Spontaneous subunit exchange and biochemical evidence for trans-autophosphorylation in a dimer of Escherichia coli histidine kinase (EnvZ)

Sheng Jian Cai, Masayori Inouye

Research output: Contribution to journalArticlepeer-review

35 Scopus citations


The EnvZ/OmpR histidyl-aspartyl phosphorelay (HAP) system in Escherichia coli regulates the expression of ompF and ompC, the major outer membrane porin genes, in response to environmental osmolarity changes. Here, we report that dimers of EnvZc, the cytoplasmic domain of EnvZ, undergo spontaneous subunit exchange in solution. By introducing a cysteine substitution (S260C) in the dimerization domain of EnvZc, we were able to crosslink the two subunits in a dimer and trap the heterodimer formed between two different mutant EnvZc. By using a complementing system with two autophosphorylation-defective EnvZc mutants, one containing the H243V mutation at the autophosphorylation site and the other containing the G405A mutation in the ATP-binding domain, we demonstrated that an EnvZc(G405A) subunit can be phosphorylated by an EnvZc(H243V) subunit only when a heterodimer is formed. The rate of subunit exchange is concentration-dependent, with higher rates at higher concentrations of protein. The disulfide-crosslinked EnvZc(G405A) homodimer could not be phosphorylated by EnvZc(H243V), since the heterodimer formation between the two mutant proteins was blocked, indicating that autophosphorylation cannot occur by dimer-dimer interaction. By using MBP-ΔL-EnvZc(S260C) fusion protein (ΔL: the linker region, spanning residues 180-222, was deleted), it was found that in the disulfide-crosslinked MBP-ΔL-EnvZc(S260C)/ΔL-EnvZc(S260C/G405A) heterodimer, only the ΔL-EnvZc(S260C/G405A) subunit was phosphorylated but not the MBP-ΔL-EnvZc(S260C) subunit. Together, the present results provide biochemical evidence that EnvZ autophosphorylation occurs in trans and only within an EnvZ dimer.

Original languageEnglish (US)
Pages (from-to)495-503
Number of pages9
JournalJournal of molecular biology
Issue number3
StatePublished - Jun 6 2003

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Molecular Biology


  • Disulfide cross-linking
  • Histidine kinase
  • Osmoregulation
  • Signal transduction
  • Subunit exchange

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