Mechanistic insights into global suppressors of protein folding defects

Abstract

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 five global suppressors of the bacterial toxin CcdB, the known M182T global suppressor of TEM-1 β-lactamase, the N239Y global suppressor of p53-DBD and three suppressors of the SARS-CoV-2 spike Receptor Binding Domain. The suppressors both alone, and in conjunction with inactive mutants, stabilise the protein both thermodynamically and kinetically in-vitro, predominantly through acceleration of the refolding rate parameters. When coupled to inactive mutants they promote increased in-vivo solubilities as well as regain-of-function phenotypes. Our study also demonstrates that the global suppressor approach can be used to consistently stabilise wild-type proteins, including for downstream translational applications.

Identification of stabilizing point mutations through mutagenesis of destabilized protein libraries

“…7 B ), and a proper interpretation is only possible in the context of a high-resolution structure. In another study, where we have carried out detailed mechanistic studies to understand the mechanistic basis for global suppressors in multiple protein systems, we have solved the structures of a few individual stabilizing mutations (S12G, V46L, and S60E) identified in the present study ( 40 ). In the case of S12G, we found that two additional water molecules were present in the place of S12 and formed hydrogen bonds with the main chain of residue E11 and R13.…”

Stabilizing proteins through saturation suppressor mutagenesis

“…Most of the stabilizing mutations occur in surface exposed loop regions (Figure 7B) and a proper interpretation is only possible in the context of a high resolution structure. In another study, where we have carried out detailed mechanistic studies to understand the mechanistic basis for global suppressors in multiple protein systems, we have solved the structures of a few individual stabilizing mutations (S12G, V46L and S60E) identified in the present study (40). In the case of S12G, we found that two additional water molecules were present in the place of S12 and formed hydrogen bonds with the main chain of residue E11 and R13.…”

Ter-Seq: A high-throughput method to stabilize transient ternary complexes and measure associated kinetics

“…The affinities of all CcdB mutants with GyrA14 were also measured by SPR as described previously (Chattopadhyay et al, 2021) (Figure S11-S14, Table S7-S9). The rejuvenation rate constants measured by SPR correlated well with rejuvenation rate constants determined from YSD.…”