Segmental Motions, Not a Two-State Concerted Switch, Underlie Allostery in CheY

The switch between an inactive and active conformation is an important transition for signaling proteins, yet the mechanisms underlying such switches are not clearly understood. Escherichia coli CheY, a response regulator protein from the two-component signal transduction system that regulates bacterial chemotaxis, is an ideal protein for the study of allosteric mechanisms. By using 15N CPMG relaxation dispersion experiments, we monitored the inherent dynamic switching of unphosphorylated CheY. We show that CheY does not undergo a two-state concerted switch between the inactive and active conformations. Interestingly, partial saturation of Mg2+ enhances the intrinsic allosteric motions. Taken together with chemical shift perturbations, these data indicate that the μs-ms timescale motions underlying CheY allostery are segmental in nature. We propose an expanded allosteric network of residues, including W58, that undergo asynchronous, local switching between inactive and active-like conformations as the primary basis for the allosteric mechanism. [Display omitted] ► NMR dynamics data reveal CheY does not undergo concerted, two-state switching ► Asynchronous, local switching of a network of residues facilitates CheY activation ► Mg2+ enhances the dynamics associated with the inactive-to-active transition Allosteric proteins are extremely important in signaling, yet their switching mechanism is not well understood. Here, McDonald et al. show that CheY intrinsically samples alternate conformations and that, rather than switching in a concerted fashion, CheY utilizes segmental motions to asynchronously reach active state.