Quantitative Characterization of Configurational Space Sampled by HIV‐1 Nucleocapsid Using Solution NMR, X‐ray Scattering and Protein Engineering. Issue 11 (16th March 2016)

• Record Type: Journal Article
• Authors: Deshmukh, Lalit; Schwieters, Charles D.; Grishaev, Alexander; Clore, G. Marius
• Is Part Of: Chemphyschem. Volume 17:Issue 11(2016)
• Page Start: 1548
• Page End: 1552
• Publication Date: 2016-03-16
• Subjects: HIV-1 -- nuclear magnetic resonance -- nucleocapsid -- protein engineering -- X-ray scattering; Chemistry, Physical and theoretical -- Periodicals; 541.05
• DOI: 10.1002/cphc.201600212
• Languages: English
• ISSNs: 1439-4235

Abstract

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. Abstract : Unliganded HIV‐1 nucleocapsid is investigated using solution NMR–residual dipolar couplings, small‐angle X‐ray scattering, and protein engineering, coupled with ensemble‐simulated annealing. The configurational space sampled by the two zinc knuckles can be described by three clusters, two major (40–50 % each) and one minor (≈10 %), while the termini are intrinsically disordered. One of the major clusters is close to the conformation found in nucleocapsid–nucleic acid complexes.