RESUMO
Peptide engineering efforts have delivered drugs for diverse human diseases. Side chain alteration is among the most common approaches to designing new peptides for specific applications. The peptide backbone can be modified as well, but this strategy has received relatively little attention. Here we show that new and favorable contacts between a His side chain on a target protein and an aromatic side chain on a synthetic peptide ligand can be engineered by rational and coordinated side chain modification and backbone extension. Side chain modification alone was unsuccessful. Binding measurements, high-resolution structural studies and pharmacological outcomes all support the synergy between backbone and side chain modification in engineered ligands of the parathyroid hormone receptor-1, which is targeted by osteoporosis drugs. These results should motivate other structure-based designs featuring coordinated side chain modification and backbone extension to enhance the engagement of peptide ligands with target proteins.
Assuntos
Histidina , Peptídeos , Humanos , Histidina/química , Sequência de Aminoácidos , Ligantes , Peptídeos/química , ProteínasRESUMO
The highly automated macromolecular crystallography beamline AMX/17-ID-1 is an undulator-based high-intensity (>5 × 1012â photonsâ s-1), micro-focus (7â µm × 5â µm), low-divergence (1â mrad × 0.35â mrad) energy-tunable (5-18â keV) beamline at the NSLS-II, Brookhaven National Laboratory, Upton, NY, USA. It is one of the three life science beamlines constructed by the NIH under the ABBIX project and it shares sector 17-ID with the FMX beamline, the frontier micro-focus macromolecular crystallography beamline. AMX saw first light in March 2016 and started general user operation in February 2017. At AMX, emphasis has been placed on high throughput, high capacity, and automation to enable data collection from the most challenging projects using an intense micro-focus beam. Here, the current state and capabilities of the beamline are reported, and the different macromolecular crystallography experiments that are routinely performed at AMX/17-ID-1 as well as some plans for the near future are presented.
Assuntos
Síncrotrons , Cristalografia por Raios X , Substâncias Macromoleculares/químicaRESUMO
The lower respiratory tract infections affecting children worldwide are in large part caused by the parainfluenza viruses (HPIVs), particularly HPIV3, along with human metapneumovirus and respiratory syncytial virus, enveloped negative-strand RNA viruses. There are no vaccines for these important human pathogens, and existing treatments have limited or no efficacy. Infection by HPIV is initiated by viral glycoprotein-mediated fusion between viral and host cell membranes. A viral fusion protein (F), once activated in proximity to a target cell, undergoes a series of conformational changes that first extend the trimer subunits to allow insertion of the hydrophobic domains into the target cell membrane and then refold the trimer into a stable postfusion state, driving the merger of the viral and host cell membranes. Lipopeptides derived from the C-terminal heptad repeat (HRC) domain of HPIV3 F inhibit infection by interfering with the structural transitions of the trimeric F assembly. Clinical application of this strategy, however, requires improving the in vivo stability of antiviral peptides. We show that the HRC peptide backbone can be modified via partial replacement of α-amino acid residues with ß-amino acid residues to generate α/ß-peptides that retain antiviral activity but are poor protease substrates. Relative to a conventional α-lipopeptide, our best α/ß-lipopeptide exhibits improved persistence in vivo and improved anti-HPIV3 antiviral activity in animals.
