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1.
J Biol Chem ; 298(9): 102323, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35931112

RESUMO

Integrin α5ß1 mediates cell adhesion to the extracellular matrix by binding fibronectin (Fn). Selectivity for Fn by α5ß1 is achieved through recognition of an RGD motif in the 10th type III Fn domain (Fn10) and the synergy site in the ninth type III Fn domain (Fn9). However, details of the interaction dynamics are unknown. Here, we compared synergy-site and Fn-truncation mutations for their α5ß1-binding affinities and stabilities. We also interrogated binding of the α5ß1 ectodomain headpiece fragment to Fn using hydrogen-deuterium exchange (HDX) mass spectrometry to probe binding sites and sites of integrin conformational change. Our results suggest the synergistic effect of Fn9 requires both specific residues and a folded domain. We found some residues considered important for synergy are required for stability. Additionally, we show decreases in fibronectin HDX are localized to a synergy peptide containing contacting residues in two ß-strands, an intervening loop in Fn9, and the RGD-containing loop in Fn10, indicative of binding sites. We also identified binding sites in the α5-subunit ß-propeller domain for the Fn9 synergy site and in the ß1-subunit ßI domain for Fn10 based on decreases in α5ß1 HDX. Interestingly, the dominant effect of Fn binding was an increase in α5ß1 deuterium exchange distributed over multiple sites that undergo changes in conformation or solvent accessibility and appear to be sites where energy is stored in the higher-energy, open-integrin conformation. Together, our results highlight regions important for α5ß1 binding to Fn and dynamics associated with this interaction.


Assuntos
Fibronectinas , Integrina alfa5beta1 , Domínios e Motivos de Interação entre Proteínas , Sítios de Ligação , Adesão Celular , Medição da Troca de Deutério , Fibronectinas/química , Fibronectinas/genética , Integrina alfa5beta1/química , Mutação , Oligopeptídeos/química , Solventes
2.
Nature ; 542(7639): 55-59, 2017 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-28117447

RESUMO

Integrins are adhesion receptors that transmit force across the plasma membrane between extracellular ligands and the actin cytoskeleton. In activation of the transforming growth factor-ß1 precursor (pro-TGF-ß1), integrins bind to the prodomain, apply force, and release the TGF-ß growth factor. However, we know little about how integrins bind macromolecular ligands in the extracellular matrix or transmit force to them. Here we show how integrin αVß6 binds pro-TGF-ß1 in an orientation biologically relevant for force-dependent release of TGF-ß from latency. The conformation of the prodomain integrin-binding motif differs in the presence and absence of integrin binding; differences extend well outside the interface and illustrate how integrins can remodel extracellular matrix. Remodelled residues outside the interface stabilize the integrin-bound conformation, adopt a conformation similar to earlier-evolving family members, and show how macromolecular components outside the binding motif contribute to integrin recognition. Regions in and outside the highly interdigitated interface stabilize a specific integrin/pro-TGF-ß orientation that defines the pathway through these macromolecules which actin-cytoskeleton-generated tensile force takes when applied through the integrin ß-subunit. Simulations of force-dependent activation of TGF-ß demonstrate evolutionary specializations for force application through the TGF-ß prodomain and through the ß- and not α-subunit of the integrin.


Assuntos
Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Integrinas/química , Integrinas/metabolismo , Fator de Crescimento Transformador beta1/agonistas , Fator de Crescimento Transformador beta1/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Evolução Molecular , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Fator de Crescimento Transformador beta1/metabolismo
3.
EMBO J ; 37(3): 384-397, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29343545

