RESUMO
SARS-CoV-2-neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) or the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated FcγR-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Three of 46 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo, increased lung inflammation can rarely occur in SARS-CoV-2 antibody-infused macaques.
Assuntos
Anticorpos Neutralizantes/imunologia , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Antivirais/imunologia , Líquido da Lavagem Broncoalveolar/química , COVID-19/patologia , COVID-19/virologia , Citocinas/metabolismo , Feminino , Haplorrinos , Humanos , Pulmão/patologia , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Domínios Proteicos , RNA Guia de Cinetoplastídeos/metabolismo , Receptores de IgG/metabolismo , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química , Carga Viral , Replicação ViralRESUMO
Aided by extensive spike protein mutation, the SARS-CoV-2 Omicron variant overtook the previously dominant Delta variant. Spike conformation plays an essential role in SARS-CoV-2 evolution via changes in receptor-binding domain (RBD) and neutralizing antibody epitope presentation, affecting virus transmissibility and immune evasion. Here, we determine cryo-EM structures of the Omicron and Delta spikes to understand the conformational impacts of mutations in each. The Omicron spike structure revealed an unusually tightly packed RBD organization with long range impacts that were not observed in the Delta spike. Binding and crystallography revealed increased flexibility at the functionally critical fusion peptide site in the Omicron spike. These results reveal a highly evolved Omicron spike architecture with possible impacts on its high levels of immune evasion and transmissibility.
Assuntos
COVID-19 , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2 , Humanos , Mutação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/químicaRESUMO
Sulfahydantoin-based molecules may provide a means to counteract antibiotic resistance, which is on the rise. These molecules may act as inhibitors of ß-lactamase enzymes, which are key in some resistance mechanisms. In this paper, we report on the synthesis of 6 novel sulfahydantoin derivatives by the key reaction of chlorosulfonyl isocyanate to form α-amino acid derived sulfamides, and their cyclization into sulfahydantoins. The synthesis is rapid and provides the target compounds in 8 steps. We investigated their potential as ß-lactamase inhibitors using two common Class A ß-lactamases, TEM-1 and the prevalent extended-spectrum TEM-15. Two compounds, 3 and 6, show substantial inhibition of the ß-lactamases with IC50 values between 130 and 510 µM and inferred Ki values between 32 and 55 µM.
Assuntos
Desenvolvimento de Medicamentos , Compostos de Enxofre/farmacologia , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismo , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Compostos de Enxofre/síntese química , Compostos de Enxofre/química , Inibidores de beta-Lactamases/síntese química , Inibidores de beta-Lactamases/químicaRESUMO
The 12-kDa FK506-binding protein (FKBP12) is the target of the commonly used immunosuppressive drug FK506. The FKBP12-FK506 complex binds to calcineurin and inhibits its activity, leading to immunosuppression and preventing organ transplant rejection. Our recent characterization of crystal structures of FKBP12 proteins in pathogenic fungi revealed the involvement of the 80's loop residue (Pro90) in the active site pocket in self-substrate interaction providing novel evidence on FKBP12 dimerization in vivo. The 40's loop residues have also been shown to be involved in reversible dimerization of FKBP12 in the mammalian and yeast systems. To understand how FKBP12 dimerization affects FK506 binding and influences calcineurin function, we generated Aspergillus fumigatus FKBP12 mutations in the 40's and 50's loop (F37 M/L; W60V). Interestingly, the mutants exhibited variable FK506 susceptibility in vivo indicating differing dimer strengths. In comparison to the 80's loop P90G and V91C mutants, the F37 M/L and W60V mutants exhibited greater FK506 resistance, with the F37M mutation showing complete loss in calcineurin binding in vivo. Molecular dynamics and pulling simulations for each dimeric FKBP12 protein revealed a two-fold increase in dimer strength and significantly higher number of contacts for the F37M, F37L, and W60V mutations, further confirming their varying degree of impact on FK506 binding and calcineurin inhibition in vivo.
