Biophysical Correlates of Enhanced Immunogenicity of a Stabilized Variant of the Receptor Binding Domain of SARS-CoV-2.

The receptor binding domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We have previously reported the design and characterization of a mammalian cell expressed RBD derivative, mRBD1-3.2, that has higher thermal stability and greatly enhanced immunogenicity relative to the wild type mRBD. The protein is highly thermotolerant and immunogenic and is being explored for use in room temperature stable Covid-19 vaccine formulations. In the current study, we have investigated the folding pathway of both WT and stabilized RBD. It was found that chemical denaturation of RBD proceeds through a stable equilibrium intermediate. Thermal and chemical denaturation is reversible, as assayed by binding to the receptor ACE2. Unusually, in its native state, RBD binds to the hydrophobic probe ANS, and enhanced ANS binding is observed for the equilibrium intermediate state. Further characterization of the folding of mRBD1-3.2, both in solution and after reconstitution of lyophilized protein stored for a month at 37 °C, revealed a higher stability represented by higher Cm, faster refolding, slower unfolding, and enhanced resistance to proteolytic cleavage relative to WT. In contrast to WT RBD, the mutant showed decreased interaction with the hydrophobic moiety linoleic acid. Collectively, these data suggest that the enhanced immunogenicity results from reduced conformational fluctuations that likely enhance in vivo half-life as well as reduce the exposure of irrelevant non-neutralizing epitopes to the immune system.

Mechanistic insights into global suppressors of protein folding defects.

Most amino acid substitutions in a protein either lead to partial loss-of-function or are near neutral. Several studies have shown the existence of second-site mutations that can rescue defects caused by diverse loss-of-function mutations. Such global suppressor mutations are key drivers of protein evolution. However, the mechanisms responsible for such suppression remain poorly understood. To address this, we characterized multiple suppressor mutations both in isolation and in combination with inactive mutants. We examined six global suppressors of the bacterial toxin CcdB, the known M182T global suppressor of TEM-1 ß-lactamase, the N239Y global suppressor of p53-DBD and three suppressors of the SARS-CoV-2 spike Receptor Binding Domain. When coupled to inactive mutants, they promote increased in-vivo solubilities as well as regain-of-function phenotypes. In the case of CcdB, where novel suppressors were isolated, we determined the crystal structures of three such suppressors to obtain insight into the specific molecular interactions responsible for the observed effects. While most individual suppressors result in small stability enhancements relative to wildtype, which can be combined to yield significant stability increments, thermodynamic stabilisation is neither necessary nor sufficient for suppressor action. Instead, in diverse systems, we observe that individual global suppressors greatly enhance the foldability of buried site mutants, primarily through increase in refolding rate parameters measured in vitro. In the crowded intracellular environment, mutations that slow down folding likely facilitate off-pathway aggregation. We suggest that suppressor mutations that accelerate refolding can counteract this, enhancing the yield of properly folded, functional protein in vivo.