RESUMO

Growth differentiation factor 8 (GDF8)/myostatin is a latent TGF-ß family member that potently inhibits skeletal muscle growth. Here, we compared the conformation and dynamics of precursor, latent, and Tolloid-cleaved GDF8 pro-complexes to understand structural mechanisms underlying latency and activation of GDF8. Negative stain electron microscopy (EM) of precursor and latent pro-complexes reveals a V-shaped conformation that is unaltered by furin cleavage and sharply contrasts with the ring-like, cross-armed conformation of latent TGF-ß1. Surprisingly, Tolloid-cleaved GDF8 does not immediately dissociate, but in EM exhibits structural heterogeneity consistent with partial dissociation. Hydrogen-deuterium exchange was not affected by furin cleavage. In contrast, Tolloid cleavage, in the absence of prodomain-growth factor dissociation, increased exchange in regions that correspond in pro-TGF-ß1 to the α1-helix, latency lasso, and ß1-strand in the prodomain and to the ß6'- and ß7'-strands in the growth factor. Thus, these regions are important in maintaining GDF8 latency. Our results show that Tolloid cleavage activates latent GDF8 by destabilizing specific prodomain-growth factor interfaces and primes the growth factor for release from the prodomain.


Assuntos
Músculo Esquelético/crescimento & desenvolvimento , Miostatina/metabolismo , Precursores de Proteínas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Linhagem Celular , Medição da Troca de Deutério , Drosophila , Ativação Enzimática/fisiologia , Furina/metabolismo , Células HEK293 , Humanos , Microscopia Eletrônica , Metaloproteases Semelhantes a Toloide/metabolismo
4.
Nat Methods ; 16(7): 595-602, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31249422

RESUMO

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.


Assuntos
Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Análise de Dados , Concentração de Íons de Hidrogênio
5.
J Pept Sci ; 24(1)2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29322650

RESUMO

Alzheimer disease is a neurodegenerative disease affecting an increasing number of patients worldwide. Current therapeutic strategies are directed to molecules capable to block the aggregation of the ß-amyloid(1-42) (Aß) peptide and its shorter naturally occurring peptide fragments into toxic oligomers and amyloid fibrils. Aß-specific antibodies have been recently developed as powerful antiaggregation tools. The identification and functional characterization of the epitope structures of Aß antibodies contributes to the elucidation of their mechanism of action in the human organism. In previous studies, the Aß(4-10) peptide has been identified as an epitope for the polyclonal anti-Aß(1-42) antibody that has been shown capable to reduce amyloid deposition in a transgenic Alzheimer disease mouse model. To determine the functional significance of the amino acid residues involved in binding to the antibody, we report here the effects of alanine single-site mutations within the Aß-epitope sequence on the antigen-antibody interaction. Specific identification of the essential affinity preserving mutant peptides was obtained by exposing a Sepharose-immobilized antibody column to an equimolar mixture of mutant peptides, followed by analysis of bound peptides using high-resolution MALDI-Fourier transform-Ion Cyclotron Resonance mass spectrometry. For the polyclonal antibody, affinity was preserved in the H6A, D7A, S8A, and G9A mutants but was lost in the F4, R5, and Y10 mutants, indicating these residues as essential amino acids for binding. Enzyme-linked immunosorbent assays confirmed the binding differences of the mutant peptides to the polyclonal antibody. In contrast, the mass spectrometric analysis of the mutant Aß(4-10) peptides upon affinity binding to a monoclonal anti-Aß(1-17) antibody showed complete loss of binding by Ala-site mutation of any residue of the Aß(4-10) epitope. Surface plasmon resonance affinity determination of wild-type Aß(1-17) to the monoclonal Aß antibody provided a binding constant KD in the low nanomolar range. These results provide valuable information in the elucidation of the binding mechanism and the development of Aß-specific antibodies with improved therapeutic efficacy.


Assuntos
Alanina/genética , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/imunologia , Anticorpos Monoclonais/imunologia , Epitopos/química , Alanina/imunologia , Peptídeos beta-Amiloides/genética , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Epitopos/imunologia , Humanos , Espectrometria de Massas , Mutação
6.
Nature ; 463(7280): 501-6, 2010 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-20072125

