RESUMEN
Considerable debate has focused on whether sampling of molecular dynamics trajectories restrained by crystallographic data can be used to develop realistic ensemble models for proteins in their natural, solution state. For the SARS-CoV-2 main protease, Mpro, we evaluated agreement between solution residual dipolar couplings (RDCs) and various recently reported multi-conformer and dynamic-ensemble crystallographic models. Although Phenix-derived ensemble models showed only small improvements in crystallographic Rfree, substantially improved RDC agreement over fits to a conventionally refined 1.2-Å X-ray structure was observed, in particular for residues with above average disorder in the ensemble. For a set of six lower resolution (1.55-2.19 Å) Mpro X-ray ensembles, obtained at temperatures ranging from 100 to 310 K, no significant improvement over conventional two-conformer representations was found. At the residue level, large differences in motions were observed among these ensembles, suggesting high uncertainties in the X-ray derived dynamics. Indeed, combining the six ensembles from the temperature series with the two 1.2-Å X-ray ensembles into a single 381-member "super ensemble" averaged these uncertainties and substantially improved agreement with RDCs. However, all ensembles showed excursions that were too large for the most dynamic fraction of residues. Our results suggest that further improvements to X-ray ensemble refinement are feasible, and that RDCs provide a sensitive benchmark in such endeavors. Remarkably, a weighted ensemble of 350 PDB Mpro X-ray structures provided slightly better cross-validated agreement with RDCs than any individual ensemble refinement, implying that differences in lattice confinement also limit the fit of RDCs to X-ray coordinates.
Asunto(s)
Proteasas 3C de Coronavirus , SARS-CoV-2 , Humanos , Resonancia Magnética Nuclear Biomolecular/métodos , Conformación Proteica , SARS-CoV-2/enzimología , Proteasas 3C de Coronavirus/química , Cristalografía por Rayos XRESUMEN
The 68-kDa homodimeric 3C-like protease of SARS-CoV-2, Mpro (3CLpro /Nsp5), is a key antiviral drug target. NMR spectroscopy of this large system proved challenging and resonance assignments have remained incomplete. Here we present the near-complete (>97 %) backbone assignments of a C145A variant of Mpro (Mpro C145A ) both with, and without, the N-terminal auto-cleavage substrate sequence, in its native homodimeric state. We also present SILLY (Selective Inversion of thioL and Ligand for NOESY), a simple yet effective pseudo-3D NMR experiment that utilizes NOEs to identify interactions between Cys-thiol or aliphatic protons, and their spatially proximate backbone amides in a perdeuterated protein background. High protection against hydrogen exchange is observed for 10 of the 11 thiol groups in Mpro C145A , even those that are partially accessible to solvent. A combination of SILLY methods and high-resolution triple-resonance NMR experiments reveals site-specific interactions between Mpro , its substrate peptides, and other ligands, which present opportunities for competitive binding studies in future drug design efforts.
Asunto(s)
COVID-19 , Protones , Amidas , Antivirales/química , Proteasas 3C de Coronavirus , Cisteína Endopeptidasas/metabolismo , Humanos , Ligandos , Espectroscopía de Resonancia Magnética , Péptidos/metabolismo , Inhibidores de Proteasas , SARS-CoV-2 , Solventes , Compuestos de SulfhidriloRESUMEN
Understanding the factors that underpin the enormous catalytic proficiencies of enzymes is fundamental to catalysis and enzyme design. Enzymes are, in part, able to achieve high catalytic proficiencies by utilizing the binding energy derived from nonreacting portions of the substrate. In particular, enzymes with substrates containing a nonreacting phosphodianion group coordinated in a distal site have been suggested to exploit this binding energy primarily to facilitate a conformational change from an open inactive form to a closed active form, rather than to either induce ground state destabilization or stabilize the transition state. However, detailed structural evidence for the model is limited. Here, we use ß-phosphoglucomutase (ßPGM) to investigate the relationship between binding a phosphodianion group in a distal site, the adoption of a closed enzyme form, and catalytic proficiency. ßPGM catalyzes the isomerization of ß-glucose 1-phosphate to glucose 6-phosphate via phosphoryl transfer reactions in the proximal site, while coordinating a phosphodianion group of the substrate(s) in a distal site. ßPGM has one of the largest catalytic proficiencies measured and undergoes significant domain closure during its catalytic cycle. We find that side chain substitution at the distal site results in decreased substrate binding that destabilizes the closed active form but is not sufficient to preclude the adoption of a fully closed, near-transition state conformation. Furthermore, we reveal that binding of a phosphodianion group in the distal site stimulates domain closure even in the absence of a transferring phosphoryl group in the proximal site, explaining the previously reported ß-glucose 1-phosphate inhibition. Finally, our results support a trend whereby enzymes with high catalytic proficiencies involving phosphorylated substrates exhibit a greater requirement to stabilize the closed active form.
