RESUMO
Mutations in human isocitrate dehydrogenase 1 (IDH1) drive tumor formation in a variety of cancers by replacing its conventional activity with a neomorphic activity that generates an oncometabolite. Little is understood of the mechanistic differences among tumor-driving IDH1 mutants. We previously reported that the R132Q mutant unusually preserves conventional activity while catalyzing robust oncometabolite production, allowing an opportunity to compare these reaction mechanisms within a single active site. Here, we employ static and dynamic structural methods and observe that, compared to R132H, the R132Q active site adopts a conformation primed for catalysis with optimized substrate binding and hydride transfer to drive improved conventional and neomorphic activity over R132H. This active site remodeling reveals a possible mechanism of resistance to selective mutant IDH1 therapeutic inhibitors. This work enhances our understanding of fundamental IDH1 mechanisms while pinpointing regions for improving inhibitor selectivity.
Assuntos
Domínio Catalítico , Isocitrato Desidrogenase , Mutação , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Humanos , Cinética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores Enzimáticos/farmacologiaRESUMO
Mutations in human isocitrate dehydrogenase 1 (IDH1) drive tumor formation in a variety of cancers by replacing its conventional activity with a neomorphic activity that generates an oncometabolite. Little is understood of the mechanistic differences among tumor-driving IDH1 mutants. We previously reported that the R132Q mutant uniquely preserves conventional activity while catalyzing robust oncometabolite production, allowing an opportunity to compare these reaction mechanisms within a single active site. Here, we employed static and dynamic structural methods and found that, compared to R132H, the R132Q active site adopted a conformation primed for catalysis with optimized substrate binding and hydride transfer to drive improved conventional and neomorphic activity over R132H. This active site remodeling revealed a possible mechanism of resistance to selective mutant IDH1 therapeutic inhibitors. This work enhances our understanding of fundamental IDH1 mechanisms while pinpointing regions for improving inhibitor selectivity.
RESUMO
Mutations in human isocitrate dehydrogenase 1 (IDH1) drive tumor formation in a variety of cancers by replacing its conventional activity with a neomorphic activity that generates an oncometabolite. Little is understood of the mechanistic differences among tumor-driving IDH1 mutants. We previously reported that the R132Q mutant uniquely preserves conventional activity while catalyzing robust oncometabolite production, allowing an opportunity to compare these reaction mechanisms within a single active site. Here, we employed static and dynamic structural methods and found that, compared to R132H, the R132Q active site adopted a conformation primed for catalysis with optimized substrate binding and hydride transfer to drive improved conventional and neomorphic activity over R132H. This active site remodeling revealed a possible mechanism of resistance to selective mutant IDH1 therapeutic inhibitors. This work enhances our understanding of fundamental IDH1 mechanisms while pinpointing regions for improving inhibitor selectivity.
RESUMO
Proteins gain optimal fitness such as foldability and function through evolutionary selection. However, classical studies have found that evolutionarily designed protein sequences alone cannot guarantee foldability, or at least not without considering local contacts associated with the initial folding steps. We previously showed that foldability and function can be restored by removing frustration in the folding energy landscape of a model WW domain protein, CC16, which was designed based on Statistical Coupling Analysis (SCA). Substitutions ensuring the formation of five local contacts identified as "on-path" were selected using the closest homolog native folded sequence, N21. Surprisingly, the resulting sequence, CC16-N21, bound to Group I peptides, while N21 did not. Here, we identified single-point mutations that enable N21 to bind a Group I peptide ligand through structure and dynamic-based computational design. Comparison of the docked position of the CC16-N21/ligand complex with the N21 structure showed that residues at positions 9 and 19 are important for peptide binding, whereas the dynamic profiles identified position 10 as allosterically coupled to the binding site and exhibiting different dynamics between N21 and CC16-N21. We found that swapping these positions in N21 with matched residues from CC16-N21 recovers nature-like binding affinity to N21. This study validates the use of dynamic profiles as guiding principles for affecting the binding affinity of small proteins.
