Optimization of Conformational Dynamics in an Epistatic Evolutionary Trajectory

Abstract

The understanding of protein evolution depends on the ability to relate the impact of mutations on molecular traits to organismal fitness. Biological activity and robustness have been regarded as important features in shaping protein evolutionary landscapes. Conformational dynamics, which is essential for protein function, has received little attention in the context of evolutionary analyses. Here we employ NMR spectroscopy, the chief experimental tool to describe protein dynamics at atomic level in solution at room temperature, to study the intrinsic dynamic features of a metallo- Β: -lactamase enzyme and three variants identified during a directed evolution experiment that led to an expanded substrate profile. We show that conformational dynamics in the catalytically relevant microsecond to millisecond timescale is optimized along the favored evolutionary trajectory. In addition, we observe that the effects of mutations on dynamics are epistatic. Mutation Gly262Ser introduces slow dynamics on several residues that surround the active site when introduced in the wild-type enzyme. Mutation Asn70Ser removes the slow dynamics observed for few residues of the wild-type enzyme, but increases the number of residues that undergo slow dynamics when introduced in the Gly262Ser mutant. These effects on dynamics correlate with the epistatic interaction between these two mutations on the bacterial phenotype. These findings indicate that conformational dynamics is an evolvable trait, and that proteins endowed with more dynamic active sites also display a larger potential for promoting evolution.

Insights into an evolutionary strategy leading to antibiotic resistance

“…For BcII a recent study using NMR indeed demonstrated that successful evolution is epistatic and increases protein loop dynamics on a timescale related to catalytic turnover rates ( i.e . micro- to milliseconds)29, and it is very likely that in variant #5–1 of AIM-1 increased loop dynamics are also introduced by the three mutations.…”

“…Since AIM-1 has a broad specificity towards a wide range of substrates we employed in vitro evolution techniques to identify residues that may be essential for the function of this enzyme and to demonstrate how AIM-1 can adapt to β-lactam substrates. While this is the first study to probe the adaptability of a MBL from the B3 subgroup similar techniques have been employed to probe the mechanism and evolution of the well-characterised B1-type MBLs IMP24252627 and BcII28293031, as well as the carbapenem-specific B2-type MBL CphA3233. Specifically, we used site-saturation mutagenesis (SSM) to generate mutant forms of AIM-1 and screened the mutants for enhanced activity and altered substrate specificity.…”

Distinct Roles for Conformational Dynamics in Protein-Ligand Interactions

“…These data provide insight into the potential mechanism by which these two features couple to support protein evolution. In particular, lowered stability – whether global or local – supports access to a wide range of conformational substates that facilitate populating conformations relevant for novel specificity (Gonzalez et al, 2016). Thus, the QM variant might be regarded as a key intermediate in the evolutionary trajectory between the Tiam1 and Tiam2 PDZ domains.…”

Conformational dynamics and enzyme evolution

“…Further principal component analysis of the conformational dynamics of these enzymes demonstrated that the ancestral b-lactamases form a cluster that is distinct from the more rigid modern TEM-1 lactamase [101]. An unrelated study by Vila and co-workers [103] employed NMR spectroscopy in order to explore the intrinsic dynamic features of different variants of a metallo-b-lactamase, metallo-b-lactamase II (BcII). The authors focused on the wild-type enzymes as well as three variants with expanded substrate scope obtained during a directed evolution trajectory and demonstrated that the enzyme has optimized the microto-millisecond timescale dynamics along the evolutionary trajectory, and that the effect of individual mutations on the dynamics is epistatic.…”