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1.
Proc Natl Acad Sci U S A ; 118(17)2021 04 27.
Artículo en Inglés | MEDLINE | ID: mdl-33888588

RESUMEN

The Src-homology-2 domain-containing phosphatase SHP2 is a critical regulator of signal transduction, being implicated in cell growth and differentiation. Activating mutations cause developmental disorders and act as oncogenic drivers in hematologic cancers. SHP2 is activated by phosphopeptide binding to the N-SH2 domain, triggering the release of N-SH2 from the catalytic PTP domain. Based on early crystallographic data, it has been widely accepted that opening of the binding cleft of N-SH2 serves as the key "allosteric switch" driving SHP2 activation. To test the putative coupling between binding cleft opening and SHP2 activation as assumed by the allosteric switch model, we critically reviewed structural data of SHP2, and we used extensive molecular dynamics (MD) simulation and free energy calculations of isolated N-SH2 in solution, SHP2 in solution, and SHP2 in a crystal environment. Our results demonstrate that the binding cleft in N-SH2 is constitutively flexible and open in solution and that a closed cleft found in certain structures is a consequence of crystal contacts. The degree of opening of the binding cleft has only a negligible effect on the free energy of SHP2 activation. Instead, SHP2 activation is greatly favored by the opening of the central ß-sheet of N-SH2. We conclude that opening of the N-SH2 binding cleft is not the key allosteric switch triggering SHP2 activation.


Asunto(s)
Regulación Alostérica , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Dominio Catalítico , Simulación de Dinámica Molecular
2.
Hum Mutat ; 41(6): 1171-1182, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32112654

RESUMEN

Germline PTPN11 mutations cause Noonan syndrome (NS), the most common disorder among RASopathies. PTPN11 encodes SHP2, a protein tyrosine-phosphatase controlling signaling through the RAS-MAPK and PI3K-AKT pathways. Generally, NS-causing PTPN11 mutations are missense changes destabilizing the inactive conformation of the protein or enhancing its binding to signaling partners. Here, we report on two PTPN11 variants resulting in the deletion or duplication of one of three adjacent glutamine residues (Gln255 -to-Gln257 ). While p.(Gln257dup) caused a typical NS phenotype in carriers of a first family, p.(Gln257del) had incomplete penetrance in a second family. Missense mutations involving Gln256 had previously been reported in NS. This poly-glutamine stretch is located on helix B of the PTP domain, a region involved in stabilizing SHP2 in its autoinhibited state. Molecular dynamics simulations predicted that changes affecting this motif perturb the SHP2's catalytically inactive conformation and/or substrate recognition. Biochemical data showed that duplication and deletion of Gln257 variably enhance SHP2's catalytic activity, while missense changes involving Gln256 affect substrate specificity. Expression of mutants in HEK293T cells documented their activating role on MAPK signaling, uncoupling catalytic activity and modulation of intracellular signaling. These findings further document the relevance of helix B in the regulation of SHP2's function.


Asunto(s)
Síndrome de Noonan/genética , Péptidos/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Adolescente , Niño , Preescolar , Femenino , Glutamina/genética , Células HEK293 , Humanos , Lactante , Masculino , Persona de Mediana Edad , Dominios Proteicos , Transducción de Señal
3.
J Chem Inf Model ; 60(6): 3157-3171, 2020 06 22.
Artículo en Inglés | MEDLINE | ID: mdl-32395997