RESUMO

In an effort to find new pharmacological modalities to overcome resistance to ATP-binding-site inhibitors of Bcr-Abl, we recently reported the discovery of GNF-2, a selective allosteric Bcr-Abl inhibitor. Here, using solution NMR, X-ray crystallography, mutagenesis and hydrogen exchange mass spectrometry, we show that GNF-2 binds to the myristate-binding site of Abl, leading to changes in the structural dynamics of the ATP-binding site. GNF-5, an analogue of GNF-2 with improved pharmacokinetic properties, when used in combination with the ATP-competitive inhibitors imatinib or nilotinib, suppressed the emergence of resistance mutations in vitro, displayed additive inhibitory activity in biochemical and cellular assays against T315I mutant human Bcr-Abl and displayed in vivo efficacy against this recalcitrant mutant in a murine bone-marrow transplantation model. These results show that therapeutically relevant inhibition of Bcr-Abl activity can be achieved with inhibitors that bind to the myristate-binding site and that combining allosteric and ATP-competitive inhibitors can overcome resistance to either agent alone.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Fusão bcr-abl/química , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Animais , Antineoplásicos/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica , Benzamidas , Sítios de Ligação , Transplante de Medula Óssea , Linhagem Celular Tumoral , Cristalização , Modelos Animais de Doenças , Feminino , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Concentração Inibidora 50 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Masculino , Espectrometria de Massas , Camundongos , Modelos Moleculares , Mutação/genética , Piperazinas/química , Piperazinas/farmacologia , Estrutura Terciária de Proteína , Pirimidinas/química , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Transplante Heterólogo
7.
Expert Rev Proteomics ; 12(2): 159-69, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25711416

RESUMO

IL-23 is an important therapeutic target for the treatment of inflammatory diseases. Adnectins are targeted protein therapeutics that are derived from domain III of human fibronectin and have a similar protein scaffold to antibodies. Adnectin 2 was found to bind to IL-23 and compete with the IL-23/IL-23R interaction, posing a potential protein therapeutic. Hydrogen/deuterium exchange mass spectrometry and computational methods were applied to probe the binding interactions between IL-23 and Adnectin 2 and to determine the correlation between the two orthogonal methods. This review summarizes the current structural knowledge about IL-23 and focuses on the applicability of hydrogen/deuterium exchange mass spectrometry to investigate the higher order structure of proteins, which plays an important role in the discovery of new and improved biotherapeutics.


Assuntos
Terapia Biológica , Deutério/química , Hidrogênio/química , Interleucina-23/química , Biologia Computacional , Humanos , Interleucina-23/metabolismo , Espectrometria de Massas/métodos , Ligação Proteica , Conformação Proteica , Receptores de Interleucina/química
8.
Nature ; 462(7276): 1070-4, 2009 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-20033049

RESUMO

The clinical efficacy of epidermal growth factor receptor (EGFR) kinase inhibitors in EGFR-mutant non-small-cell lung cancer (NSCLC) is limited by the development of drug-resistance mutations, including the gatekeeper T790M mutation. Strategies targeting EGFR T790M with irreversible inhibitors have had limited success and are associated with toxicity due to concurrent inhibition of wild-type EGFR. All current EGFR inhibitors possess a structurally related quinazoline-based core scaffold and were identified as ATP-competitive inhibitors of wild-type EGFR. Here we identify a covalent pyrimidine EGFR inhibitor by screening an irreversible kinase inhibitor library specifically against EGFR T790M. These agents are 30- to 100-fold more potent against EGFR T790M, and up to 100-fold less potent against wild-type EGFR, than quinazoline-based EGFR inhibitors in vitro. They are also effective in murine models of lung cancer driven by EGFR T790M. Co-crystallization studies reveal a structural basis for the increased potency and mutant selectivity of these agents. These mutant-selective irreversible EGFR kinase inhibitors may be clinically more effective and better tolerated than quinazoline-based inhibitors. Our findings demonstrate that functional pharmacological screens against clinically important mutant kinases represent a powerful strategy to identify new classes of mutant-selective kinase inhibitors.


Assuntos
Antineoplásicos/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Mutação/genética , Inibidores de Proteínas Quinases/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Pulmão/efeitos dos fármacos , Camundongos , Modelos Químicos , Modelos Moleculares , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/toxicidade
9.
J Biol Chem ; 288(8): 5443-50, 2013 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-23316053

RESUMO

The c-abl proto-oncogene encodes a unique protein-tyrosine kinase (Abl) distinct from c-Src, c-Fes, and other cytoplasmic tyrosine kinases. In normal cells, Abl plays prominent roles in cellular responses to genotoxic stress as well as in the regulation of the actin cytoskeleton. Abl is also well known in the context of Bcr-Abl, the oncogenic fusion protein characteristic of chronic myelogenous leukemia. Selective inhibitors of Bcr-Abl, of which imatinib is the prototype, have had a tremendous impact on clinical outcomes in chronic myelogenous leukemia and revolutionized the field of targeted cancer therapy. In this minireview, we focus on the structural organization and dynamics of Abl kinases and how these features influence inhibitor sensitivity.