Assuntos
Mutação com Ganho de Função , Proteínas , Ligantes , Domínios WW , Sequência de Aminoácidos , Proteínas/química , Peptídeos/química , Dobramento de ProteínaRESUMO
PLEKHA7 (pleckstrin homology domain containing family A member 7) plays key roles in intracellular signaling, cytoskeletal organization, and cell adhesion, and is associated with multiple human cancers. The interactions of its pleckstrin homology (PH) domain with membrane phosphatidyl-inositol-phosphate (PIP) lipids are critical for proper cellular localization and function, but little is known about how PLEKHA7 and other PH domains interact with membrane-embedded PIPs. Here we describe the structural basis for recognition of membrane-bound PIPs by PLEHA7. Using X-ray crystallography, nuclear magnetic resonance, molecular dynamics simulations, and isothermal titration calorimetry, we show that the interaction of PLEKHA7 with PIPs is multivalent, distinct from a discrete one-to-one interaction, and induces PIP clustering. Our findings reveal a central role of the membrane assembly in mediating protein-PIP association and provide a roadmap for understanding how the PH domain contributes to the signaling, adhesion, and nanoclustering functions of PLEKHA7.
Assuntos
Proteínas de Transporte/química , Sítios de Ligação , Proteínas de Transporte/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Ligação ProteicaRESUMO
Many human diseases are the result of abnormal expression or activation of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Not surprisingly, more than 30 tyrosine kinase inhibitors (TKIs) are currently in clinical use and provide unique treatment options for many patients. PTPs on the other hand have long been regarded as "undruggable" and only recently have gained increased attention in drug discovery. Striatal-enriched tyrosine phosphatase (STEP) is a neuron-specific PTP that is overactive in Alzheimer's disease (AD) and other neurodegenerative and neuropsychiatric disorders, including Parkinson's disease, schizophrenia, and fragile X syndrome. An emergent model suggests that the increase in STEP activity interferes with synaptic function and contributes to the characteristic cognitive and behavioral deficits present in these diseases. Prior efforts to generate STEP inhibitors with properties that warrant clinical development have largely failed. To identify novel STEP inhibitor scaffolds, we developed a biophysical, label-free high-throughput screening (HTS) platform based on the protein thermal shift (PTS) technology. In contrast to conventional HTS using STEP enzymatic assays, we found the PTS platform highly robust and capable of identifying true hits with confirmed STEP inhibitory activity and selectivity. This new platform promises to greatly advance STEP drug discovery and should be applicable to other PTP targets.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Proteínas Tirosina Fosfatases não Receptoras/antagonistas & inibidores , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Humanos , Estrutura MolecularRESUMO
Earlier experiments suggest that the evolutionary information (conservation and coevolution) encoded in protein sequences is necessary and sufficient to specify the fold of a protein family. However, there is no computational work to quantify the effect of such evolutionary information on the folding process. Here we explore the role of early folding steps for sequences designed using coevolution and conservation through a combination of computational and experimental methods. We simulated a repertoire of native and designed WW domain sequences to analyze early local contact formation and found that the N-terminal ß-hairpin turn would not form correctly due to strong non-native local contacts in unfoldable sequences. Through a maximum likelihood approach, we identified five local contacts that play a critical role in folding, suggesting that a small subset of amino acid pairs can be used to solve the "needle in the haystack" problem to design foldable sequences. Thus, using the contact probability of those five local contacts that form during the early stage of folding, we built a classification model that predicts the foldability of a WW sequence with 81% accuracy. This classification model was used to redesign WW domain sequences that could not fold due to frustration and make them foldable by introducing a few mutations that led to the stabilization of these critical local contacts. The experimental analysis shows that a redesigned sequence folds and binds to polyproline peptides with a similar affinity as those observed for native WW domains. Overall, our analysis shows that evolutionary-designed sequences should not only satisfy the folding stability but also ensure a minimally frustrated folding landscape.
