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 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 (Kd = 1.78 +/- 0.5 microM) similar to that observed for the intact capsid protein (Kd = 2.94 +/- 0.8 microM), suggesting that Gag self-association is not significantly influence by the P2 domain.
Newman
John L
JL
Howard Hughes Medical Institute and Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21250, USA.
Butcher
Eric W
EW
Patel
Dipti T
DT
Mikhaylenko
Yelena
Y
Summers
Michael F
MF
D036022:Capsid Proteins / Q000737:chemistry; D002499:Centrifugation, Density Gradient; D015683:Gene Products, gag / Q000737:chemistry*; D015497:HIV-1 / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D018583:Pliability; D017434:Protein Structure, Tertiary
D036022:Capsid Proteins; D015683:Gene Products, gag