Probing catalytically essential domain orientation in histidine kinase EnvZ by targeted disulfide crosslinking.
EnvZ, a dimeric transmembrane histidine kinase, belongs to the family of His-Asp phosphorelay signal transduction systems. The cytoplasmic kinase domain of EnvZ can be dissected into two independently functioning domains, A and B, whose NMR solution structures have been individually determined. Here, we examined the topological arrangement of these two domains in the EnvZ dimer, a structure that is key to understanding the mechanism underlying the autophosphorylation activity of the kinase. A series of cysteine substitution mutants were constructed to test the feasibility of chemical crosslinking between the two domains. These crosslinking data demonstrate that helix I of domain A of one subunit in the EnvZc dimer is in close proximity to domain B of the other subunit in the same dimer, while helix II of domain A of one subunit interacts with domain B of the same subunit in the EnvZc dimer. This is the first demonstration of the topological arrangement between the central dimerization domain containing the active center His residues (domain A) and the ATP-binding catalysis assisting domain (domain B) in a class I histidine kinase.
Cai
Sheng-Jian
SJ
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, NJ 08854, USA.
Khorchid
Ahmad
A
Ikura
Mitsuhiko
M
Inouye
Masayori
M
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D020134:Catalytic Domain; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000071677:Histidine Kinase; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
D001425:Bacterial Outer Membrane Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D021122:Protein Subunits; D011994:Recombinant Proteins; D011494:Protein Kinases; D000071677:Histidine Kinase; C057843:envZ protein, E coli; D003545:Cysteine