Assuntos
Aspergillus fumigatus/metabolismo , Inibidores de Calcineurina/farmacologia , Calcineurina/metabolismo , Proteínas Fúngicas/genética , Mutação/genética , Multimerização Proteica , Proteína 1A de Ligação a Tacrolimo/genética , Tacrolimo/farmacologia , Sequência de Aminoácidos , Simulação por Computador , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Proteína 1A de Ligação a Tacrolimo/química , Proteína 1A de Ligação a Tacrolimo/metabolismoRESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.2 sub-lineage has gained in proportion relative to BA.1. Because spike (S) protein variations may underlie differences in their pathobiology, here we determine cryoelectron microscopy (cryo-EM) structures of the BA.2 S ectodomain and compare these with previously determined BA.1 S structures. BA.2 receptor-binding domain (RBD) mutations induce remodeling of the RBD structure, resulting in tighter packing and improved thermostability. Interprotomer RBD interactions are enhanced in the closed (or 3-RBD-down) BA.2 S, while the fusion peptide is less accessible to antibodies than in BA.1. Binding and pseudovirus neutralization assays reveal extensive immune evasion while defining epitopes of two outer RBD face-binding antibodies, DH1044 and DH1193, that neutralize both BA.1 and BA.2. Taken together, our results indicate that stabilization of the closed state through interprotomer RBD-RBD packing is a hallmark of the Omicron variant and show differences in key functional regions in the BA.1 and BA.2 S proteins.
Assuntos
COVID-19 , SARS-CoV-2 , Anticorpos Antivirais , Microscopia Crioeletrônica , Humanos , Receptores Virais/metabolismo , Glicoproteína da Espícula de CoronavírusRESUMO
Aided by extensive spike protein mutation, the SARS-CoV-2 Omicron variant overtook the previously dominant Delta variant. Spike conformation plays an essential role in SARS-CoV-2 evolution via changes in receptor binding domain (RBD) and neutralizing antibody epitope presentation affecting virus transmissibility and immune evasion. Here, we determine cryo-EM structures of the Omicron and Delta spikes to understand the conformational impacts of mutations in each. The Omicron spike structure revealed an unusually tightly packed RBD organization with long range impacts that were not observed in the Delta spike. Binding and crystallography revealed increased flexibility at the functionally critical fusion peptide site in the Omicron spike. These results reveal a highly evolved Omicron spike architecture with possible impacts on its high levels of immune evasion and transmissibility.
RESUMO
New SARS-CoV-2 variants that have accumulated multiple mutations in the spike (S) glycoprotein enable increased transmission and resistance to neutralizing antibodies. Here, we study the antigenic and structural impacts of the S protein mutations from four variants, one that was involved in transmission between minks and humans, and three that rapidly spread in human populations and originated in the United Kingdom, Brazil or South Africa. All variants either retained or improved binding to the ACE2 receptor. The B.1.1.7 (UK) and B.1.1.28 (Brazil) spike variants showed reduced binding to neutralizing NTD and RBD antibodies, respectively, while the B.1.351 (SA) variant showed reduced binding to both NTD- and RBD-directed antibodies. Cryo-EM structural analyses revealed allosteric effects of the mutations on spike conformations and revealed mechanistic differences that either drive inter-species transmission or promotes viral escape from dominant neutralizing epitopes. HIGHLIGHTS: Cryo-EM structures reveal changes in SARS-CoV-2 S protein during inter-species transmission or immune evasion.Adaptation to mink resulted in increased ACE2 binding and spike destabilization.B.1.1.7 S mutations reveal an intricate balance of stabilizing and destabilizing effects that impact receptor and antibody binding.E484K mutation in B.1.351 and B.1.1.28 S proteins drives immune evasion by altering RBD conformation.S protein uses different mechanisms to converge upon similar solutions for altering RBD up/down positioning.