RESUMEN

SH2 domain-containing tyrosine phosphatase 2 (SHP2), encoded by PTPN11, plays a fundamental role in the modulation of several signaling pathways. Germline and somatic mutations in PTPN11 are associated with different rare diseases and hematologic malignancies, and recent studies have individuated SHP2 as a central node in oncogenesis and cancer drug resistance. The SHP2 structure includes two Src homology 2 domains (N-SH2 and C-SH2) followed by a catalytic protein tyrosine phosphatase (PTP) domain. Under basal conditions, the N-SH2 domain blocks the active site, inhibiting phosphatase activity. Association of the N-SH2 domain with binding partners containing short amino acid motifs comprising a phosphotyrosine residue (pY) leads to N-SH2/PTP dissociation and SHP2 activation. Considering the relevance of SHP2 in signaling and disease and the central role of the N-SH2 domain in its allosteric regulation mechanism, we performed microsecond-long molecular dynamics (MD) simulations of the N-SH2 domain complexed to 12 different peptides to define the structural and dynamical features determining the binding affinity and specificity of the domain. Phosphopeptide residues at position -2 to +5, with respect to pY, have significant interactions with the SH2 domain. In addition to the strong interaction of the pY residue with its conserved binding pocket, the complex is stabilized hydrophobically by insertion of residues +1, +3, and +5 in an apolar groove of the domain and interaction of residue -2 with both the pY and a protein surface residue. Additional interactions are provided by hydrogen bonds formed by the backbone of residues -1, +1, +2, and +4. Finally, negatively charged residues at positions +2 and +4 are involved in electrostatic interactions with two lysines (Lys89 and Lys91) specific for the SHP2 N-SH2 domain. Interestingly, the MD simulations illustrated a previously undescribed conformational flexibility of the domain, involving the core ß sheet and the loop that closes the pY binding pocket.


Asunto(s)
Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Dominios Homologos src , Humanos , Simulación de Dinámica Molecular , Fosfopéptidos/metabolismo , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Transducción de Señal
4.
Hum Mutat ; 39(7): 959-964, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29737001

RESUMEN

Primrose syndrome (PS) is a rare disorder characterized by macrocephaly, tall stature, intellectual disability, autistic traits, and disturbances of glucose metabolism with insulin-resistant diabetes and distal muscle wasting occurring in adulthood. The disorder is caused by functional dysregulation of ZBTB20, a transcriptional repressor controlling energetic metabolism and developmental programs. ZBTB20 maps in a genomic region that is deleted in the 3q13.31 microdeletion syndrome, which explains the clinical overlap between the two disorders. A narrow spectrum of amino acid substitutions in a restricted region of ZBTB20 encompassing the first and second zinc-finger motifs have been reported thus far. Here, we characterize clinically and functionally the first truncating mutation [(c.1024delC; p.(Gln342Serfs*42)] and a missense change affecting the third zinc-finger motif of the protein [(c.1931C > T; p.(Thr644Ile)]. Our data document that both mutations have dominant negative impact on wild-type ZBTB20, providing further evidence of the specific behavior of PS-causing mutations on ZBTB20 function.


Asunto(s)
Anomalías Múltiples/genética , Calcinosis/genética , Enfermedades del Oído/genética , Predisposición Genética a la Enfermedad , Discapacidad Intelectual/genética , Atrofia Muscular/genética , Proteínas del Tejido Nervioso/genética , Factores de Transcripción/genética , Anomalías Múltiples/fisiopatología , Calcinosis/fisiopatología , Niño , Preescolar , Deleción Cromosómica , Cromosomas Humanos Par 3/genética , Hibridación Genómica Comparativa , Enfermedades del Oído/fisiopatología , Femenino , Humanos , Discapacidad Intelectual/fisiopatología , Masculino , Atrofia Muscular/fisiopatología , Mutación Missense/genética , Dedos de Zinc/genética
5.
Am J Hum Genet ; 96(5): 816-25, 2015 May 07.
Artículo en Inglés | MEDLINE | ID: mdl-25865493

RESUMEN

Transcription factors operate in developmental processes to mediate inductive events and cell competence, and perturbation of their function or regulation can dramatically affect morphogenesis, organogenesis, and growth. We report that a narrow spectrum of amino-acid substitutions within the transactivation domain of the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog (MAF), a leucine zipper-containing transcription factor of the AP1 superfamily, profoundly affect development. Seven different de novo missense mutations involving conserved residues of the four GSK3 phosphorylation motifs were identified in eight unrelated individuals. The distinctive clinical phenotype, for which we propose the eponym Aymé-Gripp syndrome, is not limited to lens and eye defects as previously reported for MAF/Maf loss of function but includes sensorineural deafness, intellectual disability, seizures, brachycephaly, distinctive flat facial appearance, skeletal anomalies, mammary gland hypoplasia, and reduced growth. Disease-causing mutations were demonstrated to impair proper MAF phosphorylation, ubiquitination and proteasomal degradation, perturbed gene expression in primary skin fibroblasts, and induced neurodevelopmental defects in an in vivo model. Our findings nosologically and clinically delineate a previously poorly understood recognizable multisystem disorder, provide evidence for MAF governing a wider range of developmental programs than previously appreciated, and describe a novel instance of protein dosage effect severely perturbing development.


