Regulation of Response Regulator Autophosphorylation through Interdomain Contacts

Abstract

DNA-binding response regulators (RRs) of the OmpR/PhoB subfamily alternate between inactive and active conformational states, with the latter having enhanced DNA-binding affinity. Phosphorylation of an aspartate residue in the receiver domain, usually via phosphotransfer from a cognate histidine kinase, stabilizes the active conformation. Many of the available structures of inactive OmpR/PhoB family proteins exhibit extensive interfaces between the N-terminal receiver and C-terminal DNA-binding domains. These interfaces invariably involve the α4-β5-α5 face of the receiver domain, the locus of the largest differences between inactive and active conformations and the surface that mediates dimerization of receiver domains in the active state. Structures of receiver domain dimers of DrrB, DrrD, and MtrA have been determined, and phosphorylation kinetics were analyzed. Analysis of phosphotransfer from small molecule phosphodonors has revealed large differences in autophosphorylation rates among OmpR/PhoB RRs. RRs with substantial domain interfaces exhibit slow rates of phosphorylation. Rates are greatly increased in isolated receiver domain constructs. Such differences are not observed between autophosphorylation rates of full-length and isolated receiver domains of a RR that lacks interdomain interfaces, and they are not observed in histidine kinase-mediated phosphotransfer. These findings suggest that domain interfaces restrict receiver domain conformational dynamics, stabilizing an inactive conformation that is catalytically incompetent for phosphotransfer from small molecule phosphodonors. Inhibition of phosphotransfer by domain interfaces provides an explanation for the observation that some RRs cannot be phosphorylated by small molecule phosphodonors in vitro and provides a potential mechanism for insulating some RRs from small molecule-mediated phosphorylation in vivo.

Phosphorylation-dependent conformational changes and domain rearrangements in Staphylococcus aureus VraR activation

“…2A). Receiver and DNA-binding domain packing arrangements are not conserved in available structures of inactive NarL/FixJ subfamily response regulators (12,13), suggesting that a variety of closed conformations have evolved as has been observed in the more widely characterized OmpR/PhoB winged-helix transcription factor response regulator subfamily (14). The association of the isolated VraR receiver domain (VraR REC ) and DNA-binding domain (VraR DBD ) is relatively weak, with a dissociation constant of 134 μM (Table 1).…”

Sensor–response regulator interactions in a cross-regulated signal transduction network

“…Receiver domain inhibition by the effector domain stabilizes the inactive state for diverse response regulators that function as transcriptional factors (Barbieri et al, 2010). This functions to inhibit phosphorylation by metabolites such as acetyl phosphate, without influencing sensormediated phosphorylation (Barbieri et al, 2010;Da Re et al, 1999).…”

“…This functions to inhibit phosphorylation by metabolites such as acetyl phosphate, without influencing sensormediated phosphorylation (Barbieri et al, 2010;Da Re et al, 1999). Indeed, NarX robustly phosphorylates NarL both in vivo and in vitro , so the relatively small effect on NarX-NarL interaction implied by the BACTH analysis, if genuine, would not meaningfully influence phosphoryl transfer between this cognate pair.…”

Structure and dynamics of the polymyxin-resistance-associated response regulator PmrA in complex with the promoter DNA

“…The recognition helixes of PrrA 9 and MtrA 10 are occluded by the REC-DBD interactions, which prevents them from binding to DNA in the inactive state. However, there is no interaction between REC and DBD in the DrrD 12 inactive structure 11 .…”

“…Structural studies of inactive, unphosphorylated response regulators showed diverse interactions between REC and DBD. DrrB 8 , PrrA 9 and MtrA 10 have extensive interfaces between REC and DBD that inhibit phosphorylation of these response regulators by small-molecule phosphodonors 11 . The recognition helixes of PrrA 9 and MtrA 10 are occluded by the REC-DBD interactions, which prevents them from binding to DNA in the inactive state.…”