RESUMO
Calcineurin is a critical enzyme in fungal pathogenesis and antifungal drug tolerance and, therefore, an attractive antifungal target. Current clinically accessible calcineurin inhibitors, such as FK506, are immunosuppressive to humans, so exploiting calcineurin inhibition as an antifungal strategy necessitates fungal specificity in order to avoid inhibiting the human pathway. Harnessing fungal calcineurin-inhibitor crystal structures, we recently developed a less immunosuppressive FK506 analog, APX879, with broad-spectrum antifungal activity and demonstrable efficacy in a murine model of invasive fungal infection. Our overarching goal is to better understand, at a molecular level, the interaction determinants of the human and fungal FK506-binding proteins (FKBP12) required for calcineurin inhibition in order to guide the design of fungus-selective, nonimmunosuppressive FK506 analogs. To this end, we characterized high-resolution structures of the Mucor circinelloides FKBP12 bound to FK506 and of the Aspergillus fumigatus, M. circinelloides, and human FKBP12 proteins bound to the FK506 analog APX879, which exhibits enhanced selectivity for fungal pathogens. Combining structural, genetic, and biophysical methodologies with molecular dynamics simulations, we identify critical variations in these structurally similar FKBP12-ligand complexes. The work presented here, aimed at the rational design of more effective calcineurin inhibitors, indeed suggests that modifications to the APX879 scaffold centered around the C15, C16, C18, C36, and C37 positions provide the potential to significantly enhance fungal selectivity. IMPORTANCE Invasive fungal infections are a leading cause of death in the immunocompromised patient population. The rise in drug resistance to current antifungals highlights the urgent need to develop more efficacious and highly selective agents. Numerous investigations of major fungal pathogens have confirmed the critical role of the calcineurin pathway for fungal virulence, making it an attractive target for antifungal development. Although FK506 inhibits calcineurin, it is immunosuppressive in humans and cannot be used as an antifungal. By combining structural, genetic, biophysical, and in silico methodologies, we pinpoint regions of the FK506 scaffold and a less immunosuppressive analog, APX879, centered around the C15 to C18 and C36 to C37 positions that could be altered with selective extensions and/or deletions to enhance fungal selectivity. This work represents a significant advancement toward realizing calcineurin as a viable target for antifungal drug discovery.
Assuntos
Antifúngicos/química , Inibidores de Calcineurina/química , Calcineurina/química , Proteínas Fúngicas/química , Mucor/metabolismo , Mucormicose/microbiologia , Tacrolimo/química , Sequência de Aminoácidos , Antifúngicos/farmacologia , Calcineurina/genética , Calcineurina/metabolismo , Inibidores de Calcineurina/farmacologia , Desenho de Fármacos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Mucor/efeitos dos fármacos , Mucor/genética , Mucormicose/tratamento farmacológico , Mucormicose/genética , Mucormicose/metabolismo , Alinhamento de Sequência , Tacrolimo/farmacologia , Proteína 1A de Ligação a Tacrolimo/química , Proteína 1A de Ligação a Tacrolimo/genética , Proteína 1A de Ligação a Tacrolimo/metabolismoRESUMO
The severe acute respiratory coronavirus 2 (SARS-CoV-2) spike (S) protein is the target of vaccine design efforts to end the coronavirus disease 2019 (COVID-19) pandemic. Despite a low mutation rate, isolates with the D614G substitution in the S protein appeared early during the pandemic and are now the dominant form worldwide. Here, we explore S conformational changes and the effects of the D614G mutation on a soluble S ectodomain construct. Cryoelectron microscopy (cryo-EM) structures reveal altered receptor binding domain (RBD) disposition; antigenicity and proteolysis experiments reveal structural changes and enhanced furin cleavage efficiency of the G614 variant. Furthermore, furin cleavage alters the up/down ratio of the RBDs in the G614 S ectodomain, demonstrating an allosteric effect on RBD positioning triggered by changes in the SD2 region, which harbors residue 614 and the furin cleavage site. Our results elucidate SARS-CoV-2 S conformational landscape and allostery and have implications for vaccine design.
Assuntos
Peptídeo Hidrolases/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , COVID-19/patologia , COVID-19/virologia , Microscopia Crioeletrônica , Humanos , Imunogenicidade da Vacina , Simulação de Dinâmica Molecular , Mutação , Domínios Proteicos , Estabilidade Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas/metabolismo , Proteólise , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with multiple spike mutations enable increased transmission and antibody resistance. We combined cryo-electron microscopy (cryo-EM), binding, and computational analyses to study variant spikes, including one that was involved in transmission between minks and humans, and others that originated and spread in human populations. All variants showed increased angiotensin-converting enzyme 2 (ACE2) receptor binding and increased propensity for receptor binding domain (RBD)-up states. While adaptation to mink resulted in spike destabilization, the B.1.1.7 (UK) spike balanced stabilizing and destabilizing mutations. A local destabilizing effect of the RBD E484K mutation was implicated in resistance of the B.1.1.28/P.1 (Brazil) and B.1.351 (South Africa) variants to neutralizing antibodies. Our studies revealed allosteric effects of mutations and mechanistic differences that drive either interspecies transmission or escape from antibody neutralization.
