Chemistry & Biochemistry
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Item DECIPHERING THE MOLECULAR MECHANISM BEHIND THE SARS-COV-2 FUSION DOMAIN(2024) Birtles, Daniel; Lee, Jinwoo; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)SARS-CoV-2 is an extremely infectious virus, yet despite a plethora of research the viral lifecycle is still not well understood, particularly the process of membrane fusion. The traditional means by which viral glycoproteins facilitate fusion is that of the six-helix bundle, within which a short, conserved sequence known as the fusion domain (FD) initiates the process as it embeds within and perturbs the target cell membrane, in turn lowering the energetic barrier necessary to coalesce two opposing membranes. Furthermore, the highly conserved coronavirus FD is found to be available on the SARS-CoV-2 spike protein surface, which along with its integral role within the viral lifecycle makes it an ideal therapeutic target. However, limited knowledge of the exact molecular mechanism by which the SARS-COV-2 FD conducts its role within the fusion process has prevented the production of antiviral treatments. Here we describe the elucidation of key molecular details regarding how the SARS-CoV-2 FD initiates the process of membrane fusion. Firstly, the FD was found to contain a unique assembly of fusogenic regions, known as a fusion peptide (FP) and fusion loop (FL), which operate in synergy to elicit efficient fusion. This was followed by the discovery of a preference for the FD to fuse within conditions akin to the late endosomal membrane, with both pH and lipid composition significantly impacting fusion. It was found that the endosomal resident anionic lipid BMP imparts a negative impact on lipid packing within the membrane, which positively correlates with fusion. The unique mechanism by which the coronavirus FD initiates fusion was cemented when we uncovered the importance of several positively charged residues towards the FDs function. This also led to unearthing a mutant of the FD (K825A), which if found to have naturally occurred within the full spike protein, has the potential to produce a more virulent strain of SARS-CoV-2.Item Exploring the pH dependence of the SARS-CoV-2 complete fusion domain and the role of its unique structural features(Wiley, 2022-08-11) Birtles, Daniel; Oh, Anna E.; Lee, JinwooSARS-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.