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. 2015 Feb;1848(2):721-30.
doi: 10.1016/j.bbamem.2014.11.025. Epub 2014 Dec 2.

NMR Structures and Localization of the Potential Fusion Peptides and the Pre-Transmembrane Region of SARS-CoV: Implications in Membrane Fusion

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Free PMC article

NMR Structures and Localization of the Potential Fusion Peptides and the Pre-Transmembrane Region of SARS-CoV: Implications in Membrane Fusion

Mukesh Mahajan et al. Biochim Biophys Acta. .
Free PMC article

Abstract

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) poses a serious public health hazard. The S2 subunit of the S glycoprotein of SARS-CoV carries out fusion between the virus and the host cells. However, the exact mechanism of the cell fusion process is not well understood. Current model suggests that a conformational transition, upon receptor recognition, of the two heptad core regions of S2 may expose the hydrophobic fusogenic peptide or fusion peptide for membrane insertion. Three regions of the S2 subunit have been proposed to be involved in cell-cell fusion. The N-terminal fusion peptide (FP, residues 770-788), an internal fusion peptide (IFP, residues 873-888) and the pre-transmembrane region (PTM, residues 1185-1202) demonstrated interactions with model lipid membranes and potentially involved in the fusion process. Here, we have determined atomic resolution structures of these three peptides in DPC detergent micelles by solution NMR. FP assumes α-helical conformation with significant distortion at the central Gly residues; enabling a close packing among sidechains of aromatic residues including W, Y and F. The 3-D structure of PMT is characterized by a helix-loop-helix with extensive aromatic interactions within the helices. IFP adopts a rather straight α-helical conformation defined by packing among sidechains of aromatic and aliphatic residues. Paramagnetic spin labeled NMR has demonstrated surface localization of PMT whereas FP and IFP inserted into the micelles. Collectively, data presented in this study will aid in understanding fusion mechanism of SARS-CoV.

Keywords: Cell fusion; Fusion peptide; Fusion protein; NMR; SARS-CoV; Structure.

Figures

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Fig. 1
Fig. 1
Spike glycoprotein of SARS-CoV is composed of two domains S1 and S2 separated by a protease cleavage site. The N-terminal domain has a signal peptide (SP) and a transmembrane domain (TM) is present at the C-terminus. Other regions are receptor binding domain (RBD), two heptad repeat (HR1 and HR2) sequences and putative fusion peptides named as fusion peptide (FP), internal fusion peptide (IFP) and pre transmembrane domain (PTM).
Fig. 2
Fig. 2
Fingerprint regions of 1H–1H 2-D NOESY spectra of SARS-CoV fusion peptides showing sequence specific resonance assignments of FP (panel A), IFP (panel B) and PTM (panel C) in DPC micelles.
Fig. 3
Fig. 3
Sections of 1H–1H 2-D NOESY spectra of FP (panel A), IFP (panel B) and PTM (panel C) showing NOEs contacts of low-field shifted aromatic ring proton resonances (6.5–7.5 ppm) with up-field shifted aliphatic resonances (4.5–0.8 ppm).
Fig. 4
Fig. 4
Bar diagram summarizing number and types of NOEs for FP (panel A), IFP (panel B) and PTM (panel C). Intra-residue, sequential and medium range NOEs are marked as white, light gray and dark bars, respectively.
Fig. 5
Fig. 5
(A) Backbone superposition of twenty lowest energy structures of FP in the presence of DPC micelles. (B) A representative structure of the micelle bound FP showing backbone folding as ribbon and sidechain disposition as stick. (C) A surface representation of FP showing localization of polar amino acids (blue color) and hydrophobic residues (yellow). Figures were generated using molecular visualization software Molmol and PyMol.
Fig. 6
Fig. 6
(A) Backbone superposition of 20 lowest energy structures of IFP in DPC micelles. (B) A representative structure of the micelle bound IFP showing backbone folding as ribbon and sidechain disposition as stick. (C) A surface representation of IFP showing localization of polar amino acids (blue color) and hydrophobic residues (yellow). Figures were generated using molecular visualization softwares Molmol and PyMol.
Fig. 7
Fig. 7
(A) Backbone superposition of 20 lowest energy structures of PTM in DPC micelles. (B) A representative structure of the micelle bound PTM showing backbone folding as ribbon and sidechain disposition as stick. (C) A surface representation of PTM showing localization of polar amino acids (blue and red color) and hydrophobic residues (yellow and green). Figures were generated using molecular visualization softwares Molmol and PyMol.
Fig. 8
Fig. 8
Bar diagrams showing the % attenuation of CαH/NH cross peaks upon additions of paramagnetic probes, 5-DSA and 16-DSA, for FP (panel A), IFP (panel B) and PTM (panel C). Signal attenuation cannot be obtained for few residues; these are indicated by a filled circle.
Fig. 9
Fig. 9
A hypothetical model of membrane fusion between SARS-CoV and host cells showing involvement of the three putative fusion peptides, FP, IFP and PTM. Upon activation by receptor binding of the S1 domain, the FP (blue color) and IFP (red color) of the S2 domain of the fusion protein insert into host cells causing folding back of the HR1 (green color) and HR2 (yellow color) into six helix bundle. The aromatic residue rich PTM domain (magenta color) and the TM domain residing on the viral membrane provide a contiguous track of hydrophobic helical structures with HR domains and fusion peptides. Such an arrangement may facilitate the mixing of lipid molecules between the two membranes leading to the fusion of the viral and host cells.

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