Assuntos
Lipopeptídeos/farmacologia , Vírus da Parainfluenza 3 Humana/efeitos dos fármacos , Infecções Respiratórias/patologia , Sequência de Aminoácidos , Aminoácidos/química , Aminoácidos/metabolismo , Animais , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Linhagem Celular , Colesterol/química , Desenho de Fármacos , Humanos , Lipopeptídeos/química , Lipopeptídeos/metabolismo , Vírus da Parainfluenza 3 Humana/isolamento & purificação , Multimerização Proteica , Ratos , Infecções Respiratórias/virologia , Distribuição Tecidual , Temperatura de Transição , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus/efeitos dos fármacosRESUMO
Human parainfluenza virus 3 (HPIV3) and respiratory syncytial virus (RSV) cause lower respiratory infection in infants and young children. There are no vaccines for these pathogens, and existing treatments have limited or questionable efficacy. Infection by HPIV3 or RSV requires fusion of the viral and cell membranes, a process mediated by a trimeric fusion glycoprotein (F) displayed on the viral envelope. Once triggered, the pre-fusion form of F undergoes a series of conformational changes that first extend the molecule to allow for insertion of the hydrophobic fusion peptide into the target cell membrane and then refold the trimeric assembly into an energetically stable post-fusion state, a process that drives the merger of the viral and host cell membranes. Peptides derived from defined regions of HPIV3 F inhibit infection by HPIV3 by interfering with the structural transitions of the trimeric F assembly. Here we describe lipopeptides derived from the C-terminal heptad repeat (HRC) domain of HPIV3 F that potently inhibit infection by both HPIV3 and RSV. The lead peptide inhibits RSV infection as effectively as does a peptide corresponding to the RSV HRC domain itself. We show that the inhibitors bind to the N-terminal heptad repeat (HRN) domains of both HPIV3 and RSV F with high affinity. Co-crystal structures of inhibitors bound to the HRN domains of HPIV3 or RSV F reveal remarkably different modes of binding in the N-terminal segment of the inhibitor.
Assuntos
Lipopeptídeos/farmacologia , Vírus da Parainfluenza 3 Humana/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Inibidores de Proteínas Virais de Fusão/farmacologia , Proteínas Virais de Fusão/farmacologia , Sequência de Aminoácidos , Cristalografia por Raios X , Humanos , Lipopeptídeos/metabolismo , Testes de Sensibilidade Microbiana , Vírus da Parainfluenza 3 Humana/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Mucosa Respiratória/virologia , Vírus Sinciciais Respiratórios/química , Inibidores de Proteínas Virais de Fusão/metabolismo , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus/efeitos dos fármacosRESUMO
Racemic crystallography has been used to elucidate the secondary and tertiary structures of peptides and small proteins that are recalcitrant to conventional crystallization. It is unclear, however, whether racemic crystallography can capture native quaternary structure, which could be disrupted by heterochiral associations. We are exploring the use of racemic crystallography to characterize the self-assembly behavior of membrane-associated peptides, very few of which have been crystallized. We report a racemic crystal structure of the membrane-active peptide melittin; the new structure allows comparison with a previously reported crystal structure of L-melittin. The tetrameric assembly observed in crystalline L-melittin has been proposed to represent the tetrameric state detected in solution for this peptide. This tetrameric assembly is precisely reproduced in the racemic crystal, which strengthens the conclusion that the tetramer is biologically relevant. More broadly, these findings suggest that racemic crystallography can provide insight on native quaternary structure.
Assuntos
Meliteno/química , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Estrutura Quaternária de Proteína , EstereoisomerismoRESUMO
The structural principles that govern interactions between l- and d-peptides are not well understood. Among natural proteins, coiled-coil assemblies formed between or among α-helices are the most regular feature of tertiary and quaternary structures. We recently reported the first high-resolution structures for heterochiral coiled-coil dimers, which represent a starting point for understanding associations of l- and d-polypeptides. These structures were an unexpected outcome from crystallization of a racemic peptide corresponding to the transmembrane domain of the influenza A M2 protein (M2-TM). The reported structures raised the possibility that heterochiral coiled-coil dimers prefer an 11-residue (hendecad) sequence repeat, in contrast to the 7-residue (heptad) sequence repeat that is dominant among natural coiled coils. To gain insight on sequence repeat preferences of heterochiral coiled-coils, we have examined three M2-TM variants containing substitutions intended to minimize steric clashes between side chains at the coiled-coil interface. In each of the three new crystal structures, we observed heterochiral coiled-coil associations that closely match a hendecad sequence motif, which strengthens the conclusion that this motif is intrinsic to the pairing of α-helices with opposite handedness. In each case, the presence of a hendecad motif was established by comparing the observed helical frequency to that of an ideal hendecad. This comparison revealed that decreasing the size of the amino acid side chain at positions that project toward the superhelical axis produces tighter packing, as determined by the size of the coiled-coil radius. These results provide a basis for future design of heterochiral coiled-coil pairings.
