Mechanism of metal ion-induced activation of a two-component sensor kinase.

HHS Public Access Author manuscript Author Manuscript Biochem J. Author manuscript; available in PMC 2019 May 08. Published in final edited form as: Biochem J. ; 476(1): 115–135. doi:10.1042/BCJ20180577. Mechanism of Metal Ion-Induced Activation of a Two-Component Sensor Kinase Trisiani Affandi‡,1 and Megan M. McEvoy* ‡Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721 *Institute for Society and Genetics, Department of Microbiology, Immunology, and Molecular Author Manuscript Genetics, and the Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095 Abstract Author Manuscript Two-component systems are essential for bacteria to sense, respond, and adapt to changing environments, such as elevation of Cu(I)/Ag(I) ions in the periplasm. In Escherichia coli, the CusS-CusR two-component system upregulates the cusCFBA genes under increased periplasmic Cu(I)/Ag(I) concentrations to help maintain metal ion homeostasis. The CusS histidine kinase is a homodimeric integral membrane protein that binds to periplasmic Cu(I)/Ag(I) and transduces a signal to its cytoplasmic kinase domain. However, the mechanism of how metal binding in the periplasm activates autophosphorylation in the cytoplasm is unknown. Here, we report that only one of the two metal ion binding sites in CusS enhances dimerization of the sensor domain. Utilizing nanodisc technology to study full-length CusS, we show that metal-induced dimerization in the sensor domain triggers kinase activity in the cytoplasmic domain. We also investigated autophosphorylation in the cytoplasmic domain of CusS and phosphotransfer between CusS and CusR. In vitro analyses show that CusS autophosphorylates its conserved H271 residue at the N1 position of the histidine imidazole. The phosphoryl group is removed by the response regulator CusR in a reaction that requires a conserved aspartate at position 51. Functional analyses in vivo of CusS and CusR variants with mutations in the autophosphorylation or phosphoacceptor residues suggest that the phosphotransfer event is essential for metal resistance in E. coli. Biochemical analysis shows that the CusS dimer autophosphorylates using a cis mechanism. Our results support a signal transduction model in which rotation and bending movements in the cytoplasmic domain maintain the mode of autophosphorylation. Author Manuscript INTRODUCTION Bacteria live in a variety of environments and therefore have developed mechanisms to respond to changing conditions. Two-component systems (TCSs) are the predominant Correspondence: Megan M. McEvoy () . 1Present address: Department of Craniofacial Biology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045 Author Contribution TA and MMM conceived the idea for the project. TA conducted the experiments and analyzed the data. TA and MMM wrote the paper. Competing Interests The Authors declare there are no competing interests associated with the manuscript. Affandi and McEvoy Page 2 Author Manuscript Author Manuscript signaling systems used in prokaryotes to sense, respond, and adapt to a variety of stimuli in the environment (1,2). A prototypical TCS is comprised of an integral membrane histidine kinase (HK) and its cognate cytoplasmic response regulator (RR) (3). Typically, HKs have a variable extracellular sensor domain that is connected to the conserved cytoplasmic kinase domain through transmembrane helices. Upon sensing of environmental stimuli by the extracellular sensor domain, a signal is transmitted to the cytoplasmic domain initiating autophosphorylation on a conserved and specific His residue. In most prokaryotic systems, the RRs are the terminal component of the signaling pathway that function as phosphorylation-activated switches to adapt to stimuli changes. The RR catalyzes the phosphoryl transfer from the phosphohistidine of the HK to its conserved Asp in the regulatory domain. The signaling pathway consists of three phosphotransfer reactions. First, a HK homodimer uses ATP to autophosphorylate a conserved histidine residue. Second, the phosphoryl group on the phosphorylated HK (HK~P) is transferred to a conserved aspartate residue on a cognate RR. Third, the phosphorylated RR (RR~P) is dephosphorylated by an intrinsic or HK-induced RR~P autophosphatase activity. When phosphorylated, the RR~P interacts with genes or protein targets triggering cellular response to the stimuli (1,4). The mechanism of signal propagation from the sensor domain to the kinase domain resulting in changes in activity has been an active area of study. Author Manuscript While copper is an essential element for most organisms, only a small amount of intracellular copper is needed and excess amounts can be toxic even at low levels due to its redox properties (5–8). Silver is not required for any biological process, but it shares similar chemical and ligand binding properties with Cu(I), and it is toxic at even lower concentrations (9). Therefore, organisms have developed mechanisms to maintain copper and silver ion homeostasis in the intracellular environment through acquisition, sequestration, and efflux of metal ions. E. coli survives copper and silver stress in part by pumping out excess metals through the CusCFBA efflux pump; the CusS-CusR TCS regulates the expression of cusCFBA genes involved in maintaining metal ion homeostasis in cells (7,10). Author Manuscript CusS is a histidine kinase with a periplasmic sensor domain, two transmembrane (TM) αhelices, and a cytoplasmic domain. The periplasmic sensor domain of CusS senses increased levels of Cu(I)/Ag(I) and transmits a signal to the cytoplasmic kinase domain upon binding Cu(I)/Ag(I). The cytoplasmic domain of CusS consists of three domains: a “histidine kinase, adenylyl cyclases, methyl-accepting proteins, phosphatases” (HAMP) domain, a dimerization and histidine phosphotransfer (DHp) domain, and a catalytic and ATP binding domain (CA) (1). The HAMP domain consists of two amphipathic helices with coiled-coil properties that form a homodimeric, four-helical, parallel coiled-coil structure (11,12). The DHp domain includes two α-helices that mediate homodimerization by forming a four-helix bundle (1,13). The CA domain typically forms an α/β sandwich that binds ATP and catalyzes autophosphorylation of a conserved His residue in the DHp domain (14). The H271 residue of CusS corresponds to the highly conserved histidine residue found in all histidine kinases, and is the likely site of phosphorylation in this kinase, but its functional role has not yet been demonstrated in any in vitro or in vivo studies. CusR is the downstream response regulator of CusS, which consists of a receiver domain and an effector domain. Upon phosphorylation of the conserved aspartate, D51, in the receiver domain, CusR is Biochem J. Author manuscript; available in PMC 2019 May 08. Affandi and McEvoy Page 3 Author Manuscript activated and functions (...truncated)