Assuntos
SARS-CoV-2/química , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Substituição de Aminoácidos , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , COVID-19/transmissão , COVID-19/veterinária , COVID-19/virologia , Microscopia Crioeletrônica , Adaptação ao Hospedeiro , Humanos , Evasão da Resposta Imune , Vison/virologia , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Receptores de Coronavírus/metabolismo , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
The SARS-CoV-2 spike (S) protein, a primary target for COVID-19 vaccine development, presents its receptor binding domain in two conformations, the receptor-accessible 'up' or receptor-inaccessible 'down' states. Here we report that the commonly used stabilized S ectodomain construct '2P' is sensitive to cold temperatures, and this cold sensitivity is abrogated in a 'down' state-stabilized ectodomain. Our findings will impact structural, functional and vaccine studies that use the SARS-CoV-2 S ectodomain.
Assuntos
Glicoproteína da Espícula de Coronavírus/química , Anticorpos Antivirais/química , Vacinas contra COVID-19/química , Temperatura Baixa , Microscopia Crioeletrônica , Ensaio de Imunoadsorção Enzimática , Humanos , Desnaturação Proteica , Domínios Proteicos , Estabilidade Proteica , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Ressonância de Plasmônio de SuperfícieRESUMO
SARS-CoV-2 neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) and the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV-1 infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infection in vitro , while five non-neutralizing NTD antibodies mediated FcγR-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Nonetheless, three of 31 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo , increased lung inflammation can occur in SARS-CoV-2 antibody-infused macaques.
RESUMO
The coronavirus (CoV) spike (S) protein, involved in viral-host cell fusion, is the primary immunogenic target for virus neutralization and the current focus of many vaccine design efforts. The highly flexible S-protein, with its mobile domains, presents a moving target to the immune system. Here, to better understand S-protein mobility, we implemented a structure-based vector analysis of available ß-CoV S-protein structures. Despite an overall similarity in domain organization, we found that S-proteins from different ß-CoVs display distinct configurations. Based on this analysis, we developed two soluble ectodomain constructs for the SARS-CoV-2 S-protein, in which the highly immunogenic and mobile receptor binding domain (RBD) is either locked in the all-RBDs 'down' position or adopts 'up' state conformations more readily than the wild-type S-protein. These results demonstrate that the conformation of the S-protein can be controlled via rational design and can provide a framework for the development of engineered CoV S-proteins for vaccine applications.
Assuntos
Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Microscopia Eletrônica/métodos , Modelos Moleculares , Mutação , Conformação Proteica , Domínios Proteicos , Subunidades Proteicas/química , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
The SARS-CoV-2 spike (S) protein, a primary target for COVID-19 vaccine development, presents its Receptor Binding Domain in two conformations: receptor-accessible "up" or receptor-inaccessible "down" conformations. Here, we report that the commonly used stabilized S ectodomain construct "2P" is sensitive to cold temperature, and that this cold sensitivity is resolved in a "down" state stabilized spike. Our results will impact structural, functional and vaccine studies that use the SARS-CoV-2 S ectodomain.
