Evolution of Conformational Dynamics Determines the Conversion of a Promiscuous Generalist into a Specialist Enzyme

Abstract

β-Lactamases are produced by many modern bacteria as a mechanism of resistance toward β-lactam antibiotics, the most common antibiotics in use. β-Lactamases, however, are ancient enzymes that originated billions of years ago. Recently, proteins corresponding to 2- to 3-Gy-old Precambrian nodes in the evolution of Class A β-lactamases have been prepared and shown to be moderately efficient promiscuous catalysts, able to degrade a variety of antibiotics with catalytic efficiency levels similar to those of an average modern enzyme. Remarkably, there are few structural differences (in particular at the active-site regions) between the resurrected enzymes and a penicillin-specialist modern β-lactamase. Here, we propose that the ancestral promiscuity originates from conformational dynamics. We investigate the differences in conformational dynamics of the ancient and extant β-lactamases through MD simulations and quantify the contribution of each position to functionally related dynamics through Dynamic Flexibility Index. The modern TEM-1 lactamase shows a comparatively rigid active-site region, likely reflecting adaptation for efficient degradation of a specific substrate (penicillin), whereas enhanced deformability at the active-site neighborhood in the ancestral resurrected proteins likely accounts for the binding and subsequent degradation of antibiotic molecules of different size and shape. Clustering of the conformational dynamics on the basis of Principal Component Analysis is in agreement with the functional divergence, as the ancient β-lactamases cluster together, separated from their modern descendant. Finally, our analysis leads to testable predictions, as sites of potential relevance for the evolution of dynamics are identified and mutations at those sites are expected to alter substrate-specificity.

Conformational dynamics and enzyme evolution

“…It should be noted that the ancestral lactamases were experimentally characterized and demonstrated to be able to promiscuously catalyse the degradation of a range of antibiotics with activity levels that are similar to those of the average modern enzyme [84]. A combination of molecular dynamics simulations [101], with analysis through the 'Dynamic Flexibility Index' [102] (an approach which allows for the quantification of the contribution of each position in a protein to functionally related dynamics) showed clear changes in the conformational dynamics of the enzyme across evolutionary time. That is, while the active site of the modern lactamase was shown to be comparatively rigid, the ancestral lactamases showed much greater flexibility, in particular, in residues near the active site of the enzyme.…”

“…That is, while the active site of the modern lactamase was shown to be comparatively rigid, the ancestral lactamases showed much greater flexibility, in particular, in residues near the active site of the enzyme. 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).…”

Structure-Encoded Global Motions and Their Role in Mediating Protein-Substrate Interactions

“…Our systematic study of 34 families of enzymes showed that the correlations become even more pronounced when focusing on the mobilities in the global modes (3). In a series of studies, the dynamic flexibility index determined from ENMs as a measure of mobility is used to understand the sequence evolution and functional enhancement (52,(71)(72)(73).…”

The Role of Conformational Dynamics and Allostery in the Disease Development of Human Ferritin

“…These specific DFI patterns suggest that the protein's native state is actually an ensemble of conformations displaying the structural variability in the active site region required for efficient binding of substrates of different sizes and shapes. On the other hand, DFI analysis of modern TEM-1 lactamase shows a comparatively rigid active-site region, likely reflecting adaptation for the efficient degradation of a specific substrate, penicillin (43). Principal component analysis of the ancestral b-lactamases and the extant TEM-1 lactamase reveal the special dynamics associated with substrate promiscuity and is in agreement with the functional divergence, as the highest-order modes of the ancient b-lactamases cluster together, separated from their modern descendant.…”