Assuntos
Dobramento de Proteína , Proteínas , Sequência de Aminoácidos , Funções Verossimilhança , Modelos Moleculares , Proteínas/genéticaRESUMO
Bcl-xL is a major inhibitor of apoptosis, a fundamental homeostatic process of programmed cell death that is highly conserved across evolution. Because it plays prominent roles in cancer, Bcl-xL is a major target for anticancer therapy and for studies aimed at understanding its structure and activity. Although Bcl-xL is active primarily at intracellular membranes, most studies have focused on soluble forms of the protein lacking both the membrane-anchoring C-terminal tail and the intrinsically disordered loop, and this has resulted in a fragmented view of the protein's biological activity. Here, we describe the conformation of full-length Bcl-xL. Using NMR spectroscopy, molecular dynamics simulations, and isothermal titration calorimetry, we show how the three structural elements affect the protein's structure, dynamics, and ligand-binding activity in both its soluble and membrane-anchored states. The combined data provide information about the molecular basis for the protein's functionality and a view of its complex molecular mechanisms.
Assuntos
Apoptose , Simulação de Dinâmica Molecular , Espectroscopia de Ressonância Magnética , Conformação Proteica , Proteína bcl-XRESUMO
The human IκB Kinase (IKK) is a multisubunit protein complex of two kinases and one scaffolding subunit that controls induction of transcription factor NF-κB activity. IKK behaves as an entity of aberrantly high apparent molecular weight in solution. Recent X-ray crystallographic and cryo-electron microscopy structures of individual catalytic subunits (IKK1/IKKα and IKK2/IKKß) reveal that they are both stably folded dimeric proteins that engage in extensive homo-oligomerization through unique surfaces that are required for activation of their respective catalytic activities. The NEMO/IKKγ subunit is a predominantly coiled coil protein that is required for activation of IKK through the canonical NF-κB signaling pathway. Here we report size-exclusion chromatography, multi-angle light scattering, analytical centrifugation, and thermal denaturation analyses of full-length human recombinant NEMO as well as deletion and disease-linked variants. We observe that NEMO is predominantly a dimer in solution, although by virtue of its modular coiled coil regions NEMO exhibits complicated solution dynamics involving portions that are mutually antagonistic toward homodimerization. This behavior causes NEMO to behave as a significantly larger sized particle in solution. Analyses of NEMO in complex with IKK2 indicate that NEMO preserves this structurally dynamic character within the multisubuit complex and provides the complex-bound IKK2 further propensity toward homo-oligomerization. These observations provide critical information on the structural plasticity of NEMO subunit dimers which helps clarify its role in diseases and in IKK regulation through oligomerization-dependent phosphorylation of catalytic IKK2 subunit dimers.
Assuntos
Quinase I-kappa B/química , Complexos Multiproteicos/química , Multimerização Proteica , Humanos , Hidrodinâmica , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Mutantes , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteínas Recombinantes , Soluções , Relação Estrutura-AtividadeRESUMO
Isocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP+-dependent conversion of isocitrate to α-ketoglutarate (αKG) to provide critical cytosolic substrates and drive NADPH-dependent reactions like lipid biosynthesis and glutathione regeneration. In biochemical studies, the forward reaction is studied at neutral pH, while the reverse reaction is typically characterized in more acidic buffers. This led us to question whether IDH1 catalysis is pH-regulated, which would have functional implications under conditions that alter cellular pH, like apoptosis, hypoxia, cancer, and neurodegenerative diseases. Here, we show evidence of catalytic regulation of IDH1 by pH, identifying a trend of increasing kcat values for αKG production upon increasing pH in the buffers we tested. To understand the molecular determinants of IDH1 pH sensitivity, we used the pHinder algorithm to identify buried ionizable residues predicted to have shifted pKa values. Such residues can serve as pH sensors, with changes in protonation states leading to conformational changes that regulate catalysis. We identified an acidic residue buried at the IDH1 dimer interface, D273, with a predicted pKa value upshifted into the physiological range. D273 point mutations had decreased catalytic efficiency and, importantly, loss of pH-regulated catalysis. Based on these findings, we conclude that IDH1 activity is regulated, at least in part, by pH. We show this regulation is mediated by at least one buried acidic residue â¼12 Å from the IDH1 active site. By establishing mechanisms of regulation of this well-conserved enzyme, we highlight catalytic features that may be susceptible to pH changes caused by cell stress and disease.
