Fully hydrophobic HIV gp41 adopts a hemifusion-like conformation in phospholipid bilayers

Abstract

Edited by Karen G. Fleming The HIV envelope glycoprotein mediates virus entry into target cells by fusing the virus lipid envelope with the cell membrane. This process requires large-scale conformational changes of the fusion protein gp41. Current understanding of the mechanisms with which gp41 induces membrane merger is limited by the fact that the hydrophobic N-terminal fusion peptide (FP) and C-terminal transmembrane domain (TMD) of the protein are challenging to characterize structurally in the lipid bilayer. Here we have expressed a gp41 construct that contains both termini, including the FP, the fusion peptide-proximal region (FPPR), the membrane-proximal external region (MPER), and the TMD. These hydrophobic domains are linked together by a shortened water-soluble ectodomain. We reconstituted this "short NC" gp41 into a virus-mimetic lipid membrane and conducted solid-state NMR experiments to probe the membrane-bound conformation and topology of the protein. 13 C chemical shifts indicate that the C-terminal MPER-TMD is predominantly ␣-helical, whereas the N-terminal FP-FPPR exhibits ␤-sheet character. Water and lipid 1 H polarization transfer to the protein revealed that the TMD is well-inserted into the lipid bilayer, whereas the FPPR and MPER are exposed to the membrane surface. Importantly, correlation signals between the FP-FPPR and the MPER are observed, providing evidence that the ectodomain is sufficiently collapsed to bring the N-and C-terminal hydrophobic domains into close proximity. These results support a hemifusion-like model of the short NC gp41 in which the ectodomain forms a partially folded hairpin that places the FPPR and MPER on the opposing surfaces of two lipid membranes. HIV-1 enters sensitive cells using its envelope glycoprotein complex, gp160. The protein is biosynthesized as a homotrimer and is transported to the cell surface after proteolytic cleavage into two subunits, gp120 and gp41 (1, 2). Receptor binding and pH changes trigger a cascade of protein conformational

Cholesterol Interaction with the Trimeric HIV Fusion Protein gp41 in Lipid Bilayers Investigated by Solid-State NMR Spectroscopy and Molecular Dynamics Simulations

“…We hypothesize that the extended membrane-surface MPER helices, together with the trimeric TMD stalk, create a strongly asymmetric wedge shape that should incur local membrane curvature [18,87]. This local curvature and disorder prime the virus envelope to bend and merge with the opposing target cell membrane, which is bound by the N-terminal FP and fusion-peptide proximal region [88]. Second, cholesterol binding of gp41 may act as a mechanism for lateral partitioning of gp41 to the edge of the cholesterol-rich microdomain.…”

Virus Structures and Dynamics by Magic-Angle Spinning NMR

“…The Retroviridae HIV-1 is the causative agent of acquired immunodeficiency syndrome and the associated global pandemic that has claimed more than 35 million lives so far. HIV-1 protein assemblies have been studied by several MAS NMR research groups focusing on the Gag polyprotein and mature CA assemblies (14,27,35,40,(66)(67)(68)(69)(70)(71)(72)(73)(74)(75)(76)(77)(78)(79)(80), viral protein U (17,(81)(82)(83), and glycoprotein 41 (gp41) (26,(84)(85)(86).…”

Mechanistic insights of host cell fusion of SARS-CoV-1 and SARS-CoV-2 from atomic resolution structure and membrane dynamics

“…Biophysical methods, fluorescence resonance energy transfer (FRET) and optical spectroscopy (CD, FTIR) techniques are extensively used examining binding and perturbation of membranes of several important viral FPs [ [39] , [40] , [41] , [42] , [43] ]. Solution and solid-state NMR methods are playing vital roles in gaining atomistic information of FPs in complex with model membrane systems [ [44] , [45] , [46] , [47] , [48] , [49] , [50] , [51] , [52] , [53] , [54] ]. The primary structures of FPs contain a high proportion of hydrophobic amino acids and FPs of type I fusion proteins e.g.…”