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Published online before print July 6, 2004, 10.1110/ps.04614804
Protein Science (2004), 13:2101-2107. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Flexibility in the P2 domain of the HIV-1 Gag polyprotein

John L. Newman, Eric W. Butcher, Dipti T. Patel, Yelena Mikhaylenko and Michael F. Summers

Howard Hughes Medical Institute (HHMI) and Department of Chemistry and Biochemistry, University of Maryland Baltimore County (UMBC), Baltimore, Maryland 21250, USA

(RECEIVED January 7, 2004; FINAL REVISION April 27, 2004; ACCEPTED April 27, 2004)

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 (CACTD) through the NC domain of Gag (CACTD-p2-NC, Gag residues 276–431) for NMR relaxation and sedimentation equilibrium (SE) studies. The CACTD and NC domains are folded as expected, but residues of the p2 segment, and the adjoining thirteen C-terminal residues of CACTD 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. CACTD-p2-NC forms dimers and self-associates with an equilibrium constant (Kd = 1.78 ± 0.5 µM) similar to that observed for the intact capsid protein (Kd = 2.94 ± 0.8 µM), suggesting that Gag self-association is not significantly influence by the P2 domain.

Keywords: NMR; protein structure and dynamics; HIV-1; p2

Abbreviations: HIV-1, human immunodeficiency virus type-1 • CACTD, capsid C-terminal domain protein • CA p24, full-length capsid protein • p2, 14-residue protein within Gag • NC, nucleocapsid protein • MA, matrix protein • SE, sedimentation equilibrium • Kd, equilibrium dissociation constant • NMR, nuclear magnetic resonance • HSQC, heteronuclear single quantum correlation • HNCA, triple resonance experiment • HN(CO)CA, triple resonance experiment • 2D, two-dimensional • 3D, three-dimensional • NOESY, nuclear Overhauser effect spectroscopy • R1 (T1), longitudinal relaxation rate (time) • R2 (T2), transversal relaxation (time) • XNOE, heteronuclear 15N{1H} nuclear Overhauser effect • TFE, 2,2,2-trifluoroethanol • CSI, chemical shift index

Reprint requests to: Michael F. Summers, Howard Hughes Medical Institute and Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, MD 21250, USA; e-mail: summers{at}hhmi.umbc.edu; fax: (410) 455-1174.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04614804.


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