REFINED SOLUTION STRUCTURE AND BACKBONE DYNAMICS OF HIV-1 NEF

The tendency of HTV-1 Nef to form aggregates in solution, particularly at pH values below 8, together with its large fraction of highly mobi le residues seriously complicated determination of its three-dimension al structure, both for heteronuclear solution NMR (Grzesiek et al., 19 96a, Nat Struct Biol 3:340-345) and for X-ray crystallography (Lee et al., 1996, Cell 85:931-932). Methods used to determine the Nef structu re by NMR at pH 8 and 0.6 mM concentration are presented, together wit h a detailed description of Nef's secondary and tertiary structure. Th e described techniques have general applicability for the NMR structur e determination of proteins that are aggregating and/or have limited s tability at low pH values. Extensive chemical shift assignments are re ported for backbone and side chain H-1, C-13, and N-15 resonances of t he HIV-1 Nef deletion mutants NEFDelta Z2-39, NEFDelta 2-39,Delta 159- 173, and of NEFDelta 2-39,Delta 159-173 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 310 helix, and a f ive-stranded anti-parallel beta-sheet. The analysis of N-15 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 am ide protons provides evidence for the stability of the Nef core even o n the time scale of hours. Significant internal motions on the ps to u s time scale are detected for residues 60 to 71 and for residues 149 t o 180, which form solvent-exposed loops. The residues of the HIV-1 pro tease cleavage site (W57/L58) do not exhibit large amplitude motions o n the sub-nanosecond time scale, and their side chains insert themselv es into a hydrophobic crevice formed between the C-terminus of helix 1 and the N-terminus of helix 2. A refined structure has been determine d based on additional constraints for side-chain and backbone dihedral angles derived from a large number of three-bond J-couplings and ROE data.