Two‐component signaling in the AAA+ ATPase DctD: binding Mg²⁺and BeF₃selects between alternative dimeric states of the receiver domain

Abstract

A Crystallogral structure is described for the Mg2+-BeF3--bound receiver domain of Sinorhizobium meliloti DctD bearing amino acid substitution E121K. Differences between the apo- and ligand-bound active sites are similar to those reported for other receiver domains. However, the off and on states of the DctD receiver domain are characterized by dramatically different dimeric structures, which supports the following hypothesis of signal transduction. In the off state, the receiver domain and coiled-coil linker form a dimer that inhibits oligomerization of the AAA+ ATPase domain. In this conformation, the receiver domain cannot be phosphorylated or bind Mg2+ and BeF3-. Instead, these modifications stabilize an alternative dimeric conformation that repositions the subunits by approximately 20 A, thus replacing the a4-b5-a5 interface with an a4-b5 interface. Reoriented receiver domains permit the ATPase domain to oligomerize and stimulate open complex formation by the s54 form of RNA polymerase. NtrC, which shares 38% sequence identity with DctD, works differently. Its activated receiver domain must facilitate oligomerization of its ATPase domain. Significant differences exist in the signaling surfaces of the DctD and NtrC receiver domains that may help explain how triggering the common two-component switch can variously regulate assembly of a AAA+ ATPase domain.

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

“…N-terminus insertions producing constitutively active proteins were found at the interface between the N-and C-terminal domains, and in adjoining loops and helices. 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.…”

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.…”

“…Interestingly, in the NtrC/ DctD subfamily, which lacks conservation in the α4-β5-α5 face, different members have been shown to use distinctly different surfaces of the regulatory domain for multimerization. 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 structural basis for regulated assembly and function of the transcriptional activator NtrC

“…In their inactive states these proteins are usually dimers that bind to pairs of tandem sites in the enhancer elements. Upon activation via the regulatory domains, they oligomerize into ATPase-active rings that use the energy from ATP hydrolysis to physically remodel closed complexes of 54 holoenzyme and promoter DNA (Rombel et al 1998;Neuwald et al 1999;Chaney et al 2001;Lee et al 2003).Crystal or NMR structures are available for several isolated domains and truncation constructs of EBPs including NtrC, DctD, PspF, ZraR, and NtrC1 (Volkman et al 1995;Kern et al 1999;Pelton et al 1999;Meyer et al 2001;Park et al 2002;Hastings et al 2003;Lee et al 2003;Doucleff et al 2005;Rappas et al 2005;Sallai and Tucker 2005). Existing structural information on DctD and NtrC1 has been used to provide a model of how two-component signal transduction can regulate assembly of their AAA+ ATPase domains ), but this model fails to explain regulation in the closely related protein NtrC (40% sequence identity, 60% sequence similarity) Hastings et al 2003).…”