Structure and function of locked conformation of the SARS-CoV-2 spike protein identified

By Jocelyn Solis-MoreiraMar 11 2021

A new preprint research paper, posted to the bioRxiv* server, uncovered more information on the different structural conformations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The findings suggest the spike protein turns into a locked state to stabilize the spike protein during virus egress.

There are three prefusion conformations for SARS-CoV-2 — two of which are shared with another coronavirus called SARS-CoV-1 S. However, the locked conformation is new, and there are limited data regarding its role in the virus’ life cycle.

SARS-CoV-2 virus particle release and the accompanying structural transitions of the surface spike protein. a, schematic diagram illustrating the release of nascent SARS-CoV-2 virus particles from the cell. Labeled boxes indicate the predicted conformational states of spike proteins as the virus travels through the acidic intracellular compartment before it was released into the neutral extracellular environment. b, the three prefusion conformational states observed for the SARS-CoV-2 spike protein and factors that influence the structural transitions between the conformational states.

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources

Characterizing lock conformation

The research team created a SARS-CoV-2 S protein construct called S-R/x3 that uses a disulfide pair to stabilize the spike into the locked conformation. About 77% of S-R/x3 particles conformed to a locked state, while 23% remained in a closed state.

Structure of S-R/x3

The locked conformation reconstruction helped the researchers find the receptor-binding domain (RBD) tightly clustered around a three-fold symmetry axis, linoleic acid bound in an RBD pocket, and residues 833-855 folded in a helix-loop-helix fusion peptide proximal region (FPPR) motif. In addition, a substantial disordered loop was discovered between residues 617 to 633.

These observations support previous research on locked conformation, which suggests the structure remains in a closed state because the folding of the FPPR motif prevents RBD from opening up.

Structural characteristics of a locked conformation with the D614G substitution

The team found that having the D614G substitution reduced the amount of locked conformation to approximately 20% of total particles. In contrast, over 80% of particles were in the closed conformation.

Similar to the S-R/x3 structure, there continued to be a presence of the three-fold axis, linoleic acid in the RBD, and the folded motif. However, the structure changes when observing domain D. The domain D showed a disordered loop structure between residues 617 to 641 that refolded into two small a-helices connected by a loop in the locked S-R/x3/D614G state.

Two locked states in the spike protein

The researchers observed two different locked conformations: 614D (S-R/x3), which the authors refer to as “locked-1,” and the 614G (S-R/x3/D614G) labeled “locked-2”.

Having the D614G mutation changed the structure of domain D in residues 617 to 641.

“Due to this structural perturbation, there are differences in the FPPR motif and in residues 316-325 which are located in the hinge region connecting domain C and D. In the 614D locked-1 structure, the long sidechains of R634 and Y636 interdigitate with the sidechains of F318 and Y837. In this interaction, R634 in domain D is sandwiched between F318 of the hinge region and Y837 of the FPPR motif, bridging these three structural features,” described the researchers.

They suggest the zip-locking interaction helps to sustain the loop structure observed in residues 617-641.

Another change with the inclusion of the D614 substitution is the absence of a salt bridge in the FPPR motif in the second locked conformation. Instead, the refolding into two small alpha-helices suggests a D614 mutation alters electrostatic interaction involving binding.

Moving from a locked to a closed state

Moving from the first locked state to the second locked state breaks linkages between residues in domain D and the FPPR motif. To move the locked state to a closed state would require further movement at the hinge region of the spike protein to allow the opening of the RBD.

The lack of interactions with R634, Y636, F318 and Y837 in the S-R/x3/D614G structures may help explain how D614G promotes RBD opening.

Additionally, low pH levels maintain the RBD to a locked conformation. But when pH levels increase to 7.4, the metastability transitions it to a closed conformation. This opens the receptor-binding domain of the spike protein. Moving to a neutral pH also reverses the locked-2 conformation.

The researchers found removing an electrostatic interaction between D614 and R634 in the D614G spike protein destabilizes domain D, causing a locked-2 conformation. They speculate the different conformations in domain D are involved in regulating RBD opening and receptor binding.

“Given that low pH and lipid binding both favor locked conformations, we speculate that during virus assembly, both the 614D and D614G spikes are in their respective locked-1 and locked-2 conformations and that this provides a mechanism to prevent premature transition into open or post-fusion conformations during virus assembly. Once viruses are released into the neutral pH environment outside of the cell, over time, the spike will transition to the closed conformation where the RBD can open and mediate binding to target cells.”

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources