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HIV-1 Nef protein: Purification, crystallizations, and preliminary X-ray diffraction studies

✍ Scribed by P. Franken; S. Arold; A. Padilla; E Hoh; M. P. Strub; M. Boyer; M. Jullien; C. Dumas; M. Bodeus; R. Benarous

Publisher
Cold Spring Harbor Laboratory Press
Year
2008
Tongue
English
Weight
304 KB
Volume
6
Category
Article
ISSN
0961-8368

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✦ Synopsis

Abstract

Human immunodeficiency virus Nef protein accelerates virulent progression of AIDS by its interaction with specific cellular proteins involved in cellular activation and signal transduction. Here we report the purification and crystallization of the conserved core of HTV‐1~LAI~ Nef protein in the unliganded form and in complex with the wild‐type SH3 domain of the p59^fyn^ protein‐tyrosine kinase. One‐dimensional NMR experiments show that full‐length protein and truncated fragment corresponding to the product of HIV‐1 protease cleavage have a well‐folded compact tertiary structure. The ligand‐free HIV‐1 Nef~core~ protein forms cubic crystals belonging to space group P23 with unit cell dimensions of a = b = c = 86.4 Å. The Nef‐Fyn SH3 cocrystals belong to the space group P6~1~22 or its enantiomorph, P6~5~22, with unit cell dimensions of a = b = 108.2 Å and c = 223.7 Å. Both crystal forms diffract to a resolution limit of 3.0 Å resolution using synchrotron radiation, and are thus suitable for X‐ray structure determination.

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