Asunto(s)
Catarata/genética , Sordera/genética , Glucógeno Sintasa Quinasa 3/genética , Discapacidad Intelectual/genética , Proteínas Proto-Oncogénicas c-maf/genética , Catarata/patología , Síndrome de Down/genética , Síndrome de Down/patología , Humanos , Discapacidad Intelectual/patología , Mutación , Fenotipo , Fosforilación , Convulsiones/genética , Convulsiones/patología
6.
Hum Mutat ; 38(4): 451-459, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-28074573

RESUMEN

Germline mutations in PTPN11, the gene encoding the Src-homology 2 (SH2) domain-containing protein tyrosine phosphatase (SHP2), cause Noonan syndrome (NS), a relatively common, clinically variable, multisystem disorder. Here, we report on the identification of five different PTPN11 missense changes affecting residues Leu261 , Leu262 , and Arg265 in 16 unrelated individuals with clinical diagnosis of NS or with features suggestive for this disorder, specifying a novel disease-causing mutation cluster. Expression of the mutant proteins in HEK293T cells documented their activating role on MAPK signaling. Structural data predicted a gain-of-function role of substitutions at residues Leu262 and Arg265 exerted by disruption of the N-SH2/PTP autoinhibitory interaction. Molecular dynamics simulations suggested a more complex behavior for changes affecting Leu261 , with possible impact on SHP2's catalytic activity/selectivity and proper interaction of the PTP domain with the regulatory SH2 domains. Consistent with that, biochemical data indicated that substitutions at codons 262 and 265 increased the catalytic activity of the phosphatase, while those affecting codon 261 were only moderately activating but impacted substrate specificity. Remarkably, these mutations underlie a relatively mild form of NS characterized by low prevalence of cardiac defects, short stature, and cognitive and behavioral issues, as well as less evident typical facial features.


Asunto(s)
Predisposición Genética a la Enfermedad/genética , Mutación , Síndrome de Noonan/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas/genética , Modelos Moleculares , Mutación Missense , Síndrome de Noonan/patología , Unión Proteica , Dominios Proteicos , Proteína Tirosina Fosfatasa no Receptora Tipo 11/química , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Dominios Homologos src
7.
Biomacromolecules ; 15(8): 3083-92, 2014 Aug 11.
Artículo en Inglés | MEDLINE | ID: mdl-25029480

RESUMEN

Sulfated glycosaminoglycans (GAGs) can direct cellular processes by interacting with proteins of the extracellular matrix (ECM). In this study we characterize the interaction profiles of chemically sulfated hyaluronan (HA) and chondroitin sulfate (CS) derivatives with bone morphogenetic protein-2 (BMP-2) and investigate their relevance for complex formation with the receptor BMPR-IA. These goals were addressed by surface plasmon resonance (SPR) and ELISA in combination with molecular modeling and dynamics simulation. We found not only the interaction of BMP-2 with GAGs to be dependent on the type and sulfation of GAGs but also BMP-2/GAG/BMPR-IA complex formation. The conformational plasticity of the BMP-2 N-termini plays a key role in the structural and thermodynamic characteristics of the BMP-2/GAG/BMPR-IA system. Hence we propose a model that provides direct insights into the importance of the structural and dynamical properties of the BMP-2/BMPR-IA system for its regulation by sulfated GAGs, in which structural asymmetry plays a key role.


Asunto(s)
Proteína Morfogenética Ósea 2/química , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/química , Glicosaminoglicanos/química , Materiales Biocompatibles/química , Técnicas Biosensibles , Sulfatos de Condroitina/química , Biología Computacional , Matriz Extracelular/química , Humanos , Ácido Hialurónico/química , Conformación Proteica , Termodinámica , Ingeniería de Tejidos
8.
Comput Struct Biotechnol J ; 23: 1169-1180, 2024 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-38510972