RESUMO
Invasive fungal infections are a leading cause of death in immunocompromised patients and remain difficult to treat since fungal pathogens, like mammals, are eukaryotes and share many orthologous proteins. As a result, current antifungal drugs have limited clinical value, are sometimes toxic, can adversely affect human reaction pathways and are increasingly ineffective due to emerging resistance. One potential antifungal drug, FK506, establishes a ternary complex between the phosphatase, calcineurin, and the 12-kDa peptidyl-prolyl isomerase FK506-binding protein, FKBP12. It has been well established that calcineurin, highly conserved from yeast to mammals, is necessary for invasive fungal disease and is inhibited when in complex with FK506/FKBP12. Unfortunately, FK506 is also immunosuppressive in humans, precluding its usage as an antifungal drug, especially in immunocompromised patients. Whereas the homology between human and fungal calcineurin proteins is > 80%, the human and fungal FKBP12s share 48-58% sequence identity, making them more amenable candidates for drug targeting efforts. Here we report the backbone and sidechain NMR assignments of recombinant FKBP12 proteins from the pathogenic fungi Mucor circinelloides and Aspergillus fumigatus in the apo form and compare these to the backbone assignments of the FK506 bound form. In addition, we report the backbone assignments of the apo and FK506 bound forms of the Homo sapiens FKBP12 protein for evaluation against the fungal forms. These data are the first steps towards defining, at a residue specific level, the impacts of FK506 binding to fungal and mammalian FKBP12 proteins. Our data highlight differences between the human and fungal FKBP12s that could lead to the design of more selective anti-fungal drugs.
Assuntos
Aspergillus fumigatus/química , Proteínas Fúngicas/química , Mucor/química , Ressonância Magnética Nuclear Biomolecular , Proteína 1A de Ligação a Tacrolimo/química , Sequência de Aminoácidos , Isótopos de Carbono , Isótopos de Nitrogênio , ProteínasRESUMO
Understanding the principles of protein dynamics will help guide engineering of protein function: altering protein motions may be a barrier to success or may be an enabling tool for protein engineering. The impact of dynamics on protein function is typically reported over a fraction of the full scope of motional timescales. If motional patterns vary significantly at different timescales, then only by monitoring motions broadly will we understand the impact of protein dynamics on engineering functional proteins. Using an integrative approach combining experimental and in silico methodologies, we elucidate protein dynamics over the entire span of fast to slow timescales (ps to ms) for a laboratory-engineered system composed of five interrelated ß-lactamases: two natural homologs and three laboratory-recombined variants. Fast (ps-ns) and intermediate (ns-µs) dynamics were mostly conserved. However, slow motions (µs-ms) were few and conserved in the natural homologs yet were numerous and widely dispersed in their recombinants. Nonetheless, modified slow dynamics were functionally tolerated. Crystallographic B-factors from high-resolution X-ray structures were partly predictive of the conserved motions but not of the new slow motions captured in our solution studies. Our inspection of protein dynamics over a continuous range of timescales vividly illustrates the complexity of dynamic impacts of protein engineering as well as the functional tolerance of an engineered enzyme system to new slow motions.
Assuntos
Modelos Moleculares , Conformação Proteica , Engenharia de Proteínas , beta-Lactamases/química , Sítios de Ligação , Cristalografia por Raios X , Cinética , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , beta-Lactamases/metabolismoRESUMO
Calcineurin is important for fungal virulence and a potential antifungal target, but compounds targeting calcineurin, such as FK506, are immunosuppressive. Here we report the crystal structures of calcineurin catalytic (CnA) and regulatory (CnB) subunits complexed with FK506 and the FK506-binding protein (FKBP12) from human fungal pathogens (Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans and Coccidioides immitis). Fungal calcineurin complexes are similar to the mammalian complex, but comparison of fungal and human FKBP12 (hFKBP12) reveals conformational differences in the 40s and 80s loops. NMR analysis, molecular dynamic simulations, and mutations of the A. fumigatus CnA/CnB-FK506-FKBP12-complex identify a Phe88 residue, not conserved in hFKBP12, as critical for binding and inhibition of fungal calcineurin. These differences enable us to develop a less immunosuppressive FK506 analog, APX879, with an acetohydrazine substitution of the C22-carbonyl of FK506. APX879 exhibits reduced immunosuppressive activity and retains broad-spectrum antifungal activity and efficacy in a murine model of invasive fungal infection.
Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/metabolismo , Inibidores de Calcineurina/farmacologia , Calcineurina/metabolismo , Cryptococcus neoformans/metabolismo , Proteína 1A de Ligação a Tacrolimo/metabolismo , Tacrolimo/farmacologia , Animais , Aspergilose/tratamento farmacológico , Aspergilose/microbiologia , Aspergillus fumigatus/efeitos dos fármacos , Sítios de Ligação , Candida albicans/efeitos dos fármacos , Candida albicans/metabolismo , Células Cultivadas , Coccidioides/efeitos dos fármacos , Coccidioides/metabolismo , Criptococose/tratamento farmacológico , Criptococose/microbiologia , Cryptococcus neoformans/efeitos dos fármacos , Cristalografia por Raios X , Descoberta de Drogas/métodos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos C57BL , Simulação de Dinâmica Molecular , Tacrolimo/metabolismoRESUMO
The widespread use of ß-lactam antibiotics has given rise to a dramatic increase in clinically-relevant ß-lactamases. Understanding the structure/function relation in these variants is essential to better address the ever-growing incidence of antibiotic resistance. We previously reported the backbone resonance assignments of a chimeric protein constituted of segments of the class A ß-lactamases TEM-1 and PSE-4 (Morin et al. in Biomol NMR Assign 4:127-130, 2010. doi: 10.1007/s12104-010-9227-8 ). That chimera, cTEM17m, held 17 amino acid substitutions relative to TEM-1 ß-lactamase, resulting in a well-folded and fully functional protein with increased dynamics. Here we report the (1)H, (13)C and (15)N backbone resonance assignments of chimera cTEM-19m, which includes 19 substitutions and exhibits increased active-site perturbation, as well as one of its deconvoluted variants, as the first step in the analysis of their dynamic behaviours.
Assuntos
Proteínas Mutantes/química , Ressonância Magnética Nuclear Biomolecular , Engenharia de Proteínas , Proteínas Recombinantes/química , beta-Lactamases/química , Isótopos de Carbono , Isótopos de Nitrogênio , Estrutura Secundária de Proteína , TrítioRESUMO
Proteins are dynamic systems, and understanding dynamics is critical for fully understanding protein function. Therefore, the question of whether laboratory engineering has an impact on protein dynamics is of general interest. Here, we demonstrate that two homologous, naturally evolved enzymes with high degrees of structural and functional conservation also exhibit conserved dynamics. Their similar set of slow timescale dynamics is highly restricted, consistent with evolutionary conservation of a functionally important feature. However, we also show that dynamics of a laboratory-engineered chimeric enzyme obtained by recombination of the two homologs exhibits striking difference on the millisecond timescale, despite function and high-resolution crystal structure (1.05 Å) being conserved. The laboratory-engineered chimera is thus functionally tolerant to modified dynamics on the timescale of catalytic turnover. Tolerance to dynamic variation implies that maintenance of native-like protein dynamics may not be required when engineering functional proteins.
Assuntos
Engenharia de Proteínas , Proteínas/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Isótopos de Nitrogênio/química , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Proteínas/química , Proteínas/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , beta-Lactamases/química , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
Enzyme engineering has been facilitated by recombination of close homologues, followed by functional screening. In one such effort, chimeras of two class-A ß-lactamases - TEM-1 and PSE-4 - were created according to structure-guided protein recombination and selected for their capacity to promote bacterial proliferation in the presence of ampicillin (Voigt et al., Nat. Struct. Biol. 2002 9:553). To provide a more detailed assessment of the effects of protein recombination on the structure and function of the resulting chimeric enzymes, we characterized a series of functional TEM-1/PSE-4 chimeras possessing between 17 and 92 substitutions relative to TEM-1 ß-lactamase. Circular dichroism and thermal scanning fluorimetry revealed that the chimeras were generally well folded. Despite harbouring important sequence variation relative to either of the two 'parental' ß-lactamases, the chimeric ß-lactamases displayed substrate recognition spectra and reactivity similar to their most closely-related parent. To gain further insight into the changes induced by chimerization, the chimera with 17 substitutions was investigated by NMR spin relaxation. While high order was conserved on the ps-ns timescale, a hallmark of class A ß-lactamases, evidence of additional slow motions on the µs-ms timescale was extracted from model-free calculations. This is consistent with the greater number of resonances that could not be assigned in this chimera relative to the parental ß-lactamases, and is consistent with this well-folded and functional chimeric ß-lactamase displaying increased slow time-scale motions.