Evidence Against the “Y–T Coupling” Mechanism of Activation in the Response Regulator NtrC

Abstract

The dominant theory on the mechanism of response regulators activation in two-component bacterial signaling systems is the ‘Y-T coupling’ mechanism, wherein the χ1 rotameric state of a highly conserved aromatic residue correlates with the activation of the protein via structural rearrangements coupled to a conserved tyrosine. In this paper we present evidence that in the receiver domain of the response regulator Nitrogen Regulatory Protein C (NtrCR) the interconversion of this tyrosine (Y101) between its rotameric states is actually faster than the rate of inactive/active conversion and is not correlated to the activation process. Data gathered from NMR relaxation dispersion experiments show that a subset of residues surrounding the conserved tyrosine sense a process that is occurring at a faster rate than the inactive/active conformational transition. We show that this process is related to χ1 rotamer exchange of Y101 and that mutation of this aromatic residue to a leucine eliminated this second faster process without affecting activation. Computational simulations of NtrCR in its active conformation further demonstrate that the rotameric state of Y101 is uncorrelated with the global conformational transition during activation. Moreover, the tyrosine does not appear to be involved in the stabilization of the active form upon phosphorylation, and is not essential in propagating the signal downstream for ATPase activity of the central domain. Our data provides experimental evidence against the generally accepted ‘Y-T coupling’ mechanism of activation in NtrCR.

A network of molecular switches controls the activation of the two-component response regulator NtrC

“…This figure provides the evidence that the Y101-T82 coupling can be either formed or broken within the active state, but is predominately broken in the inactive state. This result implies that Y-T coupling is not strictly correlated with the activation of NtrC and is in agreement with recent NMR relaxation dispersion experiments 10 , which found the interconversion of the Y101 rotameric state to be faster than the inactive/active conformational transition. Furthermore, our results in Fig.…”

“…However, because of the large size of the protein and its long activation timescales, these studies have employed biased techniques such as targeted molecular dynamics 9 , separate simulations of the active and inactive states 7,10,11 , or simulations with coarse grained representations of the protein and solvent [12][13][14][15] . These studies have shed light on various aspects of NtrC activation but have failed to provide a comprehensive view of the conformational landscape of NtrC activation.…”

Cross Talk Inhibition Nullified by a Receiver Domain Missense Substitution

“…The hydrogen bond formed by CheY residue Thr-87 stabilizes the flexible ␤4-␣4 loop active conformation in conjunction with the rotation of residue Tyr-106 (Phe in many receiver sequences). This T-loop-Y coupling is postulated to underlie the phosphorylation-induced allosteric shift (4,71,72), although the Tyr residue is inessential for NtrC, which displays a more global conformational change (73). CheY residue Ala-88 may act to monitor phosphorylation and/or metal ion binding and thereby influence motions of other residues involved in the allosteric shift (72).…”

The role of protein dynamics in allosteric effects—introduction

“…While the initial 'MWC' model for allosteric effects focussed on oligomeric proteins existing in two conformational states (Monod et al 1965), subsequent work has led to a number of extensions to the concept. First, it is clear that monomeric proteins can show functionally important allosteric effectsfor example, the regulatory proteins CheY and NtrC (Cui and Karplus 2008;McDonald et al 2012;Villali et al 2014;Volkman et al 2001). The transmission of the effects of distal mutations is analogous in many ways to the transmission of the effects of ligand binding, and is of course seen in many monomeric proteins (e.g., Clarkson et al 2006;Lee and Goodey 2011), while it has been studied in particular detail in dihydrofolate reductase (Boehr et al 2013;Wong et al 2005).…”