Assuntos
Glutaratos/metabolismo , Isocitrato Desidrogenase/metabolismo , Isocitratos/metabolismo , Mutação , Catálise , Domínio Catalítico , Glutaratos/química , Humanos , Concentração de Íons de Hidrogênio , Isocitrato Desidrogenase/química , Isocitrato Desidrogenase/genética , Isocitratos/química , Cinética , Conformação Proteica , Especificidade por SubstratoRESUMO
Point mutations in human isocitrate dehydrogenase 1 (IDH1) can drive malignancies, including lower-grade gliomas and secondary glioblastomas, chondrosarcomas, and acute myeloid leukemias. These mutations, which usually affect residue R132, ablate the normal activity of catalyzing the NADP+-dependent oxidation of isocitrate to α-ketoglutarate (αKG) while also acquiring a neomorphic activity of reducing αKG to d-2-hydroxyglutarate (D2HG). Mutant IDH1 can be selectively therapeutically targeted due to structural differences that occur in the wild type (WT) versus mutant form of the enzyme, though the full mechanisms of this selectivity are still under investigation. Here we probe the mechanistic features of the neomorphic activity and selective small molecule inhibition through a new lens, employing WaterMap and molecular dynamics simulations. These tools identified a high-energy path of water molecules connecting the inhibitor binding site with the αKG and NADP+ binding sites in mutant IDH1. This water path aligns spatially with the α10 helix from WT IDH1 crystal structures. Mutating residues at the termini of this water path specifically disrupted inhibitor binding and/or D2HG production, revealing additional key residues to consider in optimizing druglike molecules against mutant IDH1. Taken together, our findings from molecular simulations and mutant enzyme kinetic assays provide insight into how disrupting water paths through enzyme active sites can impact not only inhibitor potency but also substrate recognition and activity.
Assuntos
Isocitrato Desidrogenase/química , Isocitrato Desidrogenase/genética , Sítios de Ligação/genética , Fenômenos Biofísicos , Catálise , Domínio Catalítico/genética , Glutaratos/metabolismo , Humanos , Isocitrato Desidrogenase/antagonistas & inibidores , Isocitratos , Ácidos Cetoglutáricos/metabolismo , Cinética , Simulação de Dinâmica Molecular , Mutação/genética , Água/químicaRESUMO
The AAA+ ATPase p97 regulates ubiquitin-dependent protein homeostasis and has been pursued as a cancer drug target. The ATP-competitive inhibitor CB-5083 and allosteric inhibitor NMS-873 are the most advanced p97 inhibitors described to date. Previous studies have reported that their cytotoxicity can be readily overcome and involves single p97 mutations in the linker between the D1 and D2 ATPase domains and within D2. We report here that the proline 472 to leucine (P472L) mutation, in the D1-D2 linker and identified in CB-5083-resistant cells, desensitizes p97 to both inhibitor classes. This mutation does not disrupt the distinct D2-binding sites of the inhibitors. Instead, P472L changes ATPase domain communication within the p97 hexamer. P472L enhances cooperative D2 ATP binding and hydrolysis. This mechanism alters the function of the D1-D2 linker in the control of D2 activity involving the ATP-bound state of D1. Although increased D2 activity is sufficient to desensitize the P472L mutant to NMS-873, the mutant's desensitization to CB-5083 also requires D1 ATPase domain function. Our study highlights the remarkable adaptability of p97 ATPase domain communication that enables escape from mechanistically distinct classes of cytotoxic p97 inhibitors.
