Multimerization of Phosphorylated and Non-phosphorylated ArcA Is Necessary for the Response Regulator Function of the Arc Two-component Signal Transduction System

Abstract

To adapt to anaerobic conditions, Escherichia coli operates the Arc two-component signal transduction system, consisting of a sensor kinase, ArcB, and a response regulator, ArcA. ArcA is converted to the active form, phosphorylated ArcA (ArcA-P), by ArcB-mediated phosphorylation. The active ArcA-P binds to the promoter regions of target genes, thereby regulating their transcriptional activities. The phosphoryl group of ArcA-P is unstable with a half-life of 30 min. However, we were able to inhibit the dephosphorylation for more than 12 h by the addition of EDTA; this allowed us to characterize ArcA-P. Gel-filtration and glycerol sedimentation experiments demonstrated that ArcA exists as a homo-dimer. ArcA phosphorylated by either ArcB or carbamyl phosphate multimerizes to form a tetramer of dimers; this multimer binds to the ArcA DNA binding site. Isoelectric focusing gel electrophoresis and nitrocellulose-filter binding analyses indicated that the ArcA multimer is composed of both ArcA-P and ArcA in a ratio, 1:1. The ArcA(D54E) mutant protein was unable to be phosphorylated by ArcB. This defect resulted in the inability of ArcA(D54E) to form a multimer or to bind to the ArcA DNA binding site. These results indicate that phosphorylation of ArcA induces multimerization prior to DNA binding, and the multimerization is a prerequisite for binding. Our results suggest a novel model that phosphorylation of ArcA by ArcB regulates multimerization of ArcA, which in turn functions as a response regulator.

A two-component phosphotransfer network involving ArcB, ArcA, and RssB coordinates synthesis and proteolysis of σS (RpoS) in E. coli

“…Both phosphorylated, as well as nonphosphorylated ArcA protein bound to this rpoSp1 promoter fragment, but produced different band-shift patterns (Fig. 3A), consistent with previous similar observations at other ArcAcontrolled promoters (Lynch and Lin 1996b) and the finding that nonphosphorylated ArcA forms a dimer, whereas ArcA phosphorylation results in a higher oligomeric state (Jeon et al 2001).…”

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

“…Previous studies using electrophoretic mobility shift and DNAse I protection assays have shown that phosphorylation of ArcA is required for specific binding to target sites, that broad regions of DNA are protected by ArcA-P suggesting formation of multimers and that this multimerization can occur independently of DNA binding. 51,54,55 Solution studies, both size-exclusion chromatography and sedimentation, indicate that the isolated regulatory domain of ArcA exits exclusively as a monomer. Yet ArcA N crystallizes as a dimer in both the absence and presence of BeF 3 − .…”

“…51 Based on gel-filtration and glycerol gradient sedimentation analyses, Jeon et al concluded that unphosphorylated ArcA exists as a dimer and active ArcA exists as an octamer. Our SE and SV results also indicate that unphosphorylated ArcA exists as a dimer, but in equilibrium with a monomer with a K d ~400 μM.…”

“…The half-life of phosphorylation of ArcA is short but can be significantly increased by the removal of divalent cations. 51 Specifically, HPLC analysis of phosphorylated ArcA N (ArcA N -P) treated with 50 mM ethylene diamine tetraacetic acid (EDTA) subsequent to phosphorylation indicated >95% phosphorylation after 24 h (unpublished data), longer than the 12 h required for SV analysis. Figure 7(d) shows a c(s) distribution of ArcA N -P at concentrations ranging from 36 to 143 μM.…”

“…It has been reported that full-length ArcA exists as a dimer in solution in its inactive state and forms an octamer upon phosphorylation, with a 1:1 ratio of unphosphorylated to phosphorylated protein. 51 We have further examined the behavior of ArcA in solution by analyzing the contributions of the individual domains to oligomerization. First, we examined the phosphorylation state of ArcA and ArcA N in the presence of the small molecule phosphodonor phosphoramidate 52 by using reverse-phase high performance liquid chromatography (HPLC) to separate the unphosphorylated and phosphorylated proteins.…”