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2 results on '"Sanchaita Das"'

1. D-loop Dynamics and Near-Atomic-Resolution Cryo-EM Structure of Phalloidin-Bound F-Actin

Author

Sanchaita Das, Zeynep A. Oztug Durer, Emil Reisler, Z. Hong Zhou, Peng Ge, Elena E. Grintsevich, and Acibadem University Dspace

Subjects
Models, Molecular, Phalloidine, Cryo-electron microscopy, Phalloidin, 1.1 Normal biological development and functioning, Mutant, high-resolution cryo-EM, Biophysics, Saccharomyces cerevisiae, macromolecular substances, Article, Protein filament, 03 medical and health sciences, chemistry.chemical_compound, Models, Underpinning research, Structural Biology, Information and Computing Sciences, Microscopy, Deoxyribonuclease I, Disulfides, Molecular Biology, Actin, 030304 developmental biology, F-actin structure, 0303 health sciences, Cryoelectron Microscopy, 030302 biochemistry & molecular biology, Dynamics (mechanics), Resolution (electron density), Molecular, Biological Sciences, chemical crosslinking, Actins, Cross-Linking Reagents, chemistry, Mutation, Chemical Sciences, Generic health relevance, D-loop dynamics
Abstract

Detailed molecular information on G-actin assembly into filaments (F-actin), and their structure, dynamics, and interactions, is essential for understanding their cellular functions. Previous studies indicate that a flexible DNase I binding loop (D-loop, residues 40-50) plays a major role in actin's conformational dynamics. Phalloidin, a ``gold standard{''} for actin filament staining, stabilizes them and affects the D-loop. Using disulfide crosslinking in yeast actin D-loop mutant Q41C/V45C, light-scattering measurements, and cryoelectron microscopy reconstructions, we probed the constraints of D-loop dynamics and its contribution to F-actin formation/stability. Our data support a model of residues 41-45 distances that facilitate G- to F-actin transition. We report also a 3.3-angstrom resolution structure of phalloidin-bound F-actin in the ADP-Pi-like (ADP-BeFx) state. This shows the phalloidin-binding site on F-actin and how the relative movement between its two protofilaments is restricted by it. Together, our results provide molecular details of F-actin structure and D-loop dynamics.

Published
2020

2. Structural Organization and Dynamics of Homodimeric Cytohesin Family Arf GTPase Exchange Factors in Solution and on Membranes

Author

Osman Bilsel, Sanchaita Das, Wenhua Zhang, Sarah L. Maslen, Srinivas Chakravarthy, David G. Lambright, Mahel Zeghouf, Mark Skehel, Jacqueline Cherfils, Agata Nawrotek, Thomas C. Irving, Andrew W. Malaby, Laboratoire d'Enzymologie et Biochimie Structurales (LEBS), Centre National de la Recherche Scientifique (CNRS), Génome : structure, fonction, évolution (GSFE), Centre National de la Recherche Scientifique (CNRS)-Institut Curie-Université Paris-Sud - Paris 11 (UP11)-Institut National de la Recherche Agronomique (INRA), Laboratoire d'enzymologie et biochimie structurales (LEBS), Illinois Institute of Technology (IIT), and Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Paris-Sud - Paris 11 (UP11)-Institut National de la Recherche Agronomique (INRA)

Subjects
Models, Molecular, Phosphatidylinositol 4,5-Diphosphate, Protein Conformation, alpha-Helical, ADP ribosylation factor, [SDV]Life Sciences [q-bio], Amino Acid Motifs, Gene Expression, Receptors, Cytoplasmic and Nuclear, Guanosine triphosphate, Crystallography, X-Ray, Substrate Specificity, Mice, chemistry.chemical_compound, Structural Biology, Cloning, Molecular, GEF, 0303 health sciences, GTPase-Activating Proteins, 030302 biochemistry & molecular biology, SAXS, SEC, Recombinant Proteins, 3. Good health, Pleckstrin homology domain, Membrane, Guanosine Triphosphate, Guanine nucleotide exchange factor, Protein Binding, Endosome, Genetic Vectors, education, DLS, Allosteric regulation, NS, Guanosine Diphosphate, 03 medical and health sciences, Escherichia coli, HDX, Animals, Protein Interaction Domains and Motifs, structure, Molecular Biology, 030304 developmental biology, Binding Sites, MS, Protein Structure, Tertiary, Kinetics, chemistry, EM, Guanosine diphosphate, Liposomes, autoinhibition, Biophysics, Protein Conformation, beta-Strand, Protein Multimerization
Abstract

Summary Membrane dynamic processes require Arf GTPase activation by guanine nucleotide exchange factors (GEFs) with a Sec7 domain. Cytohesin family Arf GEFs function in signaling and cell migration through Arf GTPase activation on the plasma membrane and endosomes. In this study, the structural organization of two cytohesins (Grp1 and ARNO) was investigated in solution by size exclusion-small angle X-ray scattering and negative stain-electron microscopy and on membranes by dynamic light scattering, hydrogen-deuterium exchange-mass spectrometry and guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange assays. The results suggest that cytohesins form elongated dimers with a central coiled coil and membrane-binding pleckstrin-homology (PH) domains at opposite ends. The dimers display significant conformational heterogeneity, with a preference for compact to intermediate conformations. Phosphoinositide-dependent membrane recruitment is mediated by one PH domain at a time and alters the conformational dynamics to prime allosteric activation by Arf-GTP. A structural model for membrane targeting and allosteric activation of full-length cytohesin dimers is discussed.

Published
2019

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