Assuntos
Adenosina Trifosfatases , Indóis/farmacologia , Mutação de Sentido Incorreto , Pirimidinas/farmacologia , Proteína com Valosina , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Substituição de Aminoácidos , Células HCT116 , Humanos , Domínios Proteicos , Proteína com Valosina/antagonistas & inibidores , Proteína com Valosina/genética , Proteína com Valosina/metabolismoRESUMO
Stromal interaction molecule 1 (STIM1) monitors ER-luminal Ca2+ levels to maintain cellular Ca2+ balance and to support Ca2+ signalling. The prevailing view has been that STIM1 senses reduced ER Ca2+ through dissociation of bound Ca2+ from a single EF-hand site, which triggers a dramatic loss of secondary structure and dimerization of the STIM1 luminal domain. Here we find that the STIM1 luminal domain has 5-6 Ca2+-binding sites, that binding at these sites is energetically coupled to binding at the EF-hand site, and that Ca2+ dissociation controls a switch to a second structured conformation of the luminal domain rather than protein unfolding. Importantly, the other luminal-domain Ca2+-binding sites interact with the EF-hand site to control physiological activation of STIM1 in cells. These findings fundamentally revise our understanding of physiological Ca2+ sensing by STIM1, and highlight molecular mechanisms that govern the Ca2+ threshold for activation and the steep Ca2+ concentration dependence.
Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Molécula 1 de Interação Estromal/química , Molécula 1 de Interação Estromal/metabolismo , Animais , Sítios de Ligação , Calorimetria , Cisteína/metabolismo , Medição da Troca de Deutério , Fluorescência , Células HeLa , Humanos , Camundongos , Mutação/genética , Domínios Proteicos , Estrutura Secundária de Proteína , Solubilidade , Relação Estrutura-AtividadeRESUMO
The spread of dengue (DENV) and Zika virus (ZIKV) is a major public health concern. The primary target of antibodies that neutralize DENV and ZIKV is the envelope (E) glycoprotein, and there is interest in using soluble recombinant E (sRecE) proteins as subunit vaccines. However, the most potent neutralizing antibodies against DENV and ZIKV recognize epitopes on the virion surface that span two or more E proteins. Therefore, to create effective DENV and ZIKV vaccines, presentation of these quaternary epitopes may be necessary. The sRecE proteins from DENV and ZIKV crystallize as native-like dimers, but studies in solution suggest that these dimers are marginally stable. To better understand the challenges associated with creating stable sRecE dimers, we characterized the thermostability of sRecE proteins from ZIKV and three DENV serotypes, DENV2-4. All four proteins irreversibly unfolded at moderate temperatures (46-53 °C). At 23 °C and low micromolar concentrations, DENV2 and ZIKV were primarily dimeric, and DENV3-4 were primarily monomeric, whereas at 37 °C, all four proteins were predominantly monomeric. We further show that the dissociation constant for DENV2 dimerization is very temperature-sensitive, ranging from <1 µm at 25 °C to 50 µm at 41 °C, due to a large exothermic enthalpy of binding of -79 kcal/mol. We also found that quaternary epitope antibody binding to DENV2-4 and ZIKV sRecE is reduced at 37 °C. Our observation of reduced sRecE dimerization at physiological temperature highlights the need for stabilizing the dimer as part of its development as a subunit vaccine.
