Allosteric communication in Class A β-lactamases occurs via Cooperative Coupling of Loop Dynamics

Abstract

Allosteric effects control protein (e.g. enzyme) activity in ways that are not fully understood. Better understanding of allosteric effects, and tools to identify them, would offer promising alternative strategies to inhibitor development. Through a combination of equilibrium and nonequilibrium molecular dynamics simulations, we identify allosteric effects and communication pathways from two distant ligand binding sites to important active site structural elements that control enzymatic activity in two prototypical class A β-lactamases, TEM-1 and KPC-2. Both of these enzymes are important determinants of antibiotic resistance in widespread bacterial pathogens. The simulations show that the allosteric sites are connected to the active site in both enzymes, (e.g. affecting the conformation of the Ω-loop) highlighting how allosteric inhibitors may exert their effects. Nonequilibrium simulations reveal pathways of communication operating over distances of 30 Å or more. In these identified signaling pathways, the propagation of the signal occurs through cooperative coupling of loop dynamics. Notably, 50% or more clinically relevant amino acid substitutions in each enzyme map onto the identified signal transduction pathways. This suggests that clinically important variation may affect, or be driven by, differences in allosteric behavior, providing a mechanism by which amino acid substitutions may affect the relationship between spectrum of activity, catalytic turnover and potential allosteric behavior in this clinically important enzyme family. Simulations of the type presented here will help in identifying and analyzing such differences.

Dynamical nonequilibrium molecular dynamics reveals the structural basis for allostery and signal propagation in biomolecular systems

“…The nonequilibrium trajectories are then integrated until time t. The nature of the perturbation to be introduced in the system will depend on whatever question the user wants to address, and it can be either instantaneous or gradual. A very diverse range of perturbations has already been reported in the literature, ranging from the introduction of external electric fields and thermal gradients (e.g., [15,22,24,33]) to the switching off of radiation flows [29], ATP hydrolysis [28,35], and the removal of ligands from their binding sites [34,36,37]. The response of the system can be extracted using the Kubo-Onsager relation (Eq.…”

“…Molecules binding at allosteric sites may modulate (e.g., inhibit) the activity of the enzyme (e.g., [71,72]), by means that are generally not well understood. With this in mind, Galdadas et al [36] used the D-NEMD approach to examine effects of allosteric ligands for two clinically relevant class A β-lactamases, namely the TEM-1 and KPC-2 enzymes (Fig. 8).…”

“…Galdadas et al [36] performed 800 short nonequilibrium simulations (400 for each enzyme), and in each simulation, the allosteric ligand was removed from its binding site. The response of the system was extracted using the Kubo-Onsager relation (Eq.…”

“…4), as in the examples above. In both enzymes, the cascade of conformational changes in response to the removal of an allosteric ligand revealed the pathways by which the structural changes are transmitted from the allosteric site to the enzyme's catalytic active site [36]. In TEM-1 (Fig.…”

“…Over the last decade, improvements in forcefields, algorithms, MD software, and computer hardware have allowed for longer simulation times and made it possible to run tens/hundreds/thousands of replicates in an acceptable timeframe [30]. Recent years have seen a resurgence in the use of the D-NEMD approach, with researchers from different fields realising how powerful, generally applicable and helpful the approach is, not only to study fluid dynamics (e.g., [31][32][33]) but also biological problems such as signal transmission in proteins and allostery (e.g., [34][35][36][37]).…”