Journal
PROTEIN SCIENCE
Volume 13, Issue 8, Pages 2101-2107Publisher
WILEY
DOI: 10.1110/ps.04614804
Keywords
NMR; protein structure and dynamics; HIV-1; p2
Categories
Funding
- NIGMS NIH HHS [GM42561, R01 GM042561] Funding Source: Medline
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The HIV-1 Gag polyprotein contains a segment called p2, located between the capsid (CA) and nucleocapsid (NC) domains, that is essential for ordered virus assembly and infectivity. We subcloned, overexpressed, and purified a 156-residue polypeptide that contains the C-terminal capsid subdomain (CA(CTD)) through the NC domain of Gag (CA(CTD)-p2-NC, Gag residues 276-431) for NMR relaxation and sedimentation equilibrium (SE) studies. The CA(CTD) and NC domains are folded as expected, but residues of the p2 segment, and the adjoining thirteen C-terminal residues of CA(CTD) and thirteen N-terminal residues of NC, are flexible. Backbone NMR chemical shifts of these 40 residues deviate slightly from random coil values and indicate a small propensity toward an alpha-helical conformation. The presence of a transient coil-to-helix equilibrium may explain the unusual and necessarily slow proteolysis rate of the CA-p2 junction. CA(CTD)-p2-NC forms dimers and self-associates with an equilibrium constant (K-d = 1.78 +/- 0.5 muM) similar to that observed for the intact capsid protein (K-d = 2.94 +/- 0.8 muM), suggesting that Gag self-association is not significantly influence by the P2 domain.
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