Assuntos
Vírus da Dengue/química , Multimerização Proteica , Proteínas do Envelope Viral/química , Zika virus/química , Temperatura Corporal , Dengue/virologia , Humanos , Estabilidade Proteica , Proteínas Recombinantes/química , Vacinas de Subunidades Antigênicas/química , Vacinas Virais/química , Infecção por Zika virus/virologiaRESUMO
Obesity-associated insulin resistance plays a central role in type 2 diabetes. As such, tyrosine phosphatases that dephosphorylate the insulin receptor (IR) are potential therapeutic targets. The low-molecular-weight protein tyrosine phosphatase (LMPTP) is a proposed IR phosphatase, yet its role in insulin signaling in vivo has not been defined. Here we show that global and liver-specific LMPTP deletion protects mice from high-fat diet-induced diabetes without affecting body weight. To examine the role of the catalytic activity of LMPTP, we developed a small-molecule inhibitor with a novel uncompetitive mechanism, a unique binding site at the opening of the catalytic pocket, and an exquisite selectivity over other phosphatases. This inhibitor is orally bioavailable, and it increases liver IR phosphorylation in vivo and reverses high-fat diet-induced diabetes. Our findings suggest that LMPTP is a key promoter of insulin resistance and that LMPTP inhibitors would be beneficial for treating type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/genética , Bibliotecas de Moléculas Pequenas , Animais , Sítios de Ligação , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/genética , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Deleção de Genes , Concentração Inibidora 50 , Camundongos , Camundongos Knockout , Camundongos Obesos , Modelos Biológicos , Estrutura Molecular , Peso Molecular , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-AtividadeRESUMO
The outer membrane protein Ail (Adhesion invasion locus) is one of the most abundant proteins on the cell surface of Yersinia pestis during human infection. Its functions are expressed through interactions with a variety of human host proteins, and are essential for microbial virulence. Structures of Ail have been determined by X-ray diffraction and solution NMR spectroscopy, but those samples contained detergents that interfere with functionality, thus, precluding analysis of the structural basis for Ail's biological activity. Here, we demonstrate that high-resolution solid-state NMR spectra can be obtained from samples of Ail in detergent-free phospholipid liposomes, prepared with a lipid to protein molar ratio of 100. The spectra, obtained with 13C or 1H detection, have very narrow line widths (0.40-0.60 ppm for 13C, 0.11-0.15 ppm for 1H, and 0.46-0.64 ppm for 15N) that are consistent with a high level of sample homogeneity. The spectra enable resonance assignments to be obtained for N, CO, CA and CB atomic sites from 75 out of 156 residues in the sequence of Ail, including 80% of the transmembrane region. The 1H-detected solid-state NMR 1H/15N correlation spectra obtained for Ail in liposomes compare very favorably with the solution NMR 1H/15N TROSY spectra obtained for Ail in nanodiscs prepared with a similar lipid to protein molar ratio. These results set the stage for studies of the molecular basis of the functional interactions of Ail with its protein partners from human host cells, as well as the development of drugs targeting Ail.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Espectroscopia de Ressonância Magnética , Lipídeos de Membrana/química , Fatores de Virulência/química , Varredura Diferencial de Calorimetria , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Espectroscopia de Ressonância Magnética/métodos , Ressonância Magnética Nuclear Biomolecular , Espectroscopia de Prótons por Ressonância Magnética , SoluçõesRESUMO
Ras-interacting protein 1 (Rasip1) is an endothelial-specific Rap1 and Ras effector, important for vascular development and angiogenesis. Here, we report the crystal structure of the Rasip1 RA domain (RRA) alone, revealing the basis of dimerization, and in complex with Rap1 at 2.8 Å resolution. In contrast to most RA domains, RRA formed a dimer that can bind two Rap1 (KD = 0.9 µM) or Ras (KD = 2.2 µM) molecules. We solved the Rap1-RRA complex and found that Rasip1 binds Rap1 in the Switch I region, and Rap1 binding induces few conformation changes to Rasip1 stabilizing a ß strand and an unstructured loop. Our data explain how Rasip1 can act as a Rap1 and Ras effector and show that Rasip1 defines a subgroup of dimeric RA domains that could mediate cooperative binding to membrane-associated Ras superfamily members.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo , Sítios de Ligação , Dimerização , Humanos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas rap1 de Ligação ao GTP/química , Proteínas ras/químicaRESUMO