RESUMEN

SHP2 is a tyrosine phosphatase that plays a regulatory role in multiple intracellular signaling cascades and is known to be oncogenic in certain contexts. In the absence of effectors, SHP2 adopts an autoinhibited conformation with its N-SH2 domain blocking the active site. Given the key role of N-SH2 in regulating SHP2, this domain has been extensively studied, often by X-ray crystallography. Using a combination of structural analyses and molecular dynamics (MD) simulations we show that the crystallographic environment can significantly influence the structure of the isolated N-SH2 domain, resulting in misleading interpretations. As an orthogonal method to X-ray crystallography, we use a combination of NMR spectroscopy and MD simulations to accurately determine the conformation of apo N-SH2 in solution. In contrast to earlier reports based on crystallographic data, our results indicate that apo N-SH2 in solution primarily adopts a conformation with a fully zipped central ß-sheet, and that partial unzipping of this ß-sheet is promoted by binding of either phosphopeptides or even phosphate/sulfate ions.

9.
Methods Mol Biol ; 2705: 59-75, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37668969

RESUMEN

Src-homology 2 (SH2) domains are protein interaction domains that bind to specific peptide motifs containing phosphotyrosine. SHP2, a tyrosine phosphatase encoded by PTPN11 gene, which has been emerged as positive or negative modulator in multiple signaling pathways, contains two SH2 domains, respectively, called N-SH2 and C-SH2. These domains play a relevant role in regulating SHP2 activity, either by recognizing its binding partners or by blocking its catalytic site. Considering the multiple functions that these domains carry out in SHP2, N-SH2 and C-SH2 represent an interesting case of study. In this chapter, we present a methodology that permits, by means of the principal component analysis (PCA), to study and to rationalize the structures adopted by the SH2 domains, in terms of the conformations of their binding sites. The structures can be distinguished, grouped, classified, and reported in a diagram. This approach permits to identify the accessible conformations of the SH2 domains in different binding conditions and to eventually reveal allosteric interactions. The method further reveals that the conformation dynamics of N-SH2 and C-SH2 strongly differ, which likely reflects their distinct functional roles.


Asunto(s)
Dominios Homologos src , Dominios y Motivos de Interacción de Proteínas , Sitios de Unión , Dominio Catalítico , Fosfotirosina
10.
Commun Biol ; 6(1): 1289, 2023 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-38129686

RESUMEN

SHP2 phosphatase plays an important role in regulating several intracellular signaling pathways. Pathogenic mutations of SHP2 cause developmental disorders and are linked to hematological malignancies and cancer. SHP2 comprises two tandemly-arranged SH2 domains, a catalytic PTP domain, and a disordered C-terminal tail. Under physiological, non-stimulating conditions, the catalytic site of PTP is occluded by the N-SH2 domain, so that the basal activity of SHP2 is low. Whereas the autoinhibited structure of SHP2 has been known for two decades, its active, open structure still represents a conundrum. Since the oncogenic mutant SHP2E76K almost completely populates the active, open state, this mutant has been extensively studied as a model for activated SHP2. By molecular dynamics simulations and accurate explicit-solvent SAXS curve predictions, we present the heterogeneous atomistic ensemble of constitutively active SHP2E76K in solution, encompassing a set of conformational arrangements and radii of gyration in agreement with experimental SAXS data.


Asunto(s)
Neoplasias , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Humanos , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Mutación
11.
Chem Sci ; 14(21): 5743-5755, 2023 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-37265738

RESUMEN

SHP2 plays an important role in regulating cellular processes, and its pathogenic mutations cause developmental disorders and are linked to cancer. SHP2 is a multidomain protein, comprising two SH2 domains arranged in tandem, a catalytic PTP domain, and a disordered C-terminal tail. SHP2 is activated upon binding two linked phosphopeptides to its SH2 domains, and the peptide orientation and spacing between binding sites are critical for enzymatic activation. For decades, the tandem SH2 has been extensively studied to identify the relative orientation of the two SH2 domains that most effectively binds effectors. So far, neither crystallography nor experiments in solution have provided conclusive results. Using experiment-guided molecular simulations, we determine the heterogeneous structural ensemble of the tandem SH2 in solution in agreement with experimental data from small-angle X-ray scattering and NMR residual dipolar couplings. In the solution ensemble, N-SH2 adopts different orientations and positions relative to C-SH2. We suggest that the intrinsic structural plasticity of the tandem SH2 allows SHP2 to respond to external stimuli and is essential for its functional activity.