Assuntos
Proteínas de Fusão bcr-abl/biossíntese , Proteínas de Fusão bcr-abl/fisiologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes abl/genética , Benzamidas , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Modelos Biológicos , Modelos Químicos , Conformação Molecular , Mutação , Ácido Mirístico/química , Piperazinas/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína , Proto-Oncogene Mas , Pirimidinas/farmacologia , Domínios de Homologia de src
10.
J Biol Chem ; 288(9): 6116-29, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23303187

RESUMO

Multidomain kinases such as c-Src and c-Abl are regulated by complex allosteric interactions involving their noncatalytic SH3 and SH2 domains. Here we show that enhancing natural allosteric control of kinase activity by SH3/linker engagement has long-range suppressive effects on the kinase activity of the c-Abl core. Surprisingly, enhanced SH3/linker interaction also dramatically sensitized the Bcr-Abl tyrosine kinase associated with chronic myelogenous leukemia to small molecule inhibitors that target either the active site or the myristic acid binding pocket in the kinase domain C-lobe. Dynamics analyses using hydrogen exchange mass spectrometry revealed a remarkable allosteric network linking the SH3 domain, the myristic acid binding pocket, and the active site of the c-Abl core, providing a structural basis for the biological observations. These results suggest a rational strategy for enhanced drug targeting of Bcr-Abl and other multidomain kinase systems that use multiple small molecules to exploit natural mechanisms of kinase control.


Assuntos
Ácido Mirístico/química , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-abl/química , Sítios de Ligação , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/química , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Domínios de Homologia de src
11.
J Am Chem Soc ; 136(27): 9754-62, 2014 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-24937475

RESUMO

The hydroxylation or epoxidation of hydrocarbons by bacterial multicomponent monooxygenases (BMMs) requires the interplay of three or four protein components. How component protein interactions control catalysis, however, is not well understood. In particular, the binding sites of the reductase components on the surface of their cognate hydroxylases and the role(s) that the regulatory proteins play during intermolecular electron transfer leading to the hydroxylase reduction have been enigmatic. Here we determine the reductase binding site on the hydroxylase of a BMM enzyme, soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath). We present evidence that the ferredoxin domain of the reductase binds to the canyon region of the hydroxylase, previously determined to be the regulatory protein binding site as well. The latter thus inhibits reductase binding to the hydroxylase and, consequently, intermolecular electron transfer from the reductase to the hydroxylase diiron active site. The binding competition between the regulatory protein and the reductase may serve as a control mechanism for regulating electron transfer, and other BMM enzymes are likely to adopt the same mechanism.


Assuntos
Methylococcus capsulatus/enzimologia , Oxigenases/metabolismo , Transporte de Elétrons , Modelos Moleculares , Oxigenases/química , Solubilidade
12.
Nat Commun ; 14(1): 1885, 2023 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-37019905

RESUMO

Proteins often undergo large conformational changes when binding small molecules, but atomic-level descriptions of such events have been elusive. Here, we report unguided molecular dynamics simulations of Abl kinase binding to the cancer drug imatinib. In the simulations, imatinib first selectively engages Abl kinase in its autoinhibitory conformation. Consistent with inferences drawn from previous experimental studies, imatinib then induces a large conformational change of the protein to reach a bound complex that closely resembles published crystal structures. Moreover, the simulations reveal a surprising local structural instability in the C-terminal lobe of Abl kinase during binding. The unstable region includes a number of residues that, when mutated, confer imatinib resistance by an unknown mechanism. Based on the simulations, NMR spectra, hydrogen-deuterium exchange measurements, and thermostability measurements and estimates, we suggest that these mutations confer imatinib resistance by exacerbating structural instability in the C-terminal lobe, rendering the imatinib-bound state energetically unfavorable.


