Hinge-shift mechanism as a protein design principle for the evolution of β-lactamases from substrate promiscuity to specificity

Abstract

TEM-1 β-lactamase degrades β-lactam antibiotics with a strong preference for penicillins. Sequence reconstruction studies indicate that it evolved from ancestral enzymes that degraded a variety of β-lactam antibiotics with moderate efficiency. This generalist to specialist conversion involved more than 100 mutational changes, but conserved fold and catalytic residues, suggesting a role for dynamics in enzyme evolution. Here, we develop a conformational dynamics computational approach to rationally mold a protein flexibility profile on the basis of a hinge-shift mechanism. By deliberately weighting and altering the conformational dynamics of a putative Precambrian β-lactamase, we engineer enzyme specificity that mimics the modern TEM-1 β-lactamase with only 21 amino acid replacements. Our conformational dynamics design thus re-enacts the evolutionary process and provides a rational allosteric approach for manipulating function while conserving the enzyme active site.

Dynamic coupling of residues within proteins as a mechanistic foundation of many enigmatic pathogenic missense variants

“…This suggests that the substitutions at DARC sites are more likely to lead to genetic disease. Moreover, a comparison of DCI values of sites with disease-associated missense variants with all other protein sites supports the same observation: mutations at DARC sites, distal sites that exhibit high coupling (i.e., high DCI), are predisposed to impact function [23,24].…”

“…We used the dynamic coupling index (DCI) to identify sites strongly coupled to active sites critical for function [23,24]. We refer to them as dynamic allosteric residue coupling (DARC) sites.…”

The Interplay of Electrostatics and Chemical Positioning in the Evolution of Antibiotic Resistance in TEM β-Lactamases

“…Our findings of large electric fields in the oxyanion hole of TEM β-lactamases may help to rationalize how structural plasticity leads to substrate promiscuity. 57,60 The evolutionary history leading to TEM-1 resulted in an enzyme scaffold and active site that is capable of exerting large stabilizing electric fields necessary for TSS of chemical reactions with similar substrate geometries and mechanisms. This may be the case for hydrolases and many other enzymes, 61 which utilize pre-existing catalytic architectures, such as oxyanion holes, to enable catalysis of a large substrate scope for further improvements via directed evolution.…”

“…Nearly all crystallographic studies of TEM β-lactamases with inhibitors and substrates exhibit only a single conformation, [47][48][50][51] while MD simulations indicate conformational sampling and biasing as a mechanism for β-lactamase evolution. [37][38][52][53][54][55][56][57] In order to rationalize the observed changes over the evolutionary trajectory from TEM-1 to TEM-52 we combine the kinetic, VSE, and MD results to assess the role of electrostatic, chemical positioning, and conformational effects in modulating the activation free energy barrier (∆G ‡ from kcat and TStheory) (Figure 6, Table S2).…”

“…This interplay of electrostatic and chemical positioning to modulate the protein fitness landscape may be a general observation, as evident in the changes in active site heterogeneity in the directed evolution of de novo Kemp eliminases and ancestrally reconstructed β-lactamases. 57,[63][64] Combining the complementary approaches of MD simulations and the VSE with kinetic studies enables a more comprehensive molecular picture of the parameters modulating an enzyme's evolution towards new and/or improved function with implications in protein design, enzyme evolution, and antibiotic resistance.…”