12.
Biochim Biophys Acta ; 1808(9): 2102-10, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21658366

RESUMEN

Syringomycin E (SRE) is a member of a family of lipodepsipeptides that characterize the secondary metabolism of the plant-associated bacteria Pseudomonas syringae pv. syringae. It displays phytotoxic, antifungal and haemolytic activities, due to the membrane interaction and ion channel formation. To gain an insight into the conformation of SRE in the membrane environment, we studied the conformation of SRE bound to SDS micelle, a suitable model for the membrane-bound SRE. In fact, highly similar circular dichroism (CD) spectra were obtained for SRE bound to sodium dodecylsulphate (SDS) and to a phospholipid bilayer, indicating the conformational equivalence of SRE in these two media, at difference with the CD spectrum of SRE in water solution. The structure of SDS-bound SRE was determined by NMR spectroscopy combined with molecular dynamics calculations in octane environment. The results of this study highlight the influence of the interaction with lipids in determining the three-dimensional structure of SRE and provide the basis for further investigations on structural determinants of syringomycin E-membrane interaction.


Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Péptidos Cíclicos/química , Fosfolípidos/química , Dodecil Sulfato de Sodio/química , Antifúngicos/química , Dicroismo Circular , Hemólisis , Enlace de Hidrógeno , Canales Iónicos/química , Lípidos/química , Micelas , Simulación de Dinámica Molecular , Conformación Proteica , Rifampin/farmacología , Agua/química
13.
Comput Struct Biotechnol J ; 20: 989-1001, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35242289

RESUMEN

Protein intrinsically disordered regions (IDRs) play pivotal roles in molecular recognition and regulatory processes through structural disorder-to-order transitions. To understand and exploit the distinctive functional implications of IDRs and to unravel the underlying molecular mechanisms, structural disorder-to-function relationships need to be deciphered. The DNA site-specific recombinase system Cre/loxP represents an attractive model to investigate functional molecular mechanisms of IDRs. Cre contains a functionally dispensable disordered N-terminal tail, which becomes indispensable in the evolved Tre/loxLTR recombinase system. The difficulty to experimentally obtain structural information about this tail has so far precluded any mechanistic study on its involvement in DNA recombination. Here, we use in vitro and in silico evolution data, conformational dynamics, AI-based folding simulations, thermodynamic stability calculations, mutagenesis and DNA recombination assays to investigate how evolution and the dynamic behavior of this IDR may determine distinct functional properties. Our studies suggest that partial conformational order in the N-terminal tail of Tre recombinase and its packing to a conserved hydrophobic surface on the protein provide thermodynamic stability. Based on our results, we propose a link between protein stability and function, offering new plausible atom-detailed mechanistic insights into disorder-function relationships. Our work highlights the potential of N-terminal tails to be exploited for regulation of the activity of Cre-like tyrosine-type SSRs, which merits future investigations and could be of relevance in future rational engineering for their use in biotechnology and genomic medicine.

14.
ACS Chem Biol ; 17(8): 2099-2108, 2022 08 19.
Artículo en Inglés | MEDLINE | ID: mdl-35797699

RESUMEN

We produced a neuroglobin variant, namely, Ngb CDless, with the excised CDloop- and D-helix, directly joining the C- and E-helices. The CDless variant retained bis-His hexacoordination, and we investigated the role of the CDloop-D-helix unit in controlling the CO binding and structural dynamics by an integrative approach based on X-ray crystallography, rapid mixing, laser flash photolysis, resonance Raman spectroscopy, and molecular dynamics simulations. Rapid mixing and laser flash photolysis showed that ligand affinity was unchanged with respect to the wild-type protein, albeit with increased on and off constants for rate-limiting heme iron hexacoordination by the distal His64. Accordingly, resonance Raman spectroscopy highlighted a more open distal pocket in the CO complex that, in agreement with MD simulations, likely involves His64 swinging inward and outward of the distal heme pocket. Ngb CDless displays a more rigid overall structure with respect to the wild type, abolishing the structural dynamics of the CDloop-D-helix hypothesized to mediate its signaling role, and it retains ligand binding control by distal His64. In conclusion, this mutant may represent a tool to investigate the involvement of CDloop-D-helix in neuroprotective signaling in a cellular or animal model.


