Structure of the Escherichia coli Response Regulator NarL^,

Abstract

The crystal structure analysis of the NarL protein provides a first look at interactions between receiver and effector domains of a full-length bacterial response regulator. The N-terminal receiver domain, with 131 amino acids, is folded into a 5-strand beta sheet flanked by 5 alpha helices, as seen in CheY and in the N-terminal domain of NTRC. The C-terminal DNA-binding domain, with 62 amino acids, is a compact bundle of 4 alpha helices, of which the middle 2 form a helix-turn-helix motif closely related to that of Drosophila paired protein and other H-T-H DNA-binding proteins. The 2 domains are connected by an alpha helix of 10 amino acids and a 13-residue flexible tether that is not visible and presumably disordered in the X-ray structure. In this unphosphorylated form of NarL, the C-terminal domain is turned against the receiver domain in a manner that would preclude DNA binding. Activation of NarL via phosphorylation of Asp59 must involve transfer of information to the interdomain interface and either rotation or displacement of the DNA-binding C-terminal domain. Docking of a B-DNA duplex against the isolated C-terminal domain in the manner observed in paired protein and other H-T-H proteins suggests a stereochemical basis for DNA sequence preference: T-R-C-C-Y (high affinity) or T-R-C-T-N (low affinity), which is close to the experimentally observed consensus sequence: T-A-C-Y-N. The NarL structure is a model for other members of the FixJ or LuxR family of bacterial transcriptional activators, and possibly to the more distant OmpR and NtrC families as well.

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

“…It is not surprising therefore that many RRs utilize the α4-β5-α5 face for regulatory protein-protein interactions, the affinities of which are altered by phosphorylation. 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.…”

“…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.…”

“…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.…”

Differential regulation by the homologous response regulators NarL and NarP of Escherichia coli K‐12 depends on DNA binding site arrangement

“…Aspects of these phosphoryl-transfer reactions have been documented in vitro (Walker and DeMoss, 1993;Schrö der et al, 1994;Cavicchioli et al, 1995). Interest in the Nar regulatory system has been stimulated by the availability of a crystal structure for NarL in its unphosphorylated form (Baikalov et al, 1996).…”

Backbone Dynamics of an Atypical Orphan Response Regulator Protein, Helicobacter pylori 1043

“…RRs are typically found at the ends of autophosphotransfer pathways where they regulate transcription functioning as phosphorylation-activated switches that regulate output responses. The majority of RRs are transcription factors with effector domains that can be divided into three major subfamilies based on their DNA-binding domains: the OmpR/PhoB winged-helix domains (Kondo et al, 1997;Martinez-Hackert and Stock, 1997;Okamura et al, 2000), the NarL/FixJ four-helix domains (Baikalov et al, 1996;, and NtrC ATPase-coupled transcription factors (Pelton et al, 1999). The HP-RR is grouped into the OmpR/PhoB subfamily of response regulators, which shares structural similarities on its C-terminal effector domain with these proteins.…”