The HIV envelope gp41 is a heavily glycosylated transmembrane protein. The difficulty in producing full length recombinant gp41 necessitates an incremental approach for structural determination studies. The ectodomain region located on the outer surface of the viral membrane directly mediates membrane fusion events via an N-terminal fusion peptide (FP). The structure of a truncated gp41 ectodomain (residues 27-154) lacking FP and the C-terminal membrane anchor is a rod-like trimer comprising three parallel N-terminal alpha-helices. These are assembled as a coiled-coil in the center with three antiparallel C-terminal alpha-helices packed on the outside with highly flexible loops connecting the inner and outer helices. This so-called 6-helical bundle (6HB) is the fusion active conformation. There have been no direct structural determinations on the prefusion conformation where the N- and C-terminal helical regions are extended (pre-hairpin intermediate). The blocking or inhibition of the transition from the pre-hairpin to 6H conformation is a major target for anti-HIV drug targeting. To understand in more detail the interactions between all gp41 domains, constructs were made which included the N-terminal FP and the membrane anchor. This complex membrane protein (HIV-1 gp41 residues 1-194) was expressed in bacteria and purified as a protein-detergent complex. High resolution nuclear magnetic resolution (NMR) techniques are being used to determine the structure and conformational flexibility of gp41. This is a major undertaking given the high molecular mass of the protein - detergent complex (over 100 KDa). Detailed NMR measurements reveal a high degree of intrinsic mobility for the homotrimeric HIV-1 gp41. The fusion peptide exhibits high amplitude motion on the fast nanosecond time scale. The linker between the N and C-terminal helical regions shows both fast and slow (microsecond) motions. The findings are compatible with the protein switching rapidly between the pre-hairpin intermediate, three-helical bundle state, and the late-fusion, anti-parallel 6HB. New studies used a gp41 construct (residues 27-194) in which the fusion domain was removed in order to improve protein expression and solubility. Using advanced NMR techniques, the C-terminal domain was found to be highly mobile and dynamic. Using proteases to assess protein accessibility, the C-terminal domain was far more labile than expected and was consistent with the NMR data. The results suggest that the N-terminal domain is tightly associated in a trimeric conformation similar or close to that in the 6HB, whereas the the C-terminal domain is transitioning between extended (pre-fusion) and closed (6HB fusion active) conformations. The importance of these studies on both the full length gp41 (1-194) and the shorter 27-194 construct is that they allow the first glance at potential prefusion structures of gp41. Structural studies by NMR are also being carried out to map interaction sites of various drug and antibodies with potential therapeutic value. With the current improvements made in the protein preparations, we are also reexamining conditions for protein crystallization. These structural studies will provide insight into the fusion mechanism and so provide direction for targeted anti-HIV intervention.
|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|