory is in this case complex and includes dipolar-dipolar relaxation interference and TOCSY transfers. With simulate both of these effects and show that the interference can be exploited for detecting exchange broadening. The TOCSY effect is shown to minor, and when it is not, a solution is provided. We apply the HSQC-ROESY experiment, with a small modification to suppress ROESY crosspeaks, to a 7 kDa GB1 protein that is just 15N and 13C labeled. At 10 °C we cannot detect any conformational exchange broadening: the 1HN R 2 relaxation rates with 1.357 kHz spinlock field not larger than those recorded with a 12.136 kHz spinlock field. This means that there is no exchange broadening that can be differentially suppressed with the applied fields. Either there is no broadening, or the broadening is effectively suppressed by all fields, or the broadening cannot be suppressed by either of the fields. While initially this seems to be a disappointing result, we feel that this work establishes that the HSQC-ROESY experiment is very robust. It can indeed be utilized for proteated proteins upto about 30 kDa. This could be opening the study the milli-microsecond conformational dynamics as reported by 1HN exchange broadening for many more proteins. [ABSTRACT FROM AUTHOR]

Understanding the effects of a fertilization regime on the long-term accumulation and transformation of soil phosphorus (P) is essential for promoting the development of sustainable management of soil P. Based on a 29-year field experiment in Mollisol, the compositions and changes of P forms using a modified Hedley sequential extraction method, solution 31P-NMR and P K-edge XANES and soil properties were investigated under continuous mono maize with and without manure (NPKM and NPK). Results showed a stronger positive related coefficient between soil total P and labile P, and mid-labile P fraction was found in NPKM than in NPK treatment. It indicated NPKM could improve the availability of soil accumulated P and reduce its transformation to stable P. Accumulated inorganic P (Pi) was dominated by aluminum phosphate (Al-P) and monobasic calcium phosphate monohydrate (MCP) for NPK treatment, Al-P, MCP, and tricalcium phosphate for NPKM treatment with XANES analysis, which contributed to the P availability in Mollisol. Moreover, the proportion of IHP with XANES and ratio of orthophosphate diesters to monoesters in NPK compared to NPKM indicated the higher Po lability with NPK treatment. Pi, especially NaHCO3-Pi and NaOH-Pi, were the potential sources of resin-Pi. Soil organic matter (SOM), organic-bound iron, and alumina oxide (Fep + Alp) showed significant influence on the transformation of P forms. Our research suggested that due to the rise in SOM and Fep + Alp, the fertilization regime significantly increased most highly active soil P fractions, especially in NPKM treatment. This work gives new insight into sustainable P management, which benefits the reduction in soil P accumulation.

The calmodulin (CaM) activated α‐kinase, eukaryotic elongation factor 2 kinase (eEF‐2K), plays a central role in regulating translational elongation by phosphorylating eukaryotic elongation factor 2 (eEF‐2), thereby reducing its ability to associate with the ribosome and suppressing global protein synthesis. Using TR (for truncated), a minimal functional construct of eEF‐2K, and utilizing hydrogen/deuterium exchange mass spectrometry (HXMS), solution‐state nuclear magnetic resonance (NMR) and biochemical approaches, we investigate the conformational changes accompanying complex formation between Ca2+‐CaM and TR and the effects of autophosphorylation of the latter at Thr348, its primary regulatory site. Our results suggest that a CaM C‐lobe surface, complementary to the one involved in recognizing the calmodulin‐binding domain (CBD) of TR, provides a secondary TR‐interaction platform. CaM helix F, which is part of this secondary surface, responds to both Thr348 phosphorylation and pH changes, indicating its integration into an allosteric network that encompasses both components of the Ca2+‐CaM•TR complex. Solution NMR data suggest that CaMH107K, which carries a helix F mutation, is compromised in its ability to drive the conformational changes in TR necessary to enable efficient Thr348 phosphorylation. Biochemical studies confirm the diminished capacity of CaMH107K to induce TR autophosphorylation compared to wild‐type CaM. [ABSTRACT FROM AUTHOR]

