The human immunodeficiency virus (HIV) fusion peptide (HFP) is the N-terminal apolar region of the HIV gp41 fusion protein and interacts with target cell membranes and promotes membrane fusion. The free peptide catalyzes vesicle fusion at least to the lipid mixing stage and serves as a useful model fusion system. For gp41 constructs which lack the HFP, high-resolution structures show trimeric protein and suggest that at least three HFPs interact with the membrane with their C-termini in close proximity. In addition, previous studies have demonstrated that HFPs which are cross-linked at their C-termini to form trimers (HFPtr) catalyze fusion at a rate which is 15−40 times greater than noncross-linked HFP. In the present study, the structure of membrane-associated HFPtr was probed with solid-state nuclear magnetic resonance (NMR) methods. Chemical shift and intramolecular 13 CO-15 N distance measurements show that the conformation of the Leu-7 to Phe-11 region of HFPtr has predominant helical conformation in membranes without cholesterol and β strand conformation in membranes containing ∼30 mol% cholesterol. Interstrand 13 CO-13 CO and 13 CO-15 N distance measurements were not consistent with an in-register parallel strand arrangement but were consistent with either: (1) parallel arrangement with adjacent strands tworesidues out-of-register; or (2) antiparallel arrangement with adjacent strand crossing between Phe-8 and Leu-9. Arrangement (1) could support the rapid fusion rate of HFPtr because of placement of the apolar N-terminal regions of all strands on the same side of the oligomer while arrangement (2) could support the assembly of multiple fusion protein trimers. KeywordsHIV; fusion peptide; cholesterol; membranes; NMR; trimer Enveloped viruses such as human immunodeficiency virus (HIV) are surrounded by a membrane and infect cells through fusion between the viral membrane and the target cell membrane (1,2). This process is mediated by envelope proteins which traverse the viral membrane (3). For HIV, the gp41 envelope protein has a ∼170-residue ectodomain region † This work was supported by NIH award AI47153 to D. P. W. * To whom correspondence should be addressed. Telephone: 517−355−9715. Fax: 517−353−1793. Email: weliky@chemistry.msu.edu.. SUPPORTING INFORMATION AVAILABLE Equations are derived for determination of (ΔS/S 0 ) cor for REDOR and fpCTDQBU analyses. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 October 20. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript which lies outside the viral membrane and contains a N-terminal ∼20-residue apolar "fusion peptide" (HFP). The HFP interacts with the target cell membrane and plays an essential role in membrane fusion (4). Peptides composed of the HFP sequence induce fusion between large unilamellar vesicles (LUVs) and between erythrocytes (5,6). There are similar mutation/fusion activity relationships...
The conformational flexibility and programmed assembly of a dumbbell-shaped polyoxometalate-organic hybrid molecule comprising two Dawson-type polyoxometalates linked by a 2,2'-bipyridine unit, which can be coordinate to metal ions, in this case of Zn(2+), are described. SAXS, UV/vis, and NMR spectroscopic techniques confirm that the hybrid molecules exist as the trans dumbbell in metal-ion-free solutions and can be reversibly transformed into the cis dumbbell through coordination upon the addition of ZnCl2 into a DMSO solution containing the hybrid. Subsequent addition of EDTA reverses the switching process by extracting the Zn(2+) cations from the hybrid. During the interchange process between trans and cis dumbbells, a further reorganization of the hybrid molecules occurs through bond rotation to minimize steric clashes between the polyoxometalate subunits, in order to stabilize the corresponding dumbbell conformation. The Zn(2+)-controlled conformational transformation of the hybrid can be further utilized to manipulate the hybrid's solvophobic interaction-driven self-assembly behavior in the metal-ion driven reversible formation of 140 nm sized vesicles, studied by laser light scattering techniques.
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 α helical or β sheet FP conformation. For the β sheet population, measurements of intermolecular 13C-13C proximities in the FP are consistent with a significant fraction of intermolecular antiparallel β 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 inter-leaving 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.
Enveloped viruses such as HIV and influenza virus (IFV) are enclosed by a membrane which is obtained from an infected host cell. Infection of a new cell begins with joining or "fusion" of the viral and host cell membranes with an end result of a single membrane and the viral nucleocapsid in the host cell cytoplasm. Although membrane fusion is thermodynamically allowed, the rates of uncatalyzed membrane fusion are typically small. For this reason, enveloped viruses have fusion proteins in their membranes that bind to the host cell membranes and catalyze fusion. 1 This paper describes studies of a large and functional domain of the IFV hemagglutinin (HA) fusion protein and includes bacterial expression and isotopic labeling of the protein, characterization of its folding and fusion activity, and membrane incorporation and solid-state nuclear magnetic resonance (SSNMR) structural measurements.The HA protein is composed of HA1 and HA2 subunits. HA1 lies completely outside the virus, while HA2 has a ∼185 residue N-terminal ectodomain that lies outside the virus, a ∼25 residue transmembrane domain, and a ∼10 residue C-terminal endodomain that is inside the virus. 2 The IFV is taken into the host respiratory epithelial cell by receptor-mediated endocytosis, and the cell physiological processes lower the pH of the endosome to ∼5. The HA1 and HA2 subunits dissociate, and a large HA2 structural change results in exposure of the ∼20 residue N-terminal "fusion peptide" (IFP) region. The IFP binds to endosomal membranes, and membrane fusion occurs. There has been a pH 7.5 structure of the HA1/HA2 ectodomain complex crystallized from aqueous solution and a pH 4.4 structure of residues 34-178 of HA2 that forms the "soluble ectodomain" (SHA2) and which was also crystallized from aqueous solution. 2,3 In addition, there have been liquid-state NMR structures of IFP in detergent micelles as well as electron spin resonance measurements of motion and membrane insertion of specific residues of IFP and of a HA2 construct composed of residues 1-127. 4,5 The present work is on a "FHA2" full ectodomain construct composed of residues 1-185 of HA2 and an eight residue C-terminal tag ( Figure 1A). SSNMR has the potential for providing high-resolution structural information for FHA2 in the physiologically relevant membrane-bound state and for addressing structural effects of factors that reduce fusion activity including neutral pH and mutations.There have been some previous applications of SSNMR to other large bacterial and human membrane proteins as well as membraneassociated IFP, and our study builds on this work. 6-10 SSNMR requires efficient production of >10 mg quantities of isotopically labeled protein, and this was accomplished by FHA2 expression in Escherichia coli cells. Significant isotopic labeling requires expression in minimal media which lacks amino acids, but it was found that the purified FHA2 yield was ∼0.1 mg/L fermentation culture for E. coli grown only in minimal media. The successful approach was growth to OD 7 in a ric...
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