title: Quantitative Characterization of Configurational Space Sampled by HIV-1 Nucleocapsid Using Solution NMR, X-ray Scattering and Protein Engineering.
abstract: Nucleic-acid-related events in the HIV-1 replication cycle are mediated by nucleocapsid, a small protein comprising two zinc knuckles connected by a short flexible linker and flanked by disordered termini. Combining experimental NMR residual dipolar couplings, solution X-ray scattering and protein engineering with ensemble simulated annealing, we obtain a quantitative description of the configurational space sampled by the two zinc knuckles, the linker and disordered termini in the absence of nucleic acids. We first compute the conformational ensemble (with an optimal size of three members) of an engineered nucleocapsid construct lacking the N- and C-termini that satisfies the experimental restraints, and then validate this ensemble, as well as characterize the disordered termini, using the experimental data from the full-length nucleocapsid construct. The experimental and computational strategy is generally applicable to multidomain proteins. Differential flexibility within the linker results in asymmetric motion of the zinc knuckles which may explain their functionally distinct roles despite high sequence identity. One of the configurations (populated at a level of ≈40 %) closely resembles that observed in various ligand-bound forms, providing evidence for conformational selection and a mechanistic link between protein dynamics and function.
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, 20892-0520, USA.
authors.forename: Lalit
authors.initials: L
authors.lastname: Deshmukh
authors.affiliation: Imaging Sciences Laboratory, Center for Information and Technology, Bethesda, MD, 20892-5624, USA.
authors.forename: Charles D
authors.initials: CD
authors.lastname: Schwieters
authors.affiliation: Institute of Bioscience and Biotechnology Research, University of Maryland, Rockville, MD, 20850, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Grishaev
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, 20892-0520, USA. mariusc@mail.nih.gov.
authors.forename: G Marius
authors.initials: GM
authors.lastname: Clore
mesh_terms: D015497:HIV-1 / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D019251:Nucleocapsid / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D053838:Scattering, Small Angle; D012996:Solutions; D014961:X-Ray Diffraction
chemical_list: D012996:Solutions
keywords: HIV-1; X-ray scattering; nuclear magnetic resonance; nucleocapsid; protein engineering