Assuntos
Antineoplásicos , Piperazinas , Mesilato de Imatinib , Piperazinas/farmacologia , Pirimidinas/farmacologia , Benzamidas , Antineoplásicos/farmacologia , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fusão bcr-abl
13.
Proc Natl Acad Sci U S A ; 106(5): 1386-91, 2009 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-19164531

RESUMO

Protein dynamics are inextricably linked to protein function but there are few techniques that allow protein dynamics to be conveniently interrogated. For example, mutations and translocations give rise to aberrant proteins such as Bcr-Abl where changes in protein conformation and dynamics are believed to result in deregulated kinase activity that provides the oncogenic signal in chronic myelogeous leukemia. Although crystal structures of the down-regulated c-Abl kinase core have been reported, the conformational impact of mutations that render Abl resistant to small-molecule kinase inhibitors are largely unknown as is the allosteric interplay of the various regulatory elements of the protein. Hydrogen exchange mass spectrometry (HX MS) was used to compare the conformations of wild-type Abl with a nonmyristoylated form and with 3 clinically relevant imatinib resistance mutants (T315I, Y253H and E255V). A HX-resistant core localized to the interface between the SH2 and kinase domains, a region known to be important for maintaining the down-regulated state. Conformational differences upon demyristoylation were consistent with the SH2 domain moving to the top of the small lobe of the kinase domain as a function of activation. There were conformational changes in the T315I mutant but, surprisingly, no major changes in conformation were detected in either the Y253H or the E255V mutants. Taken together, these results provide evidence that allosteric interactions and conformational changes play a major role in Abl kinase regulation in solution. Similar analyses could be performed on any protein to provide mechanistic details about conformational changes and protein function.


Assuntos
Mutação , Proteínas Proto-Oncogênicas c-abl/química , Animais , Benzamidas , Resistência a Medicamentos/genética , Hidrogênio/química , Mesilato de Imatinib , Modelos Moleculares , Piperazinas/farmacologia , Conformação Proteica , Proteínas Proto-Oncogênicas c-abl/genética , Pirimidinas/farmacologia
14.
J Mol Biol ; 434(5): 167439, 2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-34990654

RESUMO

The 33 members of the transforming growth factor beta (TGF-ß) family are fundamentally important for organismal development and homeostasis. Family members are synthesized and secreted as pro-complexes of non-covalently associated prodomains and growth factors (GF). Pro-complexes from a subset of family members are latent and require activation steps to release the GF for signaling. Why some members are latent while others are non-latent is incompletely understood, particularly because of large family diversity. Here, we have examined representative family members in negative stain electron microscopy (nsEM) and hydrogen deuterium exchange (HDX) to identify features that differentiate latent from non-latent members. nsEM showed three overall pro-complex conformations that differed in prodomain arm domain orientation relative to the bound growth factor. Two cross-armed members, TGF-ß1 and TGF-ß2, were each latent. However, among V-armed members, GDF8 was latent whereas ActA was not. All open-armed members, BMP7, BMP9, and BMP10, were non-latent. Family members exhibited remarkably varying HDX patterns, consistent with large prodomain sequence divergence. A strong correlation emerged between latency and protection of the prodomain α1-helix from exchange. Furthermore, latency and protection from exchange correlated structurally with increased α1-helix buried surface area, hydrogen bonds, and cation-pi bonds. Moreover, a specific pattern of conserved basic and hydrophobic residues in the α1-helix and aromatic residues in the interacting fastener were found only in latent members. Thus, this first comparative survey of TGF-ß family members reveals not only diversity in conformation and dynamics but also unique features that distinguish latent members.