Assuntos
Proteínas da Matriz Viral/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , EstereoisomerismoRESUMO
Phenol-soluble modulin α3 (PSMα3) is a cytotoxic peptide secreted by virulent strains of Staphylococcus aureus. We used a stereochemical strategy to examine the mechanism of PSMα3-mediated toxicity. One hypothesis is that PSMα3 toxicity requires fibril formation; an alternative is that toxicity is caused by soluble forms of PSMα3, possibly oligomeric. We find that the unnatural enantiomer (D residues) displays cytotoxicity comparable to that of L-PSMα3. Racemic PSMα3 is similarly toxic to enantiopure PSMα3 (L or D) under some conditions, but the toxicity is lost under conditions that cause racemic PSMα3 to aggregate. A crystal structure of racemic PSMα3-NH2 displays an α-helical secondary structure and a packing pattern that is reminiscent of the cross-α arrangement recently discovered in crystals of L-PSMα3. Our data suggest that the cytotoxicity of PSMα3 does not depend on stereospecific engagement of a target protein or other chiral macromolecule, an observation that supports a mechanism based on membrane disruption. In addition, our data support the hypothesis that toxicity is exerted by a soluble form rather than an insoluble fibrillar form.
Assuntos
Toxinas Bacterianas/química , Toxinas Bacterianas/toxicidade , Células HEK293 , Humanos , Modelos Moleculares , Conformação Proteica , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
ß-Amino acids have a backbone that is expanded by one carbon atom relative to α-amino acids, and ß residues have been investigated as subunits in protein-like molecules that adopt discrete and predictable conformations. Two classes of ß residue have been widely explored in the context of generating α-helix-like conformations: ß3 -amino acids, which are homologous to α-amino acids and bear a side chain on the backbone carbon adjacent to nitrogen, and residues constrained by a five-membered ring, such the one derived from trans-2-aminocyclopentanecarboxylic acid (ACPC). Substitution of α residues with their ß3 â homologues within an α-helix-forming sequence generally causes a decrease in conformational stability. Use of a ring-constrained ß residue, however, can offset the destabilizing effect of αâß substitution. Here we extend the study of αâß substitutions, involving both ß3 and ACPC residues, to short loops within a small tertiary motif. We start from previously reported variants of the Pin1 WW domain that contain a two-, three-, or four-residue ß-hairpin loop, and we evaluate αâß replacements at each loop position for each variant. By referral to the Ï,ψ angles of the native structure, one can choose a stereochemically appropriate ACPC residue. Use of such logically chosen ACPC residues enhances conformational stability in several cases. Crystal structures of three ß-containing Pin1 WW domain variants show that a native-like tertiary structure is maintained in each case.
Assuntos
Aminoácidos/química , Cicloleucina/análogos & derivados , Proteínas/química , Cicloleucina/química , Modelos Moleculares , Estrutura Molecular , Estabilidade Proteica , TemperaturaRESUMO
Peptide-based agents derived from well-defined scaffolds offer an alternative to antibodies for selective and high-affinity recognition of large and topologically complex protein surfaces. Here, we describe a strategy for designing oligomers containing both α- and ß-amino acid residues ("α/ß-peptides") that mimic several peptides derived from the three-helix bundle "Z-domain" scaffold. We show that α/ß-peptides derived from a Z-domain peptide targeting vascular endothelial growth factor (VEGF) can structurally and functionally mimic the binding surface of the parent peptide while exhibiting significantly decreased susceptibility to proteolysis. The tightest VEGF-binding α/ß-peptide inhibits the VEGF165-induced proliferation of human umbilical vein endothelial cells. We demonstrate the versatility of this strategy by showing how principles underlying VEGF signaling inhibitors can be rapidly extended to produce Z-domain-mimetic α/ß-peptides that bind to two other protein partners, IgG and tumor necrosis factor-α. Because well-established selection techniques can identify high-affinity Z-domain derivatives from large DNA-encoded libraries, our findings should enable the design of biostable α/ß-peptides that bind tightly and specifically to diverse targets of biomedical interest. Such reagents would be useful for diagnostic and therapeutic applications.
