Truncation of the gp41 construct just past its transmembrane domain, leaving residues 1-194, results in a well-structured protein, soluble in detergent micelles. Although previously believed to adopt its natural homo-trimeric form, we have found strong evidence for a rapid dynamic equilibrium between monomeric and trimeric forms of the protein, which can be shifted to the mostly trimeric form by choice of suitable detergents and detergent: protein stoichiometry. The NOE data and 15N relaxation rates show a well structured helical conformation for residues 5-14 of the fusion domain, followed by a more disordered segment which connects it to the ecto-domain. Remarkably, resonances from the C-terminal heptad repeat (CHR) and membrane proximal region MPER) are exchange broadened to such an extent that they do not give rise to observable NMR resonances. Together with exchange broadening observed in the highly mobile immuno-dominant loop region, this points to a possible equilibrium between early and late stage 3- and 6-helical states for the ecto domain. There is no evidence for direct interaction with the transmembrane fusion domain in studies carried out at pH4, and both light scattering, SAXS, and NMR diffusion data point to a mass ratio of detergent:protein slightly above 1.0. The protein is found to be quite stable, even at temperatures of 40C. The fusion domain exhibits effective correlation times that are much shorter than for the ecto domain, indicating that the intact fusion domain helices are highly mobile within the detergent micelle/protein aggregate, while retaining their helical conformation. This excludes the previously hypothesized interaction with the gp41 transmembrane domain under our conditions of pH and detergent.

Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
2012
Total Cost
$300,635
Indirect Cost
City
State
Country
Zip Code
Roche, Julien; Louis, John M; Aniana, Annie et al. (2015) Complete dissociation of the HIV-1 gp41 ectodomain and membrane proximal regions upon phospholipid binding. J Biomol NMR 61:235-48
Lakomek, Nils-Alexander; Kaufman, Joshua D; Stahl, Stephen J et al. (2013) Internal dynamics of the homotrimeric HIV-1 viral coat protein gp41 on multiple time scales. Angew Chem Int Ed Engl 52:3911-5
Lakomek, Nils-Alexander; Ying, Jinfa; Bax, Ad (2012) Measurement of ¹⁵N relaxation rates in perdeuterated proteins by TROSY-based methods. J Biomol NMR 53:209-21