2nef

From Proteopedia

Revision as of 15:38, 15 February 2008

HIV-1 NEF (REGULATORY FACTOR), NMR, 40 STRUCTURES

Overview

The tendency of HIV-1 Nef to form aggregates in solution, particularly at, pH values below 8, together with its large fraction of highly mobile, residues seriously complicated determination of its three-dimensional, structure, both for heteronuclear solution NMR (Grzesiek et al., 1996a, Nat Struct Biol 3:340-345) and for X-ray crystallography (Lee et al., 1996, Cell 85:931-942). Methods used to determine the Nef structure by NMR, at pH 8 and 0.6 mM concentration are presented, together with a detailed, description of Nef's secondary and tertiary structure. The described, techniques have general applicability for the NMR structure determination, of proteins that are aggregating and/or have limited stability at low pH, values. Extensive chemical shift assignments are reported for backbone and, side chain 1H, 13C, and 15N resonances of the HIV-1 Nef deletion mutants, NEF delta 2-39, NEF delta 2-39, delta 159-173, and of NEF delta 2-39, delta 159-173 in complex with the SH3 domain of the Hck tyrosine protein, kinase. Besides a type II polyproline helix, Nef's structure consists of, three alpha-helices, a 3(10) helix, and a five-stranded anti-parallel, beta-sheet. The analysis of 15N relaxation parameters of the backbone, amide sites reveals that all the secondary structure elements are, non-mobile on the picosecond to nanosecond and on the millisecond time, scale. A large number of slowly exchanging amide protons provides evidence, for the stability of the Nef core even on the time scale of hours., Significant internal motions on the ps to ns time scale are detected for, residues 60 to 71 and for residues 149 to 180, which form solvent-exposed, loops. The residues of the HIV-1 protease cleavage site (W57/L58) do not, exhibit large amplitude motions on the sub-nanosecond time scale, and, their side chains insert themselves into a hydrophobic crevice formed, between the C-terminus of helix 1 and the N-terminus of helix 2. A refined, structure has been determined based on additional constraints for, side-chain and backbone dihedral angles derived from a large number of, three-bond J-coupling and ROE data.

About this Structure

2NEF is a Single protein structure of sequence from Human immunodeficiency virus type 1 (isolate bh10). This structure superseeds the now removed PDB entry 1NEF. Full crystallographic information is available from OCA.

Reference

Refined solution structure and backbone dynamics of HIV-1 Nef., Grzesiek S, Bax A, Hu JS, Kaufman J, Palmer I, Stahl SJ, Tjandra N, Wingfield PT, Protein Sci. 1997 Jun;6(6):1248-63. PMID:9194185

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