Assuntos
Peptídeos/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas/química , Sequência de Aminoácidos , Sítios de Ligação/genética , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/metabolismo , Peptídeos/farmacologia , Ligação Proteica , Proteínas/metabolismo , Homologia de Sequência de Aminoácidos , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Interactions between polypeptide chains containing amino acid residues with opposite absolute configurations have long been a source of interest and speculation, but there is very little structural information for such heterochiral associations. The need to address this lacuna has grown in recent years because of increasing interest in the use of peptides generated from d amino acids (d peptides) as specific ligands for natural proteins, e.g., to inhibit deleterious protein-protein interactions. Coiled-coil interactions, between or among α-helices, represent the most common tertiary and quaternary packing motif in proteins. Heterochiral coiled-coil interactions were predicted over 50 years ago by Crick, and limited experimental data obtained in solution suggest that such interactions can indeed occur. To address the dearth of atomic-level structural characterization of heterochiral helix pairings, we report two independent crystal structures that elucidate coiled-coil packing between l- and d-peptide helices. Both structures resulted from racemic crystallization of a peptide corresponding to the transmembrane segment of the influenza M2 protein. Networks of canonical knobs-into-holes side-chain packing interactions are observed at each helical interface. However, the underlying patterns for these heterochiral coiled coils seem to deviate from the heptad sequence repeat that is characteristic of most homochiral analogs, with an apparent preference for a hendecad repeat pattern.
Assuntos
Conformação Proteica , Proteínas/química , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
Synthetic peptides that contain backbone modifications but nevertheless adopt folded structures similar to those of natural polypeptides are of fundamental interest and may provide a basis for biomedical applications. Such molecules can, for example, mimic the ability of natural prototypes to bind to specific target macromolecules but resist degradation by proteases. We have previously shown that oligomers containing mixtures of α- and ß-amino acid residues ("α/ß-peptides") can mimic the α-helix secondary structure, and that properly designed α/ß-peptides can bind to proteins that evolved to bind to α-helical partners. Here we report fundamental studies that support the long-range goal of extending the α/ß approach to tertiary structures. We have evaluated the impact of single α â ß modifications on the structure and stability of the small and well-studied villin headpiece subdomain (VHP). The native state of this 35-residue polypeptide contains several α-helical segments packed around a small hydrophobic core. We examined α â ß substitution at four solvent-exposed positions, Asn19, Trp23, Gln26 and Lys30. In each case, both the ß(3) homologue of the natural α residue and a cyclic ß residue were evaluated. All α â ß(3) substitutions caused significant destabilization of the tertiary structure as measured by variable-temperature circular dichroism, although at some of these positions, replacing the ß(3) residue with a cyclic ß residue led to improved stability. Atomic-resolution structures of four VHP analogues were obtained via quasiracemic crystallization. These findings contribute to a fundamental α/ß-peptide knowledge-base by confirming that ß(3)-amino acid residues can serve as effective structural mimics of homologous α-amino acid residues within a natural tertiary fold, which should support rational design of functional α/ß analogues of natural poly-α-peptides.
