Structure of an Atypical Orphan Response Regulator Protein Supports a New Phosphorylation-independent Regulatory Mechanism

Abstract

Two-component signal transduction systems, commonly found in prokaryotes, typically regulate cellular functions in response to environmental conditions through a phosphorylation-dependent process. A new type of response regulator, hp1043 (HP-RR) from Helicobacter pylori, has been recently identified. HP-RR is essential for cell growth and does not require the well known phosphorelay scheme. Unphosphorylated HP-RR binds specifically to its own promoter (P 1043 ) and autoregulates the promoter of the tlpB gene (P tlpB ). We have determined the structure of HP-RR by NMR and x-ray crystallography, revealing a symmetrical dimer with two functional domains. The molecular topology resembles that of the OmpR/PhoB subfamily, however, the symmetrical dimer is stable even in the unphosphorylated state. The dimer interface, formed by three secondary structure elements (␣4-␤5-␣5), resembles that of the active, phosphorylated forms of ArcA and PhoB. Several conserved residues of the HP-RR dimeric interface deviate from the OmpR/PhoB subfamily, although there are similar salt bridges and hydrophobic patches within the interface. Our findings reveal how a new type of response regulator protein could function as a cell growth-associated regulator in the absence of post-translational modification.

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

“…The signature "switch" residues involved in rearrangements associated with phosphorylation are Ser 75 and Tyr 94 in HP-RR, located in the β4-α4 loop and β5 region, respectively. In the HP-RR structure, Ser 75 is oriented toward the active site and Tyr 94 adopts an inward position forming a hydrogen bond with the main chain nitrogen atom of Thr 79 located at the β4-α4 loop (Hong et al, 2007). According to the model-free result, the site of rearrangements associated with phosphorylation of HP-RR r showed the great conformational exchanges (Ser 75 : R ex = 3.382, Ile 95 : R ex = 5.228).…”

“…Protein expression, isotopic enrichment, and purification DNA fragments of HP-RR r (residues 1-119) and HP-RR e (residues 120-223) were previously cloned into pET-21b3-2 and pET-21a(+) vectors (GE Healthcare, Sweden) containing an IPTG inducible promoter and resistance to ampicillin (Hong et al, 2007). These vectors were used to transform the E. coli strain BL21 (DE3) (Invitrogen, USA) for fusion protein expression.…”

“…In most RRs, phosphorylation induces conformational changes of the conserved region including the β4-α4 loop and the α4 helix. The signature "switch" residues involved in rearrangements associated with phosphorylation are Ser 75 and Tyr 94 , located in the β4-α4 loop and β5 region, respectively (Hong et al, 2007). Therefore, this may be the reason why Asp 93 and Ile 95 , residues located in the vicinity of Tyr 94 , are associated with a low hNOE value and a high R 2 value with conformational exchange, re- spectively.…”

“…The β-hairpin wing between two β-sheets (β11/β12) of the C-terminus shown in Fig. 7A with pink arrows involves the minor groove DNA recognition (Hong et al, 2007). According to the chemical exchange data from the model free analysis, the centers of loop regions known as the transcription loop (Glu 175 : R ex = 6.501) and the residues at the Ala 156 and Phe 214 at the core hydrophobic packing region showed large conformational exchange, too (Fig.…”

Expression, purification, crystallization and preliminary X-ray analysis of the DNA-binding domain of aChlamydia trachomatisOmpR/PhoB-subfamily response regulator homolog, ChxR

“…Despite this fundamental difference from OmpR/PhoB response regulators, we hypothesize that the DNA-binding effector domain of ChxR (ChxR Eff ) is in a structural conformation similar to those of other OmpR/PhoB response regulators. This is supported by structural analysis of other atypical response regulators (Hong et al, 2007). In addition to demonstrating overall structural similarity, determination of the three-dimensional structure of ChxR Eff is expected to reveal the residues within and the orientation of the functional regions ( 6-7 DNA minor-groove binding wing, 3 DNA major-groove binding helix and 2-3 transactivation loop).…”