Asunto(s)
Neuroglobina/química , Animales , Hemo/química , Ligandos , Ratones , Neuroglobina/metabolismo
15.
Proteins ; 79(3): 867-79, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21287618

RESUMEN

By using multiple molecular dynamics (MD) trajectories, a quantitative description of carbon monoxide (CO) migration within crystal of L29F myoglobin mutant (L29F-Mb) was obtained. The aim was to provide a detailed model for ligand diffusion in the protein to be compared to the available L29F-Mb experimental-computational data and to the corresponding model kinetics we previously obtained for photolyzed CO within crystallized wild-type myoglobin (wt-Mb). Results suggest a clear migration pathway from distal pocket to the proximal site, similar to the one observed in wt-Mb, with a relaxation kinetics differing from the wt-Mb one essentially for the escape rate which is much higher in the mutant. Moreover MD data indicated a clear correlation between CO location within the protein and the conformation adopted by Phe29, well matching the available experimental data as obtained by time-resolved X-ray density maps. Such data, further validating the model used in the simulations, point out the subtle mutual effect between ligand diffusion and protein functional motions possibly explaining the observed dramatic variation of CO exit rate in L29F-Mb.


Asunto(s)
Mutación , Mioglobina/química , Cristalización , Cristalografía por Rayos X , Cinética , Ligandos , Modelos Moleculares , Simulación de Dinámica Molecular , Mioglobina/genética
16.
Proteins ; 78(2): 259-70, 2010 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-19714775

RESUMEN

Oxidative stress is a widespread challenge for living organisms, and especially so for parasitic ones, given the fact that their hosts can produce reactive oxygen species (ROS) as a mechanism of defense. Thus, long lived parasites, such as the flatworm Schistosomes, have evolved refined enzymatic systems capable of detoxifying ROS. Among these, glutathione peroxidases (Gpx) are a family of sulfur or selenium-dependent isozymes sharing the ability to reduce peroxides using the reducing equivalents provided by glutathione or possibly small proteins such as thioredoxin. As for other frontline antioxidant enzymatic systems, Gpxs are localized in the tegument of the Schistosomes, the outermost defense layer. In this article, we present the first crystal structure at 1.0 and 1.7 A resolution of two recombinant SmGpxs, carrying the active site mutations Sec43Cys and Sec43Ser, respectively. The structures confirm that this enzyme belongs to the monomeric class 4 (phospholipid hydroperoxide) Gpx. In the case of the Sec to Cys mutant, the catalytic Cys residue is oxidized to sulfonic acid. By combining static crystallography with molecular dynamics simulations, we obtained insight into the substrate binding sites and the conformational changes relevant to catalysis, proposing a role for the unusual reactivity of the catalytic residue.


Asunto(s)
Cristalografía por Rayos X , Glutatión Peroxidasa/química , Simulación de Dinámica Molecular , Schistosoma mansoni/enzimología , Esquistosomiasis mansoni/parasitología , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Humanos , Datos de Secuencia Molecular , Mutación Puntual , Unión Proteica , Conformación Proteica , Alineación de Secuencia
17.
J Am Chem Soc ; 132(42): 14901-9, 2010 Oct 27.
Artículo en Inglés | MEDLINE | ID: mdl-20919711

RESUMEN

The effect of structural disorder on the X-ray absorption near-edge structure (XANES) spectrum of a heme protein has been investigated using the dynamical description of the system derived from molecular dynamics (MD) simulations. The XANES spectra of neuroglobin (Ngb) and carbonmonoxy-neuroglobin (NgbCO) have been quantitatively reproduced, starting from the MD geometrical configurations, without carrying out any optimization in the structural parameter space. These results provide an important experimental validation of the reliability of the potentials used in the MD simulations and accordingly corroborate the consistency of the structural dynamic information on the metal center, related to its biological function. This analysis allowed us to demonstrate that the configurational disorder associated with the distortion of the heme plane and with the different orientations of the axial ligands can affect the XANES features at very low energy. Neglecting configurational disorder in the XANES quantitative analysis of heme proteins is a source of systematic errors in the determination of Fe coordination geometry. The combined use of XANES and MD is a novel strategy to enhance the resolution and reliability of the structural information obtained on metalloproteins, making the combination of these techniques powerful for metalloprotein investigations.