Assuntos
Proteínas da Superfamília de TGF-beta , Ligação de Hidrogênio , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Transdução de Sinais , Proteínas da Superfamília de TGF-beta/química , Proteínas da Superfamília de TGF-beta/metabolismo
15.
Elife ; 112022 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-35532124

RESUMO

Hemostasis in the arterial circulation is mediated by binding of the A1 domain of the ultralong protein von Willebrand factor (VWF) to GPIbα on platelets to form a platelet plug. A1 is activated by tensile force on VWF concatemers imparted by hydrodynamic drag force. The A1 core is protected from force-induced unfolding by a long-range disulfide that links cysteines near its N- and C-termini. The O-glycosylated linkers between A1 and its neighboring domains, which transmit tensile force to A1, are reported to regulate A1 activation for binding to GPIb, but the mechanism is controversial and incompletely defined. Here, we study how these linkers, and their polypeptide and O-glycan moieties, regulate A1 affinity by measuring affinity, kinetics, thermodynamics, hydrogen deuterium exchange (HDX), and unfolding by temperature and urea. The N-linker lowers A1 affinity 40-fold with a stronger contribution from its O-glycan than polypeptide moiety. The N-linker also decreases HDX in specific regions of A1 and increases thermal stability and the energy gap between its native state and an intermediate state, which is observed in urea-induced unfolding. The C-linker also decreases affinity of A1 for GPIbα, but in contrast to the N-linker, has no significant effect on HDX or A1 stability. Among different models for A1 activation, our data are consistent with the model that the intermediate state has high affinity for GPIbα, which is induced by tensile force physiologically and regulated allosterically by the N-linker.


Assuntos
Plaquetas , Fator de von Willebrand , Plaquetas/metabolismo , Polissacarídeos/metabolismo , Ligação Proteica , Ureia/metabolismo , Fator de von Willebrand/metabolismo
17.
J Pharm Biomed Anal ; 182: 113141, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32036298

RESUMO

The higher-order structure of a protein defines its function, and protein structural dynamics are often essential for protein binding and enzyme catalysis. Methods for protein characterization in solution are continuously being developed to understand and explore protein conformational changes with regards to function and activity. The goal of this study was to survey the use of combining HDX-MS global conformational screening with in silico modeling and continuous labeling peptide-level HDX-MS as an approach to highlight regions of interest within an enzyme required for biocatalytic processes. We surveyed in silico modeling correlated with peptide level HDX-MS experiments to characterize and localize transaminase enzyme structural dynamics at different conditions. This approach was orthogonally correlated with a global Size-Exclusion-HDX (SEC-HDX) screen for global conformational comparison and global alpha-helical content measurements by circular dichroism. Enzymatic activity and stereo-selectivity of transaminases were compared at different reaction-solution conditions that forced protein conformational changes by increasing acetonitrile concentration. The experimental peptide-level HDX-MS results demonstrated similar trends to the modeling data showing that certain regions remained folded in transaminases ATA-036 and ATA-303 with increasing acetonitrile concentration, which is also associated with shifting stereoselectivity. HDX modeling, SEC-HDX and CD experimental data showed that transaminase ATA-234 had the highest level of global unfolding with increasing acetonitrile concentration compared to the other two enzymes, which correlated with drastically reduced product conversion in transamination reaction. The combined HDX modeling/experimental workflow, based on enzymatic reactions studied at different conditions to induce changes in enzyme conformation, could be used as a tool to guide directed evolution efforts by identifying and focusing on the regions of an enzyme required for reaction product conversion and stereoselectivity.


Assuntos
Espectrometria de Massa com Troca Hidrogênio-Deutério/métodos , Peptídeos/química , Proteínas/química , Solventes/química , Dicroísmo Circular , Simulação por Computador , Enzimas/química , Simulação de Dinâmica Molecular , Conformação Proteica , Desdobramento de Proteína , Estereoisomerismo
18.
Sci Rep ; 10(1): 5324, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32210275