Assuntos
Proteínas/química , Dicroísmo Circular , Cristalização , Interações Hidrofóbicas e Hidrofílicas , Estabilidade Proteica , Estrutura Terciária de Proteína , EstereoisomerismoRESUMO
Disulfide bonds provide a convenient method for chemoselective alteration of peptide and protein structure and function. We previously reported that mild oxidation of a p53-derived bisthiol peptide (CTFANLWRLLAQNC) under dilute non-denaturing conditions led to unexpected disulfide-linked dimers as the exclusive product. The dimers were antiparallel, significantly α-helical, resistant to protease degradation, and easily reduced back to the original bisthiol peptide. Here we examine the intrinsic factors influencing peptide dimerization using a combination of amino acid substitution, circular dichroism (CD) spectroscopy, and X-ray crystallography. CD analysis of peptide variants suggests critical roles for Leu6 and Leu10 in the formation of stable disulfide-linked dimers. The 1.0 Å resolution crystal structure of the peptide dimer supports these data, revealing a leucine-rich LxxLL dimer interface with canonical knobs-into-holes packing. Two levels of higher-order oligomerization are also observed in the crystal: an antiparallel "dimer of dimers" mediated by Phe3 and Trp7 residues in the asymmetric unit and a tetramer of dimers mediated by Trp7 and Leu10. In CD spectra of Trp-containing peptide variants, minima at 227 nm provide evidence for the dimer of dimers in dilute aqueous solution. Importantly, and in contrast to the original dimer model, the canonical leucine-rich core and robust dimerization of most peptide variants suggests a tunable molecular architecture to target various proteins and evaluate how folding and oligomerization impact various properties, such as cell permeability.
RESUMO
The bacterial cyclic oligonucleotide-based antiphage signaling system (CBASS) is similar to the cGAS-STING system in humans, containing an enzyme that synthesizes a cyclic nucleotide on viral infection and an effector that senses the second messenger for the antiviral response. Cap5, containing a SAVED domain coupled to an HNH DNA endonuclease domain, is the most abundant CBASS effector, yet the mechanism by which it becomes activated for cell killing remains unknown. We present here high-resolution structures of full-length Cap5 from Pseudomonas syringae (Ps) with second messengers. The key to PsCap5 activation is a dimer-to-tetramer transition, whereby the binding of second messenger to dimer triggers an open-to-closed transformation of the SAVED domains, furnishing a surface for assembly of the tetramer. This movement propagates to the HNH domains, juxtaposing and converting two HNH domains into states for DNA destruction. These results show how Cap5 effects bacterial cell suicide and we provide proof-in-principle data that the CBASS can be extrinsically activated to limit bacterial infections.
Assuntos
Proteínas de Bactérias , Endonucleases , Pseudomonas syringae , Pseudomonas syringae/química , Pseudomonas syringae/enzimologia , Pseudomonas syringae/virologia , Proteínas de Bactérias/química , Endonucleases/química , Ligantes , Modelos Químicos , Ativação Enzimática , DNA/química , DNA/metabolismo , Nucleotídeos Cíclicos/química , Fosfatos de Dinucleosídeos/química , Apoproteínas/química , Bacteriófagos/fisiologiaRESUMO
Heme has a critical role in the chemical framework of the cell as an essential protein cofactor and signaling molecule that controls diverse processes and molecular interactions. Using a phylogenomics-based approach and complementary structural techniques, we identify a family of dimeric hemoproteins comprising a domain of unknown function DUF2470. The heme iron is axially coordinated by two zinc-bound histidine residues, forming a distinct two-fold symmetric zinc-histidine-iron-histidine-zinc site. Together with structure-guided in vitro and in vivo experiments, we further demonstrate the existence of a functional link between heme binding by Dri1 (Domain related to iron 1, formerly ssr1698) and post-translational regulation of succinate dehydrogenase in the cyanobacterium Synechocystis, suggesting an iron-dependent regulatory link between photosynthesis and respiration. Given the ubiquity of proteins containing homologous domains and connections to heme metabolism across eukaryotes and prokaryotes, we propose that DRI (Domain Related to Iron; formerly DUF2470) functions at the molecular level as a heme-dependent regulatory domain.
