Characterization of Hydrophobic Interactions of SARS-CoV-2 and MERS-CoV Spike Protein Fusion Peptides Using Single Molecule Force Measurements

Abstract

We address the challenge of understanding how hydrophobic interactions are encoded by fusion peptide sequences within coronavirus (CoV) spike proteins. Within the fusion peptides of SARS-CoV-2 and MERS-CoV, a largely conserved peptide sequence called FP1 (SFIEDLLFNK and SAIEDLLFDK in SARS-2 and MERS, respectively) has been proposed to play a key role in encoding hydrophobic interactions that drive viral-host cell membrane fusion. While a non-polar triad (LLF) is common to both FP1 sequences, and thought to dominate the encoding of hydrophobic interactions, FP1 from SARS and MERS differ in two residues (Phe 2 versus Ala 2 and Asn 9 versus Asp 9, respectively). Here we explore if single molecule force measurements can quantify hydrophobic interactions encoded by FP1 sequences, and then ask if sequence variations between FP1 from SARS and MERS lead to significant differences in hydrophobic interactions. We find that both SARS-2 and MERS wild-type FP1 generate measurable hydrophobic interactions at the single molecule level, but that SARS-2 FP1 encodes a substantially stronger hydrophobic interaction than its MERS counterpart (1.91 ± 0.03 nN versus 0.68 ± 0.03 nN, respectively). By performing force measurements with FP1 sequences with single amino acid substitutions, we determine that a single residue mutation (Phe 2 versus Ala 2) causes the almost threefold difference in the hydrophobic interaction strength generated by the FP1 of SARS-2 versus MERS, despite the presence of LLF in both sequences. Infrared spectroscopy and circular dichroism measurements support the proposal that the outsized influence of Phe 2 versus Ala 2 on the hydrophobic interaction arises from variation in the secondary structure adopted by FP1. Overall, these insights reveal how single residue diversity in viral fusion peptides, including FP1 of SARS-CoV-2 and MERS-CoV, can lead to substantial changes in intermolecular interactions proposed to play a key role in viral fusion, and hint at strategies for regulating hydrophobic interactions of peptides in a range of contexts.

Computational and comparative investigation of hydrophobic profile of spike protein of SARS-CoV-2 and SARS-CoV

“…Intricate details regarding hydrophobic profile as obtained in this research endeavor can play a role in peptide therapeutics. Results here indicate that computational approach for hydrophobic characterization helps in understanding protein folding and related aspects [39,40].…”

“…Leucine and phenylalanine, which are found inside the protein's core, are crucial for structural stability, and these occur most frequently in the whole spike protein when comparing SARS-CoV-2 and SARS-CoV [49]. These variations to the spike protein's amino acids may have an impact on how well the spike protein binds to other proteins during the viral fusion process [39]. Also, Srinivasulu and colleagues as well as Zheng and Song reported that the SARS-CoV-2 and SARS-CoV spike proteins share nearly 73.5% amino acid sequence identity, implying that the remaining 24.5% non-conserved amino acid sequences may be accountable for the antigenic variations between these two proteins which supports the findings for the hydrophobic profile of spike protein of SARS-CoV-2 and SARS-CoV [14,19,38,44], discussed here.…”

Ebola Virus Glycoprotein Strongly Binds to Membranes in the Absence of Receptor Engagement

“…37 The second option suggests a deeper binding mode, as demonstrated previously for the SARS-CoV2 Spike protein fusion loop. 38 Here, subsequent atomic force microscopy (AFM) experiments revealed that the strength of the disassociation of the protein's fusion loop from a hydrophobic surface was 1.91 nN, 39 substantially higher than for Synaptotagmin-1−membrane disassociation. In addition, a recent computational study has shown that class I fusogens have the highest membrane-binding affinities, rationalized by the deep insertion of their hydrophobic fusion loops.…”