Enzyme I (EI) is a phosphotransferase enzyme responsible for converting phosphoenolpyruvate (PEP) into pyruvate. This reaction initiates a five-step phosphorylation cascade in the bacterial phosphotransferase (PTS) transduction pathway. Under physiological conditions, EI exists in an equilibrium between a functional dimer and an inactive monomer. The monomer-dimer equilibrium is a crucial factor regulating EI activity and the phosphorylation state of the overall PTS. Experimental studies of EI's monomeric state have yet been hampered by the dimer's high thermodynamic stability, which prevents its characterization by standard structural techniques. In this study, we modified the dimerization domain of EI (EIC) by mutating three amino acids involved in the formation of intersubunit salt bridges. The engineered variant forms an active dimer in solution that can bind and hydrolyze PEP. Using hydrostatic pressure as an additional perturbation, we were then able to study the complete dissociation of the variant from 1 bar to 2.5 kbar in the absence and the presence of EI natural ligands. Backbone residual dipolar couplings collected under high-pressure conditions allowed us to determine the conformational ensemble of the isolated EIC monomeric state in solution. Our calculations reveal that three catalytic loops near the dimerization interface become unstructured upon monomerization, preventing the monomeric enzyme from binding its natural substrate. This study provides an atomic-level characterization of EI's monomeric state and highlights the role of the catalytic loops as allosteric connectors controlling both the activity and oligomerization of the enzyme. [ABSTRACT FROM AUTHOR]

LEE-encoded effector EspF (EspF) is an effector protein part of enteropathogenic Escherichia coli's (EPEC's) arsenal for intestinal infection. This intrinsically disordered protein contains three highly conserved repeats which together compose over half of the protein's complete amino acid sequence. EPEC uses EspF to hijack host proteins in order to promote infection. In the attack EspF is translocated, together with other effector proteins, to host cell via type III secretion system. Inside host EspF stimulates actin polymerization by interacting with Neural Wiskott-Aldrich syndrome protein (N-WASP), a regulator in actin polymerization machinery. It is presumed that EspF acts by disrupting the autoinhibitory state of N-WASP GTPase binding domain. In this NMR spectroscopy study, we report the 1H, 13C, and 15N resonance assignments for the complex formed by the first 47-residue repeat of EspF and N-WASP GTPase binding domain. These near-complete resonance assignments provide the basis for further studies which aim to characterize structure, interactions, and dynamics between these two proteins in solution. [ABSTRACT FROM AUTHOR]

The Betacoronavirus SARS-CoV-2 non-structural protein Nsp9 is a 113-residue protein that is essential for viral replication, and consequently, a potential target for the development of therapeutics against COVID19 infections. To capture insights into the dynamics of the protein's backbone in solution and accelerate the identification and mapping of ligand-binding surfaces through chemical shift perturbation studies, the backbone 1H, 13C, and 15N NMR chemical shifts for Nsp9 have been extensively assigned. These assignments were assisted by the preparation of an ~ 70% deuterated sample and residue-specific, 15N-labelled samples (V, L, M, F, and K). A major feature of the assignments was the "missing" amide resonances for N96-L106 in the 1H-15N HSQC spectrum, a region that comprises almost the complete C-terminal α-helix that forms a major part of the homodimer interface in the crystal structure of SARS-CoV-2 Nsp9, suggesting this region either undergoes intermediate motion in the ms to μs timescale and/or is heterogenous. These "missing" amide resonances do not unambiguously appear in the 1H-15N HSQC spectrum of SARS-CoV-2 Nsp9 collected at a concentration of 0.0007 mM. At this concentration, at the detection limit, native mass spectrometry indicates the protein is exclusively in the monomeric state, suggesting the intermediate motion in the C-terminal of Nsp9 may be due to intramolecular dynamics. Perhaps this intermediate ms to μs timescale dynamics is the physical basis for a previously suggested "fluidity" of the C-terminal helix that may be responsible for homophilic (Nsp9-Nsp9) and postulated heterophilic (Nsp9-Unknown) protein-protein interactions. [ABSTRACT FROM AUTHOR]