Integrating NMR and contact-response analysis reveals the allosteric network driving domain closure in Enzyme I

Abstract

Significance Allosteric regulation enables proteins to transmit signals over long distances, but the mechanisms linking local ligand binding to global conformational changes remain difficult to resolve. Here, we combine NMR-based chemical shift covariance analysis with computational contact-response analysis to map the allosteric network of a large multidomain enzyme, bacterial Enzyme I. We show that ligand binding reshapes a distributed network of coupled interactions that spans the active site, interdomain linker, and domain interfaces, driving domain closure. Remarkably, independent perturbations (mutations and temperature) converge on the same network, demonstrating that it reflects intrinsic features of the protein energy landscape. This integrated approach provides a general strategy for resolving allosteric mechanisms in complex biological systems.