RESUMO

Ubiquitin specific peptidase 7 (USP7) is a deubiquitinating enzyme (DUB) that removes ubiquitin tags from specific protein substrates in order to alter their degradation rate and sub-cellular localization. USP7 has been proposed as a therapeutic target in several cancers because it has many reported substrates with a role in cancer progression, including FOXO4, MDM2, N-Myc, and PTEN. The multi-substrate nature of USP7, combined with the modest potency and selectivity of early generation USP7 inhibitors, has presented a challenge in defining predictors of response to USP7 and potential patient populations that would benefit most from USP7-targeted drugs. Here, we describe the structure-guided development of XL177A, which irreversibly inhibits USP7 with sub-nM potency and selectivity across the human proteome. Evaluation of the cellular effects of XL177A reveals that selective USP7 inhibition suppresses cancer cell growth predominantly through a p53-dependent mechanism: XL177A specifically upregulates p53 transcriptional targets transcriptome-wide, hotspot mutations in TP53 but not any other genes predict response to XL177A across a panel of ~500 cancer cell lines, and TP53 knockout rescues XL177A-mediated growth suppression of TP53 wild-type (WT) cells. Together, these findings suggest TP53 mutational status as a biomarker for response to USP7 inhibition. We find that Ewing sarcoma and malignant rhabdoid tumor (MRT), two pediatric cancers that are sensitive to other p53-dependent cytotoxic drugs, also display increased sensitivity to XL177A.


Assuntos
Inibidores de Proteases/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Peptidase 7 Específica de Ubiquitina/antagonistas & inibidores , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Células MCF-7 , Inibidores de Proteases/química , Peptidase 7 Específica de Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacos
19.
Biochim Biophys Acta ; 1784(3): 530-42, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18230369

RESUMO

Human monoclonal antibodies derived from B cells of HCV-infected individuals provide information on the immune response to native HCV envelope proteins as they are recognized during infection. Monoclonal antibodies have been useful in the determination of the function and structure of specific immunogenic domains of proteins and should also be useful for the structure/function characterization of HCV E1 and E2 envelope glycoproteins. The HCV E2 envelope glycoprotein has at least three immunodistinctive conformation domains, designated A, B, and C. Conformational epitopes within domain B and C are neutralizing antibody targets on HCV pseudoparticles as well as from infectious cell culture virus. In this study, a combination of differential surface modification and mass spectrometric limited proteolysis followed by alanine mutagenesis was used to provide insight into potential conformational changes within the E2 protein upon antibody binding. The arginine guanidine groups in the E2 protein were modified with CHD in both the affinity bound and free states followed by mass spectrometric analysis, and the regions showing protection upon antibody binding were identified. This protection can arise by direct contact between the residues and the monoclonal antibody, or by antibody-induced conformational changes. Based on the mass spectrometric data, site-directed mutagenesis experiments were performed which clearly identified additional amino acid residues on E2 distant from the site of antibody interaction, whose change to alanine inhibited antibody recognition by inducing conformational changes within the E2 protein.


Assuntos
Complexo Antígeno-Anticorpo/química , Reações Antígeno-Anticorpo , Epitopos Imunodominantes/química , Proteínas do Envelope Viral/química , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Complexo Antígeno-Anticorpo/imunologia , Arginina/química , Arginina/genética , Mapeamento de Epitopos , Anticorpos Anti-Hepatite C/imunologia , Humanos , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia
20.
Nat Commun ; 10(1): 5481, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31792290

RESUMO

Integrin αVß8, which like αVß6 functions to activate TGF-ßs, is atypical. Its ß8 subunit binds to a distinctive cytoskeleton adaptor and does not exhibit large changes in conformation upon binding to ligand. Here, crystal structures, hydrogen-deuterium exchange dynamics, and affinity measurements on mutants are used to compare αVß8 and αVß6. Lack of a binding site for one of three ßI domain divalent cations and a unique ß6-α7 loop conformation in ß8 facilitate movements of the α1 and α1' helices at the ligand binding pocket toward the high affinity state, without coupling to ß6-α7 loop reshaping and α7-helix pistoning that drive large changes in ßI domain-hybrid domain orientation seen in other integrins. Reciprocal swaps between ß6 and ß8 ßI domains increase affinity of αVß6 and decrease affinity of αVß8 and define features that regulate affinity of the ßI domain and its coupling to the hybrid domain.


Assuntos
Integrinas/química , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Cadeias beta de Integrinas/química , Cadeias beta de Integrinas/genética , Cadeias beta de Integrinas/metabolismo , Integrinas/genética , Integrinas/metabolismo , Ligantes , Domínios Proteicos , Estrutura Secundária de Proteína , Alinhamento de Sequência
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