Flexibility in the P2 domain of the HIV‐1 Gag polyprotein

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 α‐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 μM) similar to that observed for the intact capsid protein (K_d = 2.94 ± 0.8 μM), suggesting that Gag self‐association is not significantly influence by the P2 domain.