Assuntos
Hemeproteínas , Synechocystis , Heme , Zinco , Histidina , Hemeproteínas/genética , Synechocystis/genética , Carbono , FerroRESUMO
The human Pin1 WW domain is a small autonomously folding protein that has been useful as a model system for biophysical studies of ß-sheet folding. This domain has resisted previous attempts at crystallization for X-ray diffraction studies, perhaps because of intrinsic conformational flexibility that interferes with the formation of a crystal lattice. Here, the crystal structure of the human Pin1 WW domain has been obtained via racemic crystallization in the presence of small-molecule additives. Both enantiomers of a 36-residue variant of the Pin1 WW domain were synthesized chemically, and the L- and D-polypeptides were combined to afford diffracting crystals. The structural data revealed packing interactions of small carboxylic acids, either achiral citrate or a D,L mixture of malic acid, with a mobile loop region of the WW-domain fold. These interactions with solution additives may explain our success in crystallization of this protein racemate. Molecular-dynamics simulations starting from the structure of the Pin1 WW domain suggest that the crystal structure closely resembles the conformation of this domain in solution. The structural data presented here should provide a basis for further studies of this important model system.
Assuntos
Excipientes/química , Peptidilprolil Isomerase/química , Sequência de Aminoácidos , Cristalização/métodos , Cristalografia por Raios X , Humanos , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Peptidilprolil Isomerase de Interação com NIMA , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
High-resolution structure elucidation has been challenging for the large group of host-defense peptides that form helices on or within membranes but do not manifest a strong folding propensity in aqueous solution. Here we report the crystal structure of an analogue of the widely studied host-defense peptide magainin 2. Magainin 2 (S8A, G13A, G18A) is a designed variant that displays enhanced antibacterial activity relative to the natural peptide. The crystal structure of magainin 2 (S8A, G13A, G18A), obtained for the racemic form, features a dimerization mode that has previously been proposed to play a role in the antibacterial activity of magainin 2 and related peptides.
Assuntos
Antibacterianos/farmacologia , Magaininas/metabolismo , Fenilalanina/química , Proteínas de Xenopus/metabolismo , Antibacterianos/síntese química , Antibacterianos/química , Bacillus subtilis/efeitos dos fármacos , Cristalografia por Raios X , Dimerização , Relação Dose-Resposta a Droga , Enterococcus faecium/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Magaininas/síntese química , Magaininas/química , Testes de Sensibilidade Microbiana , Modelos Moleculares , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade , Proteínas de Xenopus/síntese química , Proteínas de Xenopus/químicaRESUMO
Proper regulation of the bacterial cell envelope is critical for cell survival. Identification and characterization of enzymes that maintain cell envelope homeostasis is crucial, as they can be targets for effective antibiotics. In this study, we have identified a novel enzyme, called EstG, whose activity protects cells from a variety of lethal assaults in the âº-proteobacterium Caulobacter crescentus. Despite homology to transpeptidase family cell wall enzymes and an ability to protect against cell-wall-targeting antibiotics, EstG does not demonstrate biochemical activity toward cell wall substrates. Instead, EstG is genetically connected to the periplasmic enzymes OpgH and BglX, responsible for synthesis and hydrolysis of osmoregulated periplasmic glucans (OPGs), respectively. The crystal structure of EstG revealed similarities to esterases and transesterases, and we demonstrated esterase activity of EstG in vitro. Using biochemical fractionation, we identified a cyclic hexamer of glucose as a likely substrate of EstG. This molecule is the first OPG described in Caulobacter and establishes a novel class of OPGs, the regulation and modification of which are important for stress survival and adaptation to fluctuating environments. Our data indicate that EstG, BglX, and OpgH comprise a previously unknown OPG pathway in Caulobacter. Ultimately, we propose that EstG is a novel enzyme that instead of acting on the cell wall, acts on cyclic OPGs to provide resistance to a variety of cellular stresses.
