Natural and Synthetic Suppressor Mutations Defy Stability–Activity Tradeoffs

Abstract

Thermodynamic stability represents one important constraint on protein evolution, but the molecular basis for how mutations that change stability impact fitness remains unclear. Here, we demonstrate that a prevalent global suppressor mutation in TEM β-lactamase, M182T, increases fitness by reducing proteolysis in vivo. We also show that a synthetic mutation, M182S, can act as a global suppressor and suggest that its absence from natural populations is due to genetic inaccessibility rather than fundamental differences in the protein's stability or activity.

Insights into the Effects and Mechanism of Andrographolide-Mediated Recovery of Susceptibility of Methicillin-Resistant Staphylococcus aureus to β-Lactam Antibiotics

“…Studies on this class of drugs have also focused on inducing tolerance to β-lactamases and enhancement of antimicrobial activity. To date, more than 220 mutants of class A β-lactamases alone have been identified, and mutations in a few residues have been reported to cause 10-fold changes in its hydrolytic activity ( 2 – 4 ). Therefore, studies focusing on bacterial resistance should no longer be limited to the presence or absence of β-lactamases but should also begin to focus on the structural and functional characteristics of β-lactamases, their mode of action with drugs, and their alterations, in order to get a deeper understanding of the mechanistic basis for the development of antibiotic resistance.…”

Differential effects of disulfide bond formation in TEM‐1 versus CTX‐M‐9 β‐lactamase

“…The native structure represents one important aspect of the landscape, but alternative structures-including partially folded intermediates and unfolded states-and their relative energies also impact a protein's function and evolvability. For instance, low thermodynamic stability can lead to degradation or aggregation in vivo (Lee et al, 2022), and high thermodynamic stability has been shown to enable the acquisition of novel functions in in vitro evolution studies (Bloom et al, 2006). Thus, it is important to examine how sequence encodes stability, as well as structure, to develop a more complete understanding of protein evolution.…”

“…Given the potential for β‐lactamase stability to impact organismal fitness, it is possible that the conserved disulfides in class A β‐lactamases facilitate protein evolution. For instance, they might underlie the ability to broaden substrate specificity without loss of stability, avoiding so‐called stability‐activity tradeoffs (Beadle & Shoichet, 2002; Lee et al, 2022; Wang et al, 2002). By increasing protein stability, disulfides may act as buffers against deleterious mutations, enabling accelerated sequence evolution.…”

“…Interestingly, these subfamilies are unique in their ability to hydrolyze carbapenems. But although loss of the disulfide by mutation reduces or eliminates resistance to these drugs (Majiduddin & Palzkill, 2003), the disulfide itself does not play a direct role in catalysis (Smith et al, 2016); rather, it is likely destabilization leading to aggregation or degradation in the cell that causes the observed loss of antibiotic resistance, as has been demonstrated in TEM variants (Lee et al, 2022). Given the potential for β‐lactamase stability to impact organismal fitness, it is possible that the conserved disulfides in class A β‐lactamases facilitate protein evolution.…”