RESUMEN
Substrate binding to Hsp70 chaperones is involved in many biological processes, and the identification of potential substrates is important for a comprehensive understanding of these events. We present a multi-scale pipeline for an accurate, yet efficient prediction of peptides binding to the Hsp70 chaperone BiP by combining sequence-based prediction with molecular docking and MMPBSA calculations. First, we measured the binding of 15mer peptides from known substrate proteins of BiP by peptide array (PA) experiments and performed an accuracy assessment of the PA data by fluorescence anisotropy studies. Several sequence-based prediction models were fitted using this and other peptide binding data. A structure-based position-specific scoring matrix (SB-PSSM) derived solely from structural modeling data forms the core of all models. The matrix elements are based on a combination of binding energy estimations, molecular dynamics simulations, and analysis of the BiP binding site, which led to new insights into the peptide binding specificities of the chaperone. Using this SB-PSSM, peptide binders could be predicted with high selectivity even without training of the model on experimental data. Additional training further increased the prediction accuracies. Subsequent molecular docking (DynaDock) and MMGBSA/MMPBSA-based binding affinity estimations for predicted binders allowed the identification of the correct binding mode of the peptides as well as the calculation of nearly quantitative binding affinities. The general concept behind the developed multi-scale pipeline can readily be applied to other protein-peptide complexes with linearly bound peptides, for which sufficient experimental binding data for the training of classical sequence-based prediction models is not available. Proteins 2016; 84:1390-1407. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Proteínas Portadoras/química , Proteínas de Choque Térmico/química , Inmunoglobulina de Cadenas Ligeras Subrogadas/química , Péptidos/química , Factor A de Crecimiento Endotelial Vascular/química , Secuencia de Aminoácidos , Anisotropía , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Chaperón BiP del Retículo Endoplásmico , Colorantes Fluorescentes/química , Expresión Génica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Inmunoglobulina de Cadenas Ligeras Subrogadas/genética , Inmunoglobulina de Cadenas Ligeras Subrogadas/metabolismo , Isoquinolinas/química , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Péptidos/genética , Péptidos/metabolismo , Análisis por Matrices de Proteínas , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Espectrometría de Fluorescencia , Homología Estructural de Proteína , Relación Estructura-Actividad , Termodinámica , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The small heat shock protein αB-crystallin is an oligomeric molecular chaperone that binds aggregation-prone proteins. As a component of the proteostasis system, it is associated with cataract, neurodegenerative diseases, and myopathies. The structural determinants for the regulation of its chaperone function are still largely elusive. Combining different experimental approaches, we show that phosphorylation-induced destabilization of intersubunit interactions mediated by the N-terminal domain (NTD) results in the remodeling of the oligomer ensemble with an increase in smaller, activated species, predominantly 12-mers and 6-mers. Their 3D structures determined by cryo-electron microscopy and biochemical analyses reveal that the NTD in these species gains flexibility and solvent accessibility. These modulated properties are accompanied by an increase in chaperone activity in vivo and in vitro and a more efficient cooperation with the heat shock protein 70 system in client folding. Thus, the modulation of the structural flexibility of the NTD, as described here for phosphorylation, appears to regulate the chaperone activity of αB-crystallin rendering the NTD a conformational sensor for nonnative proteins.
Asunto(s)
Modelos Moleculares , Chaperonas Moleculares/química , Conformación Proteica , Cadena B de alfa-Cristalina/química , Cromatografía en Gel , Clonación Molecular , Microscopía por Crioelectrón , Electroforesis en Gel de Poliacrilamida , Proteínas HSP70 de Choque Térmico/metabolismo , Células HeLa , Humanos , Procesamiento de Imagen Asistido por Computador , Chaperonas Moleculares/metabolismo , Fosforilación , Colorantes de Rosanilina , Cadena B de alfa-Cristalina/metabolismoRESUMEN
Since the solution of the molecular structures of members of the voltage dependent anion channels (VDACs), the N-terminal α-helix has been the main focus of attention, since its strategic location, in combination with its putative conformational flexibility, could define or control the channel's gating characteristics. Through engineering of two double-cysteine mVDAC1 variants we achieved fixing of the N-terminal segment at the bottom and midpoint of the pore. Whilst cross-linking at the midpoint resulted in the channel remaining constitutively open, cross-linking at the base resulted in an "asymmetric" gating behavior, with closure only at one electric field's orientation depending on the channel's orientation in the lipid bilayer. Additionally, and while the native channel adopts several well-defined closed states (S1 and S2), the cross-linked variants showed upon closure a clear preference for the S2 state. With native-channel characteristics restored following reduction of the cysteines, it is evident that the conformational flexibility of the N-terminal segment plays indeed a major part in the control of the channel's gating behavior.