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

Abstract

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 ␣-helical conformation. The presence of a transient coil-tohelix 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 ‫ס‬ 1.78 ± 0.5 M) similar to that observed for the intact capsid protein (K d ‫ס‬ 2.94 ± 0.8 M), suggesting that Gag self-association is not significantly influence by the P2 domain.Keywords: NMR; protein structure and dynamics; HIV-1; p2The genome of the human immunodeficiency virus type-1 (HIV-1) encodes an ∼55-kDa polyprotein, called Gag, that is itself sufficient for assembly of virus-like particles (Gheysen et al. 1989;Wills and Craven 1991). Several thousand copies of Gag self-associate at lipid rafts on the plasma membrane and bud to form an immature virion. Subsequent to budding, Gag is proteolytically cleaved by the viral protease into the following mature proteins and peptide fragments (listed from N terminus to C terminus): matrix (MA), capsid (CA), p2, nucleocapsid (NC), p1, and p6 (Fig. 1A). Cleavage leads to a dramatic morphological change, termed maturation (Vogt 1996), in which the mature CA proteins assemble into the conical core particle that encapsidates two copies of the viral genome, the NC proteins coat the viral genome, and the MA proteins remain associated with the viral envelope (Coffin et al. 1997).Although the structures and functions of the mature, functional MA, CA, and NC proteins have been well characterized (Turner and Summers Abbreviations: HIV-1, human immunodeficiency virus type-1; CA CTD , 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; K d , 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; R 1 (T 1 ), longitudinal relaxation rate (time); R 2 (T 2 ), transversal relaxation (time); XNOE, heteronuclear 15 N{ 1 H} nuclear Overhauser effect; TFE, 2,2,2-trifluoroethanol; CSI, chemical shift index.Article published online ahead of...

The structural biology of HIV assembly

“…In the tomographic reconstruction of frozen-hydrated immature particles, density ascribed to the CA CTD -SP1 region was located immediately beneath the central pore of each hexamer (i.e., the bottom of the cup), suggesting that this region stabilizes the hexamer. NMR studies of isolated Gag fragments indicate that the helical propensity for the CA-SP1 junction is weak [74], but sequence analyses and mutational studies support the idea that this region may adopt an ordered helical structure in assembled virions [73,75,76]. The CA-SP1 junction was therefore modelled as a six-helix bundle in the tomographic study ( Fig.…”

Mutation of the SP1 Sequence Impairs both Multimerization and Membrane-Binding Activities of Human Immunodeficiency Virus Type 1 Gag

“…ROLE OF SP1 IN Gag ASSEMBLY 1809 the structural flexibility in the context of the CTD of CA and NC (38,51). Supporting the importance of this ␣-helix in Gag assembly, insertion of either a glycine or a proline residue into this region profoundly inhibits virus production (1).…”

“…On the other hand, the last 11 amino acids of CA together with the downstream SP1 sequence were observed as a disordered region in the crystal structure of CA 146-231 -SP1 as well as in the NMR structure of CA (CTD)-SP1-NC (38,51). One interpretation of this finding is that SP1 has a high structural flexibility, and this feature may be required by Gag in order to construct a particle for the following reasons.…”

On the Role of the SP1 Domain in HIV-1 Particle Assembly: a Molecular Switch?

“…Protein structure prediction programs suggest that the last 7 residues of the CA domain and the first 7 residues of SP1 might form an ␣-helix (1). In fact, NMR data show that the SP1 domain exhibits only a slight propensity for helix formation (52), but this tendency is dramatically enhanced in 30% trifluoroethanol (TFE), a helix-promoting solvent (50).…”