Conformational Intermediate That Controls KPC-2 Catalysis and Beta-Lactam Drug Resistance

Abstract

The KPC-2 carbapenemase enzyme is responsible for drug resistance in the majority of carbapenem-resistant gram-negative bacterial infections in the United States. A better understanding of what permits KPC-2 to hydrolyze carbapenem antibiotics and how this might be inhibited is thus of fundamental interest and great practical importance to development of better anti-infectives. By correlating molecular dynamics simulations with experimental enzyme kinetics, we have identified conformational changes that control KPC-2’s ability to hydrolyze carbapenem antibiotics. Related beta-lactamase enzymes can interconvert between catalytically permissive and catalytically nonpermissive forms of an acylenzyme intermediate critical to drug hydrolysis. Using molecular dynamics simulations, we identify a similar equilibrium in KPC-2 and analyze the determinants of this conformational change. Because the conformational dynamics of KPC-2 are complex and sensitive to allosteric changes, we develop an information-theoretic approach to identify key determinants of this change. We measure unbiased estimators of the reaction coordinate between catalytically permissive and nonpermissive states, perform information-theoretic feature selection and, using restrained molecular dynamics simulations, validate the protein conformational changes predicted to control catalytically permissive geometry. We identify two binding-pocket residues that control the conformational transitions between catalytically active and inactive forms of KPC-2. Mutations to one of these residues, Trp105, lower the stability of the catalytically permissive state in simulations and have reduced experimental kcat values that show a strong linear correlation with the simulated catalytically permissive state lifetimes. This understanding can be leveraged to predict the drug resistance of further KPC-2 mutants and help design inhibitors to combat extreme drug resistance.

β-Lactamases and β-Lactamase Inhibitors in the 21st Century

“…A common finding is that movement of Trp105, a residue that lies on one side of the active site cleft and that adopts multiple conformations in crystal structures of the unliganded enzyme [73], defines transitions between states that differ with respect to their competence for β-lactam turnover. In simulations of carbapenem acylenzyme complexes, committor analysis identifies the conformation of the Trp105 loop as one factor differentiating so-called “permissive” conformations (in which the β-lactam acylenzyme carbonyl occupies the oxyanion hole and the deacylating water is present) from “non-permissive” conformations with the carbonyl outside the oxyanion hole and the deacylating water absent [74]. In multiple-replica parallel tempering metadynamics simulations, that access timescales not available through conventional methodologies, unliganded KPC is shown to undergo motions of active site regions, including Trp105 and the omega-loop containing Glu166, that are related to hydrophobic networks in the individual α and α/β domains and that direct interactions with substrates [75].…”

Biosynthetic Incorporation of Site-Specific Isotopes in β-Lactam Antibiotics Enables Biophysical Studies

“…[46,50] The observation of multiple sets of C=O peaks is indicative of distinct conformations of the covalent PenG adduct in the enzyme active site, which was suggested previously, but could not be confirmed in the absence of isotopic labeling. [5][6][7]51] . Taking into account the different timescales of the NMR and FTIR measurements and noting the single peak in the 1D 13 C NMR spectrum, this indicates an exchange of the conformational species on the sub-ms to ps time scale (the homogeneous lifetime of most C=O vibrations is ca.…”

Predicting allostery and microbial drug resistance with molecular simulations

“…2). A simulation and feature-selection analysis was also performed, using machine learning on molecular dynamics simulations to predict off-pathway conformations in the class carbapenemase KPC-2 [51]. These approaches offer the potential to directly identify how allosteric changes alter active-site conformational equilibria and thus construct statistical tools to address a central question of allosteric modulation.…”