Structural basis for methylesterase CheB regulation by a phosphorylation-activated domain

Abstract

We report the x-ray crystal structure of the methylesterase CheB, a phosphorylation-activated response regulator involved in reversible modification of bacterial chemotaxis receptors. Methylesterase CheB and methyltransferase CheR modulate signaling output of the chemotaxis receptors by controlling the level of receptor methylation. The structure of CheB, which consists of an N-terminal regulatory domain and a C-terminal catalytic domain joined by a linker, was solved by molecular replacement methods using independent search models for the two domains. In unphosphorylated CheB, the N-terminal domain packs against the active site of the C-terminal domain and thus inhibits methylesterase activity by directly restricting access to the active site. We propose that phosphorylation of CheB induces a conformational change in the regulatory domain that disrupts the domain interface, resulting in a repositioning of the domains and allowing access to the active site. Structural similarity between the two companion receptor modification enzymes, CheB and CheR, suggests an evolutionary and͞or functional relationship. Specifically, the phosphorylated N-terminal domain of CheB may facilitate interaction with the receptors, similar to the postulated role of the N-terminal domain of CheR. Examination of surfaces in the N-terminal regulatory domain of CheB suggests that despite a common fold throughout the response regulator family, surfaces used for proteinprotein interactions differ significantly. Comparison between CheB and other response regulators indicates that analogous surfaces are used for different functions and conversely, similar functions are mediated by different molecular surfaces.

The structure–function relationship of WspR, a Pseudomonas fluorescens response regulator with a GGDEF output domain

“…These insertions presumably activated WspR via disruption of the interface between the two domains, a mechanism common to many responseregulator activation mechanisms (Eldridge et al, 2002;Park et al, 2002;Muchova et al, 2004). A comparison of the domain interaction surfaces of four full-length, two-domain response-regulator crystal structures (Robinson et al, 2003) suggests that the activation mechanisms of the different proteins, NarL (Baikalov et al, 1996), CheB (Djordjevic et al, 1998), DrrD (Buckler et al, 2002) and DrrB (Robinson et al, 2003), vary with the extent of their interdomain interfaces. For example, comparison of the DrrB and DrrD crystal structures suggests that different levels of intramolecular communication between the N-and C-terminal domains lead to significant differences in effector-domain regulation in each case (Robinson et al, 2003).…”

“…In this model, the C-terminal effector domain is inhibited in the inactive state by the N-terminal domain. Regulation through effector-domain inhibition occurs in a number of response regulators, including E. coli NarL (Baikalov et al, 1996;Eldridge et al, 2002) and S. typhimurium CheB (Djordjevic et al, 1998). Phosphorylation (Fig.…”

Structural Analysis and Solution Studies of the Activated Regulatory Domain of the Response Regulator ArcA: A Symmetric Dimer Mediated by the α4-β5-α5 Face

“…61 Another notable feature of the OmpR/PhoB subfamily is that in the inactive state the DNA recognition helix of the effector domain is freely exposed to the solvent 33,34 making it readily available for DNA binding in contrast to other RRs in which the functional regions of the effector domains are occluded by the unphosphorylated regulatory domains. 27,28 Taking into account these marked differences, it is hypothesized that members of the OmpR/PhoB subfamily use a common mechanism of regulation by dimerization.…”

“…These interactions form the basis for a variety of different inter-and intramolecular regulatory mechanisms such as the binding of CheY-P to FliM 59 and CheZ, 60 the formation of homodimers of phosphorylated FixJ, 46 the alternation between different dimeric states in DctD, 61,62 and the inhibitory contacts between the unphosphorylated regulatory and effector domains of NarL 27 and CheB. 28 An analysis of residue conservation at the α4-β5-α5 face of the three major subfamilies of RR transcription factors (OmpR/PhoB, NarL/FixJ, and NtrC/DctD) revealed important differences that distinguish the OmpR/PhoB subfamily from the others. The Consurf Server 63 (http://consurf.tau.ac.il) was used to map the conservation of residues identified in a multiple sequence alignment of members of each subfamily onto the three-dimensional structure of one of its representative members in an activated state (Fig.…”

“…The crystal structures of two full-length RRs from other subfamilies, transcription factor NarL 27 and methylesterase CheB, 28 revealed the recognition helix of NarL and the active site of CheB to be occluded by their regulatory domains in their unphosphorylated states. This suggested a mechanism of regulation in which phosphorylation of the regulatory domain relieved an inhibitory interaction with the effector domain.…”