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

Highlights

• NMR dynamics show a second process that is faster than the activation transition.

• This process is related to χ₁ rotamer exchange of Y101.

• Molecular dynamics simulations show that Y101 motion is uncorrelated with the activation transition.

• Experimental data against the dominant “Y-T coupling” mechanism of activation in NtrC are shown.

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 (NtrC^R), 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 NtrC^R 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 provide experimental evidence against the generally accepted “Y–T coupling” mechanism of activation in NtrC^R.

Introduction

Two-component systems are the predominant signaling systems in bacteria, allowing them to react to a wide variety of changes in their cellular environment. A basic two-component system consists of a histidine kinase whose conserved C-terminal transmitter domain becomes phosphorylated in response to a signal in the variable sensor domain. The histidine kinase's cognate response regulator can then catalyze the phosphoryl-transfer to its own conserved aspartate residue. In the response regulator nitrogen regulatory protein C (NtrC), which activates transcription of a number of genes under conditions of limited nitrogen supply, the receiver domain is in a pre-existing equilibrium between the active and inactive substates in the wild-type (WT) protein [1]. When its associated histidine kinase, NtrB, is phosphorylated due to low intracellular nitrogen concentrations, the subsequent transfer of the phosphate to the receiver domain of NtrC causes a shift in this pre-existing equilibrium far toward the active state through active state stabilization [2]. The structural change in the region of α-helix 3 through β-strand 5 (“3445 face”) in the receiver domain (NtrC^R) modifies its interaction with the central domain of NtrC [3], leading to oligomerization of the full-length protein [4], ATPase activity [5], and the activation of the σ⁵⁴ promoter through a DNA looping mechanism [6].

The activation mechanism of response regulators has gained much attention due to its central role in bacterial signaling and its early recognition as a classical system to study allostery in single domain proteins [7]. Because our new results presented here rule out the popular “Y–T coupling” mechanism [8], we briefly review previous publications on this topic. Crystal structures of the homologous response regulator CheY led researchers to propose the “Y–T coupling” mechanism of activation, which is reminiscent of an induced-fit view of allostery [8]. The Y and T in question are Y101 and T82 (NtrC^R numbering), which are highly conserved as either a tyrosine or a phenylalanine and as either a threonine or a serine, respectively, in response regulators [9]. A crystal structure of the receiver domain of CheY activated by the BeF₃⁻ phosphate analog showed that the position of the threonine was coupled to the χ₁ rotameric state of the tyrosine when compared to the unphosphorylated inactive protein [8]. In both low-resolution [10] and high-resolution [11] structures of CheY, the tyrosine was found to be occupying both the g+ and t rotameric states in the inactive state while the threonine moved to coordinate the phosphate group in activated CheY, leaving a gap in the side-chain packing. Tyrosine was then thought to fill this gap by going exclusively into the t state and becoming buried in the interior of the protein. This mechanism was also supported by the crystal structures of a series of mutations of the threonine and tyrosine, in which the rotameric state of the aromatic ring corresponded to whether or not the mutants were able to function properly during chemotaxis assays in Escherichia coli [12]. In 2006, Dyer and Dahlquist solved a crystal structure of unphosphorylated CheY with a fragment of its target protein, FliM [13]. They found that although the tyrosine was buried in the interior of the protein, the threonine, as well as the loop between β-strand 4 and α-helix 4 (β4/a4 loop), appeared to be only midway to the activated state. Analysis of all known structures of CheY led to the proposal that, before phosphorylation, the tyrosine was free to move independently from the threonine and the β4/a4 loop region, but upon phosphorylation, the threonine acted as a gate to the loop motion leading to a correlated movement of the tyrosine rotating to the buried t state. The authors dubbed this mode of allosteric activation “T-loop–Y coupling” [13], [14]. Importantly, activation coupled to the rotameric state of the tyrosine has been proposed to be a general mechanism of response regulator activation based on the tyrosine having distinct states in the inactive and active structures of a number of different response regulators [15], [16], [17], [18], [19].

Besides these experimental studies, many computational studies have been carried out on various receiver domains of response regulators with controversial results. Although this tyrosine was seen to be undergoing free rotameric rotation in the inactive state in molecular dynamics (MD) simulations in CheY, the motion of the β4/a4 loop and the tyrosine were reported to be correlated in simulations of the active conformation [20]. A similar result was found for FixJ where the β4/a4 loop and phenylalanine appeared to be correlated but the threonine movement was not [21]. In a more recent paper, the tyrosine was shown to be coupled to both the threonine and the β4/a4 loop using a statistical mechanics model of protein allostery in CheY [22]. In NtrC^R, elastic network modeling found that motions encompassing the aspartate that becomes phosphorylated, as well as the threonine and the tyrosine, were all correlated [23]. Pandini et al. also found support for a pre-organized network of allosteric connections between these residues and the functional site of phosphorylation [24]. Alternatively, transition path sampling in combination with potential mean force calculations found that the energy barrier of tyrosine/phenylalanine rotamer exchange was lower than the barrier involved in the inactive/active transition, making the rotameric state of the conserved aromatic residue kinetically independent from the functional conformational transition and leading them to theorize a role for the tyrosine in the thermodynamic stabilization of the active substate [25], [26].

Early NMR experiments to probe protein dynamics gave a hint that the tyrosine may not be directly coupled to the motion of the inactive/active transition. Dynamics on the microsecond to millisecond timescale were seen in residues surrounding Y101 in the phosphorylated form of NtrC^R; however, the applied model-free analysis did not allow us to quantitatively characterize conformational exchange processes [1]. Microsecond to millisecond motion has also been seen in phosphorylated CheY, with the majority of it localized to the area encompassing the β4/a4 loop and the tyrosine [27]. Despite the plethora of contradicting computational and experimental studies involving the role of the highly conserved tyrosine in the mechanism of activation of response regulators, “Y–T coupling” is the dominant description of activation for these systems [14], [17], [18], [28].

Here, we use NMR relaxation dispersion experiments in combination with MD simulations to provide evidence that Y101 in NtrC^R moves independently and more quickly than the inactive/active conformational transition both before and after the addition of the BeF₃⁻ phosphate analog. By experimentally characterizing a Y101L mutant, we demonstrate directly that Y101 is not needed for the inactive/active transition and that it does not thermodynamically stabilize the active-state structure. These data challenge the prevailing description of the activation of allosteric response regulators.