Assuntos
Caulobacter crescentus , Caulobacter , Caulobacter/metabolismo , Esterases , Membrana Celular/metabolismo , Parede Celular/metabolismo , Caulobacter crescentus/metabolismo , Antibacterianos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
One often observes small but measurable differences in the diffraction data measured from different crystals of a single protein. These differences might reflect structural differences in the protein and may reveal the natural dynamism of the molecule in solution. Partitioning these mixed-state data into single-state clusters is a critical step that could extract information about the dynamic behavior of proteins from hundreds or thousands of single-crystal data sets. Mixed-state data can be obtained deliberately (through intentional perturbation) or inadvertently (while attempting to measure highly redundant single-crystal data). To the extent that different states adopt different molecular structures, one expects to observe differences in the crystals; each of the polystates will create a polymorph of the crystals. After mixed-state diffraction data have been measured, deliberately or inadvertently, the challenge is to sort the data into clusters that may represent relevant biological polystates. Here, this problem is addressed using a simple multi-factor clustering approach that classifies each data set using independent observables, thereby assigning each data set to the correct location in conformational space. This procedure is illustrated using two independent observables, unit-cell parameters and intensities, to cluster mixed-state data from chymotrypsinogen (ChTg) crystals. It is observed that the data populate an arc of the reaction trajectory as ChTg is converted into chymotrypsin.
Assuntos
Proteínas , Modelos Moleculares , Conformação Molecular , Estrutura MolecularRESUMO
Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), threatens global public health. The world needs rapid development of new antivirals and vaccines to control the current pandemic and to control the spread of the variants. Among the proteins synthesized by the SARS-CoV-2 genome, main protease (Mpro also known as 3CLpro) is a primary drug target, due to its essential role in maturation of the viral polyproteins. In this study, we provide crystallographic evidence, along with some binding assay data, that three clinically approved anti hepatitis C virus drugs and two other drug-like compounds covalently bind to the Mpro Cys145 catalytic residue in the active site. Also, molecular docking studies can provide additional insight for the design of new antiviral inhibitors for SARS-CoV-2 using these drugs as lead compounds. One might consider derivatives of these lead compounds with higher affinity to the Mpro as potential COVID-19 therapeutics for further testing and possibly clinical trials.
Assuntos
Tratamento Farmacológico da COVID-19 , Antivirais/uso terapêutico , Proteases 3C de Coronavírus , Cisteína Endopeptidases/metabolismo , Hepacivirus/metabolismo , Humanos , Simulação de Acoplamento Molecular , Inibidores de Proteases/química , SARS-CoV-2 , Proteínas não Estruturais Virais/genéticaRESUMO
Partial replacement of α-amino acid residues with ß-amino acid residues has been established as a strategy for preserving target-engagement by helix-forming polypeptides while altering other properties. The impact of ß-residue incorporation within polypeptides that adopt less regular conformations, however, has received less attention. The C-terminal heptad repeat (HRC) domains of fusion glycoproteins from pathogenic paramyxoviruses contain a segment that must adopt an extended conformation in order to coassemble with the N-terminal heptad repeat (HRN) domain in the postfusion state and drive a merger of the viral envelope with a target cell membrane. Here, we examine the impact of single α-to-ß substitutions within this extended N-terminal segment of an engineered HRC peptide designated VIQKI. Stabilities of hexameric coassemblies formed with the native human parainfluenza virus 3 (HPIV3) HRN have been evaluated, the structures of five coassemblies have been determined, and antiviral efficacies have been measured. Many sites within the extended segment show functional tolerance of α-to-ß substitution. These results offer a basis for future development of paramyxovirus infection inhibitors with novel biological activity profiles, possibly including resistance to proteolysis.