Asunto(s)
Metales/química , Simulación de Dinámica Molecular , Proteínas/química , Dominio Catalítico , Modelos Moleculares , Análisis Espectral/métodos , Rayos X
18.
Sci Rep ; 10(1): 18530, 2020 10 28.
Artículo en Inglés | MEDLINE | ID: mdl-33116231

RESUMEN

SHP2 is a protein tyrosine phosphatase (PTP) involved in multiple signaling pathways. Mutations of SHP2 can result in Noonan syndrome or pediatric malignancies. Inhibition of wild-type SHP2 represents a novel strategy against several cancers. SHP2 is activated by binding of a phosphopeptide to the N-SH2 domain of SHP2, thereby favoring dissociation of the N-SH2 domain and exposing the active site on the PTP domain. The conformational transitions controlling ligand affinity and PTP dissociation remain poorly understood. Using molecular simulations, we revealed an allosteric interaction restraining the N-SH2 domain into a SHP2-activating and a stabilizing state. Only ligands selecting for the activating N-SH2 conformation, depending on ligand sequence and binding mode, are effective activators. We validate the model of SHP2 activation by rationalizing modified basal activity and responsiveness to ligand stimulation of several N-SH2 variants. This study provides mechanistic insight into SHP2 activation and may open routes for SHP2 regulation.


Asunto(s)
Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/fisiología , Simulación por Computador , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Mutación/genética , Unión Proteica/genética , Conformación Proteica , Dominios Proteicos/genética , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas/fisiología , Transducción de Señal/genética , Tirosina/metabolismo , Dominios Homologos src/genética
19.
Sci Rep ; 9(1): 5326, 2019 03 29.
Artículo en Inglés | MEDLINE | ID: mdl-30926858

RESUMEN

Neuroglobin (Ngb) is predominantly expressed in neurons of the central and peripheral nervous systems and it clearly seems to be involved in neuroprotection. Engineering Ngb to observe structural and dynamic alterations associated with perturbation in ligand binding might reveal important structural determinants, and could shed light on key features related to its mechanism of action. Our results highlight the relevance of the CD loop and of Phe106 as distal and proximal controls involved in ligand binding in murine neuroglobin. We observed the effects of individual and combined mutations of the CD loop and Phe106 that conferred to Ngb higher CO binding velocities, which we correlate with the following structural observations: the mutant F106A shows, upon CO binding, a reduced heme sliding hindrance, with the heme present in a peculiar double conformation, whereas in the CD loop mutant "Gly-loop", the original network of interactions between the loop and the heme was abolished, enhancing binding via facilitated gating out of the distal His64. Finally, the double mutant, combining both mutations, showed a synergistic effect on CO binding rates. Resonance Raman spectroscopy and MD simulations support our findings on structural dynamics and heme interactions in wild type and mutated Ngbs.


Asunto(s)
Neuroglobina/química , Neuroglobina/metabolismo , Sitios de Unión , Monóxido de Carbono/química , Monóxido de Carbono/metabolismo , Hemo/química , Hemo/metabolismo , Humanos , Cinética , Ligandos , Modelos Moleculares , Conformación Molecular , Mutación , Neuroglobina/genética , Unión Proteica , Relación Estructura-Actividad , Temperatura
20.
Biophys J ; 94(11): 4277-81, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18310235

RESUMEN

By using multiple molecular dynamics trajectories of photolyzed carbon monoxide (CO) within crystallized myoglobin, a quantitative description of CO diffusion and corresponding kinetics was obtained. Molecular dynamics results allowed us to construct a detailed kinetic model of the migration process, shedding light on the kinetic mechanism and relevant steps of CO migration and remarkably-well reproducing the available experimental data as provided by time-resolved Laue x-ray diffraction.


Asunto(s)
Dióxido de Carbono/química , Cristalización , Hemoglobinas/química , Modelos Químicos , Modelos Moleculares , Mioglobina/química , Sitios de Unión , Simulación por Computador , Cinética , Ligandos , Movimiento (Física) , Unión Proteica , Conformación Proteica
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