Cyanovirin-N (CV-N) is an antiviral lectin with potent activity against enveloped viruses, including HIV. The mechanism of action involves high affinity binding to mannose-rich glycans that decorate the surface of enveloped viruses. In the case of HIV, antiviral activity of CV-N is postulated to require multivalent interactions with envelope protein gp120, achieved through a pseudo-repeat of sequence that adopts two near-identical glycan-binding sites, and possibly involves a 3D-domain-swapped dimeric form of CV-N. Here, we present a covalent dimer of CV-N that increases the number of active glycan-binding sites, and we characterize its ability to recognize four glycans in solution. A CV-N variant was designed in which two native repeats were separated by the "nested" covalent insertion of two additional repeats of CV-N, resulting in four possible glycan-binding sites. The resulting Nested CV-N folds into a wild-type-like structure as assessed by circular dichroism and NMR spectroscopy, and displays high thermal stability with a Tm of 59 °C, identical to WT. All four glycan-binding domains encompassed by the sequence are functional as demonstrated by isothermal titration calorimetry, which revealed two sets of binding events to dimannose with dissociation constants Kd of 25 µM and 900 µM, assigned to domains B and B' and domains A and A' respectively. Nested CV-N displays a slight increase in activity when compared to WT CV-N in both an anti-HIV cellular assay and a fusion assay. This construct conserves the original binding specifityies of domain A and B, thus indicating correct fold of the two CV-N repeats. Thus, rational design can be used to increase multivalency in antiviral lectins in a controlled manner.
Assuntos
Antivirais/farmacologia , Proteínas de Bactérias/farmacologia , Proteínas de Transporte/farmacologia , Proteínas Recombinantes/farmacologia , Antivirais/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Polissacarídeos/metabolismo , Ligação Proteica , Dobramento de Proteína , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , TemperaturaRESUMO
Mutations in the hinge region of cyanovirin-N (CVN) dictate its preferential oligomerization state. Constructs with the Pro51Gly mutation preferentially exist as monomers, whereas wild-type cyanovirin can form domain-swapped dimers under certain conditions. Because the hinge region is an integral part of the high-affinity binding site of CVN, we investigated whether this mutation affects the shape, flexibility, and binding affinity of domain B for dimannose. Our studies indicate that the capability of monomeric wild-type CVN to resist mechanical perturbations is enhanced when compared to that of constructs in which the hinge region is more flexible. Our computational results also show that enhanced flexibility leads to blocking of the binding site by allowing different rotational isomeric states of Asn53. Moreover, at higher temperatures, this observed flexibility leads to an interaction between Asn53 and Asn42, further hindering access to the binding site. On the basis of these results, we predicted that binding affinity for dimannose would be more favorable for cyanovirin constructs containing a wild-type hinge region, whereas affinity would be impaired in the case of mutants containing Pro51Gly. Experimental characterization by isothermal titration calorimetry of a set of cyanovirin mutants confirms this hypothesis. Those possessing the Pro51Gly mutation are consistently inferior binders.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Dissacarídeos/metabolismo , Lectinas de Ligação a Manose/química , Lectinas de Ligação a Manose/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Proteínas de Transporte/genética , Lectinas de Ligação a Manose/genética , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
BCL-XL is an anti-apoptotic BCL-2 family protein found both in the cytosol and bound to intracellular membranes. Structural studies of BCL-XL have advanced by deleting its hydrophobic C-terminus and adding detergents to enhance solubility. However, since the C-terminus is essential for function and detergents strongly affect structure and activity, the molecular mechanisms controlling intracellular localization and cytoprotective activity are incompletely understood. Here we describe the conformations and ligand binding activities of water-soluble and membrane-bound BCL-XL, with its complete C-terminus, in detergent-free environments. We show that the C-terminus interacts with a conserved surface groove in the water-soluble state of the protein and inserts across the phospholipid bilayer in the membrane-bound state. Contrary to current models, membrane binding does not induce a conformational change in the soluble domain and both states bind a known ligand with affinities that are modulated by the specific state of the protein.