Exploring the pH dependence of the SARS‐CoV‐2 complete fusion domain and the role of its unique structural features

Abstract

SARS-CoV-2 may enter target cells through the process of membrane fusion at either the plasma ($pH 7.4-7.0) or endosomal ($pH 6.5-5.0) membrane in order to deliver its genetic information. The fusion domain (FD) of the spike glycoprotein is responsible for initiating fusion and is thus integral to the viral life cycle. The FD of SARS-CoV-2 is unique in that it consists of two structurally distinctive regions referred to as the fusion peptide (FP) and the fusion loop (FL); yet the molecular mechanisms behind how this FD perturbs the membrane to initiate fusion remains unclear. In this study via solution NMR, we witnessed only a slight conformational change in the FD between pH 7.4 and pH 5.0, resulting in a minor elongation of helix 1. However, we found that the FD's ability to mediate membrane fusion has a large and significant pH dependence, with fusion events being more readily induced at low pH. Interestingly, a biphasic relationship between the environmental pH and fusogenicity was discovered, suggesting a preference for the FD to initiate fusion at the late endosomal membrane. Furthermore, the conserved disulfide bond and hydrophobic motif "LLF" were found to be critical for the function of the complete FD, with minimal activity witnessed when either was perturbed. In conclusion, these findings indicate that the SARS-CoV-2 FD preferably initiates fusion at a pH similar to the late endosome through a mechanism that heavily relies on the internal disulfide bond of the FL and hydrophobic LLF motif within the FP.

C‐Terminal Lipidation of SARS‐CoV‐2 Fusion Peptide Reinstates Superior Membrane Fusion Catalytic Ability

“…The exact location of the FP is a matter of debate, but there is a wide consensus that the putative fusion peptide region resides near the cleavage site S2' of SARS-CoV-2: FP1 (816-837), FP2 (835-856, also known as the fusion loop (FL) or fusion peptide proximal region (FPPR)), FP3 (854-874), and FP4 (885-909). [14,15] Recently Santamaria et al measured the membrane binding of all the fusion regions and found that FPPR (or FP2) and FP3 weakly interacted with the membrane, whereas FP1 and FP4 bind to the membrane strongly. [14] A shorter 18-residue analog of FP1 (816-833, now onwards termed as FP) was earlier identified as the fusion peptide.…”

“…The NMR structure of FP-FPPR regions are solved in bicelles, which reports that FPPR or FP2) mostly resides at the lipid-water interface and does not embed into the membrane. [15,22,23] As a class-I fusion protein, after the cleavage at the S2' site by TMPRSS2, the FP region will be exposed to the membrane for interaction and perturbation. These observations led us to investigate the efficiency of membrane fusion by the FP.…”

“…During SARS‐CoV‐2 infection it is believed that the FP perturbs the host membrane structure. The exact location of the FP is a matter of debate, but there is a wide consensus that the putative fusion peptide region resides near the cleavage site S2′ of SARS‐CoV‐2: FP1 (816–837), FP2 (835–856, also known as the fusion loop (FL) or fusion peptide proximal region (FPPR)), FP3 (854–874), and FP4 (885–909) [14,15] . Recently Santamaria et al.…”

“…Membrane perturbation and membrane fusion studies were carried out with FP1 and its shorter analogue (FP), [16,18] FP‐FPPR (816–856), [15,19,20] and FP4 [14,21] regions to understand how the fusion peptide might catalyze lipid rearrangement to facilitate fusion. The NMR structure of FP‐FPPR regions are solved in bicelles, which reports that FPPR or FP2) mostly resides at the lipid‐water interface and does not embed into the membrane [15,22,23] . As a class‐I fusion protein, after the cleavage at the S2′ site by TMPRSS2, the FP region will be exposed to the membrane for interaction and perturbation.…”

“…[14] A shorter 18-residue analog of FP1 (816-833, now onwards termed as FP) was earlier identified as the fusion peptide. [5,16,17] Membrane perturbation and membrane fusion studies were carried out with FP1 and its shorter analogue (FP), [16,18] FP-FPPR (816-856), [15,19,20] and FP4 [14,21] regions to understand how the fusion peptide might catalyze lipid rearrangement to facilitate fusion. The NMR structure of FP-FPPR regions are solved in bicelles, which reports that FPPR or FP2) mostly resides at the lipid-water interface and does not embed into the membrane.…”