The Atypical Response Regulator Protein ChxR Has Structural Characteristics and Dimer Interface Interactions That Are Unique within the OmpR/PhoB Subfamily

Abstract

Typically as a result of phosphorylation, OmpR/PhoB response regulators form homodimers through a receiver domain as an integral step in transcriptional activation. Phosphorylation stabilizes the ionic and hydrophobic interactions between monomers. Recent studies have shown that some response regulators retain functional activity in the absence of phosphorylation and are termed atypical response regulators. The two currently available receiver domain structures of atypical response regulators are very similar to their phospho-accepting homologs, and their propensity to form homodimers is generally retained. An atypical response regulator, ChxR, from Chlamydia trachomatis, was previously reported to form homodimers; however, the residues critical to this interaction have not been elucidated. We hypothesize that the intra-and intermolecular interactions involved in forming a transcriptionally competent ChxR are distinct from the canonical phosphorylation (activation) paradigm in the OmpR/PhoB response regulator subfamily. To test this hypothesis, structural and functional studies were performed on the receiver domain of ChxR. Two crystal structures of the receiver domain were solved with the recently developed method using triiodo compound I3C. These structures revealed many characteristics unique to OmpR/PhoB subfamily members: typical or atypical. Included was the absence of two ␣-helices present in all other OmpR/ PhoB response regulators. Functional studies on various dimer interface residues demonstrated that ChxR forms relatively stable homodimers through hydrophobic interactions, and disruption of these can be accomplished with the introduction of a charged residue within the dimer interface. A gel shift study with monomeric ChxR supports that dimerization through the receiver domain is critical for interaction with DNA.

Atypical Response Regulator ChxR from Chlamydia trachomatis Is Structurally Poised for DNA Binding

“…However, the DNA binding domains of ChxR appear to have tandem symmetry (head-to-tail) based upon the SAXS analysis presented herein, as well as their monomeric state and reduced DNA binding affinity in the absence of the receiver domain. This matches the orientation of the DNA sequence motif (direct repeat) recognized by ChxR [13]. The resulting orientation is not unexpected for an OmpR/PhoB subfamily member, and was recently highlighted by Bachhawat et al [73].…”

“…The active state is stabilized by phosphorylation, which enhances DNA binding affinity, and makes dimerization through the α4–β5–α5 interface energetically favorable. Atypical RRs exist in a constitutively active, dimeric state as demonstrated by previous studies on ChxR [13], [17] and HP1043 [12], [14], [52]. Recent studies by Barbieri et al demonstrated that interdomain interfaces stabilize the inactive state and inhibit phosphotransfer-mediated activation [5].…”

Use of restrained molecular dynamics to predict the conformations of phosphorylated receiver domains in two‐component signaling systems

“…These two conserved amino acids are referred to as “switch residues” and are used as traditional indicators of conformational state for rec domains . Some studies have also implicated the aromatic residue (Phe/Tyr/His) as a factor in rec domain dimerization, due to its location in the center of a common dimerization interface . In CheY, these switch residues are Thr87 and Tyr106.…”

Atypical OmpR/PhoB Subfamily Response Regulator GlnR of Actinomycetes Functions as a Homodimer, Stabilized by the Unphosphorylated Conserved Asp-focused Charge Interactions

“…Recently, the atypical receiver domains of several orphan RRs (7,22,23) were shown to be generally similar to the typical receiver domains in their amino acid sequences and three-dimensional structures but lacked one or more residues of the highly conserved active site quintet (22,24). Because the GlnR from actinomycetes other than streptomycetes (see Fig.…”