Stabilizing the Closed SARS-CoV-2 Spike Trimer

Abstract

<jats:p>The trimeric spike (S) protein of SARS-CoV-2 is the primary focus of most vaccine design and development efforts. Due to intrinsic instability typical of class I fusion proteins, S tends to prematurely refold to the post-fusion conformation, compromising immunogenic properties and prefusion trimer yields. To support ongoing vaccine development efforts, we report the structure-based design of soluble S trimers, with increased yields and stabilities, based on introduction of single point mutations and disulfide-bridges. We identify two regions in the S-protein critical for the protein's stability: the heptad repeat region 1 of the S2 subunit and subunit domain 1 at the interface with S2. We combined a minimal selection of mostly interprotomeric mutations to create a stable S-closed variant with a 6.4-fold higher expression than the parental construct while no longer containing a heterologous trimerization domain. The cryo-EM structure reveals a correctly folded, predominantly closed pre-fusion conformation. Highly stable and well producing S protein and the increased understanding of S protein structure will support vaccine development and serological diagnostics.</jats:p>

The SARS-CoV-2 Spike mutation D614G increases entry fitness across a range of ACE2 levels, directly outcompetes the wild type, and is preferentially incorporated into trimers

“…Furthermore, developing and testing neutralizing antibodies against the Spike trimer requires the stabilization of the trimers. Other groups have achieved this by introducing mutations or exogenous trimerization peptides in the Spike protein sequence 30–32 . Such mutations may result in changes in the three-dimensional structure of the Spike trimers and alter their antigenicity.…”

Multimerization- and glycosylation-dependent receptor binding of SARS-CoV-2 spike proteins

“…This variability is important in receptor binding as only in the “up” conformation can RBD bind ACE2. Approaches to control the conformation for vaccine purposes using stabilization mutations appear to keep the RBD conformation in their down-state [11, 12], making them non preferred proteins to analyze receptor-binding properties. Also, the multimerization status of the RBD is critical to allow the analysis of ACE2 interactions.…”

Computational Analysis of Protein Stability and Allosteric Interaction Networks in Distinct Conformational Forms of the SARS-CoV-2 Spike D614G Mutant: Reconciling Functional Mechanisms through Allosteric Model of Spike Regulation

“…48 Structure-based protein design and cryo-EM structure determination established that both D614G and D614N mutations can result in the increased fusogenicity and stability which can be explained by a decrease in a premature shedding of the S1 domain. 49 The cryo-EM structure revealed a stable closed mutant conformation, suggesting that D614G/N mutations can attenuate the repulsive charge interactions at the interface between S1 and S2 providing tighter packing of the head domains against S2. 49…”

“…49 The cryo-EM structure revealed a stable closed mutant conformation, suggesting that D614G/N mutations can attenuate the repulsive charge interactions at the interface between S1 and S2 providing tighter packing of the head domains against S2. 49…”

“…These results also offer support to the experimental observations that the enhanced stability of the S-D614G mutant may be linked with the mechanism of the reduced S1 shedding. 49 and "S1-shedding" mechanism underpinning the D614G effects.…”