Solid-State NMR Spectroscopy of the HIV gp41 Membrane Fusion Protein Supports Intermolecular Antiparallel 尾 Sheet Fusion Peptide Structure in the Final Six-Helix Bundle State

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• 作者：Kelly Sackett ; Matthew J. Nethercott ; Zhaoxiong Zheng ; David P. Weliky
• 关键词：BCA ; bicinchoninic acid ; CP ; cross-polarization ; DTPC ; 1 ; 2-di-O-tetradecyl-sn-glycero-3-phosphocholine ; DTPG ; 1 ; 2-di-O-tetradecyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt ; FP ; fusion peptide ; fpCTDQBU ; finite-pulse constant-time double-quantum build-up ; MAS ; magic angle spinning ; PHI ; pre-hairpin intermediate ; REDOR ; rotational-echo double-resonance ; SHB ; six-helix bundle ; SSNMR ; solid-state NMR
• 刊名：Journal of Molecular Biology
• 出版年：6 March, 2014
• 年：2014
• 卷：426
• 期：5
• 页码：1077-1094
• 全文大小：2784 K

文摘

The HIV gp41 protein catalyzes fusion between viral and target cell membranes. Although the ~ 20-residue N-terminal fusion peptide (FP) region is critical for fusion, the structure of this region is not well characterized in large gp41 constructs that model the gp41 state at different times during fusion. This paper describes solid-state NMR (SSNMR) studies of FP structure in a membrane-associated construct (FP-Hairpin), which likely models the final fusion state thought to be thermostable trimers with six-helix bundle structure in the region C-terminal of the FP. The SSNMR data show that there are populations of FP-Hairpin with either 伪 helical or 尾 sheet FP conformation. For the 尾 sheet population, measurements of intermolecular p>13p>C-p>13p>C proximities in the FP are consistent with a significant fraction of intermolecular antiparallel 尾 sheet FP structure with adjacent strand crossing near L7 and F8. There appears to be negligible in-register parallel structure. These findings support assembly of membrane-associated gp41 trimers through interleaving of N-terminal FPs from different trimers. Similar SSNMR data are obtained for FP-Hairpin and a construct containing the 70 N-terminal residues of gp41 (N70), which is a model for part of the putative pre-hairpin intermediate state of gp41. FP assembly may therefore occur at an early fusion stage. On a more fundamental level, similar SSNMR data are obtained for FP-Hairpin and a construct containing the 34 N-terminal gp41 residues (FP34) and support the hypothesis that the FP is an autonomous folding domain.