RESUMEN
The measurement and application of residual dipolar couplings (RDCs) in solution NMR studies of biological macromolecules has become well established over the past quarter of a century. Numerous methods for generating the requisite anisotropic orientational molecular distribution have been demonstrated, each with its specific strengths and weaknesses. In parallel, an enormous number of pulse schemes have been introduced to measure the many different types of RDCs, ranging from the most widely measured backbone amide 15N-1H RDCs, to 1H-1H RDCs and couplings between low-γ nuclei. Applications of RDCs range from structure validation and refinement to the determination of relative domain orientations, the measurement of backbone and domain motions, and de novo structure determination. Nevertheless, it appears that the power of the RDC methodology remains underutilized. This review aims to highlight the practical aspects of sample preparation and RDC measurement while describing some of the most straightforward applications that take advantage of the exceptionally precise information contained in such data. Some emphasis will be placed on more recent developments that enable the accurate measurement of RDCs in larger systems, which is key to the ongoing shift in focus of biological NMR spectroscopy from structure determination toward gaining improved understanding of how molecular flexibility drives protein function.
Asunto(s)
Proteínas , Espectroscopía de Resonancia Magnética/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/químicaRESUMEN
The 68-kDa homodimeric 3C-like protease of SARS-CoV-2, Mpro (3CLpro/Nsp5), is a promising antiviral drug target. We evaluate the concordance of models generated by the newly introduced AlphaFold2 structure prediction program with residual dipolar couplings (RDCs) measured in solution for 15N-1HN and 13C'-1HN atom pairs. The latter were measured using a new, highly precise TROSY-AntiTROSY Encoded RDC (TATER) experiment. Three sets of AlphaFold2 models were evaluated: (1) MproAF, generated using the standard AlphaFold2 input structural database; (2) MproAFD, where the AlphaFold2 implementation was modified to exclude all candidate template X-ray structures deposited after Jan 1, 2020; and (3) MproAFS, which excluded all structures homologous to coronaviral Mpro. Close agreement between all three sets of AlphaFold models and experimental RDC data is found for most of the protein. For residues in well-defined secondary structure, the agreement decreases somewhat upon Amber relaxation. For these regions, MproAF agreement exceeds that of most high-resolution X-ray structures. Residues from domain 2 that comprise elements of both the active site and the homo-dimerization interface fit less well across all structures. These results indicate novel opportunities for combining experimentation with molecular dynamics simulations, where solution RDCs provide highly precise input for QM/MM simulations of substrate binding/reaction trajectories.
Asunto(s)
Proteasas 3C de Coronavirus/química , Cristalografía por Rayos X/métodos , SARS-CoV-2 , COVID-19 , Dominio Catalítico , Espectroscopía de Resonancia Magnética , Conformación Molecular , Simulación de Dinámica Molecular , Conformación Proteica , Pliegue de Proteína , Programas Informáticos , Rayos XRESUMEN
Resonance assignment and structural studies of larger proteins by nuclear magnetic resonance (NMR) can be challenging when exchange broadening, multiple stable conformations, and 1H back-exchange of the fully deuterated chain pose problems. These difficulties arise for the SARS-CoV-2 Main Protease, a homodimer of 2â¯×â¯306 residues. We demonstrate that the combination of four-dimensional (4D) TROSY-NOESY-TROSY spectroscopy and 4D NOESY-NOESY-TROSY spectroscopy provides an effective tool for delineating the 1H-1H dipolar relaxation network. In combination with detailed structural information obtained from prior X-ray crystallography work, such data are particularly useful for extending and validating resonance assignments as well as for probing structural features.
