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

Understanding how phosphoenolpyruvate (PEP) binding induces the large open-to-closed conformational transition of bacterial Enzyme I (EI) has remained a long-standing problem in structural biology. In EI, PEP binds the C-terminal EIC domain, yet catalysis requires docking of the distant N-terminal EIN domain, which carries the active-site H189 residue, onto EIC. How this local binding event is coupled to global domain rearrangement has been unclear. Here, we combine experimental Chemical Shift Covariance Analysis (CHESCA) with computational Chemically Accurate Contact Response Analysis (ChACRA) to map the allosteric network underlying EI closure. Using a library of active-site mutants that systematically tune the open-to-closed equilibrium, CHESCA identifies a dominant cluster of residues whose chemical shifts correlate with the small-angle X-ray scattering-derived population of the closed state, revealing long-range energetic coupling between the PEP-binding site and distal structural elements. To obtain atomistic resolution, ChACRA analysis of Hamiltonian replica exchange molecular dynamics simulations identifies a spatially continuous network of coupled interactions spanning the PEP-binding pocket, interdomain linker, domain interfaces, and dimer contacts. Ligand binding reshapes this network, stabilizing interactions that promote domain docking and global rearrangement. Together, these results show that EI closure is governed by an extended allosteric network rather than a direct local contact. Crucially, CHESCA and ChACRA report on different physical observables; their convergence provides experimental validation of an atomistic interaction map and atomic-resolution interpretation of sparse NMR correlations, establishing a general framework for resolving allostery in complex biomolecular systems.