RESUMEN
Enzyme regulation is vital for metabolic adaptability in living systems. Fine control of enzyme activity is often delivered through post-translational mechanisms, such as allostery or allokairy. ß-phosphoglucomutase (ßPGM) from Lactococcus lactis is a phosphoryl transfer enzyme required for complete catabolism of trehalose and maltose, through the isomerisation of ß-glucose 1-phosphate to glucose 6-phosphate via ß-glucose 1,6-bisphosphate. Surprisingly for a gatekeeper of glycolysis, no fine control mechanism of ßPGM has yet been reported. Herein, we describe allomorphy, a post-translational control mechanism of enzyme activity. In ßPGM, isomerisation of the K145-P146 peptide bond results in the population of two conformers that have different activities owing to repositioning of the K145 sidechain. In vivo phosphorylating agents, such as fructose 1,6-bisphosphate, generate phosphorylated forms of both conformers, leading to a lag phase in activity until the more active phosphorylated conformer dominates. In contrast, the reaction intermediate ß-glucose 1,6-bisphosphate, whose concentration depends on the ß-glucose 1-phosphate concentration, couples the conformational switch and the phosphorylation step, resulting in the rapid generation of the more active phosphorylated conformer. In enabling different behaviours for different allomorphic activators, allomorphy allows an organism to maximise its responsiveness to environmental changes while minimising the diversion of valuable metabolites.
Asunto(s)
Fosfotransferasas (Fosfomutasas)/metabolismo , Procesamiento Proteico-Postraduccional , Regulación Alostérica , Sitio Alostérico , Cristalografía por Rayos X , Pruebas de Enzimas , Glucosa-6-Fosfato/análogos & derivados , Glucosa-6-Fosfato/metabolismo , Glucofosfatos/metabolismo , Glucólisis , Isomerismo , Cinética , Conformación Molecular , Fosforilación , Fosfotransferasas (Fosfomutasas)/genética , Fosfotransferasas (Fosfomutasas)/aislamiento & purificación , Fosfotransferasas (Fosfomutasas)/ultraestructura , Prolina/química , Dominios Proteicos , Espectroscopía de Protones por Resonancia Magnética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructuraRESUMEN
Lysostaphin is a bacteriolytic enzyme targeting peptidoglycan, the essential component of the bacterial cell envelope. It displays a very potent and specific activity toward staphylococci, including methicillin-resistant Staphylococcus aureus. Lysostaphin causes rapid cell lysis and disrupts biofilms, and is therefore a therapeutic agent of choice to eradicate staphylococcal infections. The C-terminal SH3b domain of lysostaphin recognizes peptidoglycans containing a pentaglycine crossbridge and has been proposed to drive the preferential digestion of staphylococcal cell walls. Here we elucidate the molecular mechanism underpinning recognition of staphylococcal peptidoglycan by the lysostaphin SH3b domain. We show that the pentaglycine crossbridge and the peptide stem are recognized by two independent binding sites located on opposite sides of the SH3b domain, thereby inducing a clustering of SH3b domains. We propose that this unusual binding mechanism allows synergistic and structurally dynamic recognition of S. aureus peptidoglycan and underpins the potent bacteriolytic activity of this enzyme.
Asunto(s)
Lisostafina/química , Peptidoglicano/química , Staphylococcus aureus/química , Bacteriólisis/efectos de los fármacos , Biopelículas , Pared Celular/química , Cromatografía Líquida de Alta Presión , Análisis Mutacional de ADN , Glicina/química , Ligandos , Espectroscopía de Resonancia Magnética , Mutagénesis Sitio-Dirigida , Péptidos/química , Unión Proteica , Dominios Proteicos , Proteínas Recombinantes/química , Dominios Homologos srcRESUMEN
Brain tissue of Alzheimer's disease patients invariably contains deposits of insoluble, fibrillar aggregates of peptide fragments of the amyloid precursor protein (APP), typically 40 or 42 residues in length and referred to as Aß40 and Aß42. However, it remains unclear whether these fibrils or oligomers constitute the toxic species. Depending on sample conditions, oligomers can form in a few seconds or less. These oligomers are invisible to solution NMR spectroscopy, but they can be rapidly (<1 s) resolubilized and converted to their NMR-visible monomeric constituents by raising the hydrostatic pressure to a few kbar. Hence, utilizing pressure-jump NMR, the oligomeric state can be studied at residue-specific resolution by monitoring its signals in the monomeric state. Oligomeric states of Aß40 exhibit a high degree of order, reflected by slow longitudinal 15N relaxation (T1 > 5 s) for residues 18-21 and 31-34, whereas the N-terminal 10 residues relax much faster (T1 ≤ 1.5 s), indicative of extensive internal motions. Transverse relaxation rates rapidly increase to ca. 1000 s-1 after the oligomerization is initiated.
Asunto(s)
Péptidos beta-Amiloides/síntesis química , Resonancia Magnética Nuclear Biomolecular , Péptidos beta-Amiloides/química , Humanos , Tamaño de la Partícula , Presión , Propiedades de SuperficieRESUMEN
Phosphorus acquisition is critical for life. In low phosphate conditions, some species of bacteria have evolved mechanisms to import reduced phosphorus compounds, such as phosphite and hypophosphite, as alternative phosphorus sources. Uptake is facilitated by high-affinity periplasmic binding proteins (PBPs) that bind cargo in the periplasm and shuttle it to an ATP-binding cassette (ABC)-transporter in the bacterial inner membrane. PtxB and HtxB are the PBPs responsible for binding phosphite and hypophosphite, respectively. They recognize the P-H bond of phosphite/hypophosphite via a conserved P-H...π interaction, which confers nanomolar dissociation constants for their respective ligands. PtxB also has a low-level binding affinity for phosphate and hypophosphite, whilst HtxB can facilitate phosphite uptake in vivo. However, HtxB does not bind phosphate, thus the HtxBCDE transporter has recently been successfully exploited for biocontainment of genetically modified organisms by phosphite-dependent growth. Here we use a combination of X-ray crystallography, NMR and Microscale Thermophoresis to show that phosphite binding to HtxB depends on the protonation state of the ligand, suggesting that pH may effect the efficiency of phosphite uptake by HtxB in biotechnology applications.
Asunto(s)
Proteínas de Unión Periplasmáticas/metabolismo , Fosfitos/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/fisiología , Cristalografía por Rayos X/métodos , Ligandos , Periplasma/metabolismo , Proteínas de Unión Periplasmáticas/fisiología , Fosfatos/química , Fosfatos/metabolismo , Fosfitos/química , Fósforo/química , Fósforo/metabolismo , Unión ProteicaRESUMEN
Protein transport across the cytoplasmic membrane of bacterial cells is mediated by either the general secretion (Sec) system or the twin-arginine translocase (Tat). The Tat machinery exports folded and cofactor-containing proteins from the cytoplasm to the periplasm by using the transmembrane proton motive force as a source of energy. The Tat apparatus apparently senses the folded state of its protein substrates, a quality-control mechanism that prevents premature export of nascent unfolded or misfolded polypeptides, but its mechanistic basis has not yet been determined. Here, we investigated the innate ability of the model Escherichia coli Tat system to recognize and translocate de novo-designed protein substrates with experimentally determined differences in the extent of folding. Water-soluble, four-helix bundle maquette proteins were engineered to bind two, one, or no heme b cofactors, resulting in a concomitant reduction in the extent of their folding, assessed with temperature-dependent CD spectroscopy and one-dimensional 1H NMR spectroscopy. Fusion of the archetypal N-terminal Tat signal peptide of the E. coli trimethylamine-N-oxide (TMAO) reductase (TorA) to the N terminus of the protein maquettes was sufficient for the Tat system to recognize them as substrates. The clear correlation between the level of Tat-dependent export and the degree of heme b-induced folding of the maquette protein suggested that the membrane-bound Tat machinery can sense the extent of folding and conformational flexibility of its substrates. We propose that these artificial proteins are ideal substrates for future investigations of the Tat system's quality-control mechanism.