Nickel binding and immunological properties of the C-terminal domain of the Helicobacter pylori GroES homologue (HspA).
Helicobacter pylori synthesizes a heat-shock protein of the GroES class. The gene encoding this protein (heat-shock protein A, HspA) was recently cloned and it was shown to be unique in structure. H. pylori HspA consists of two domains: the N-terminal domain (domain A) homologous with other GroES proteins, and a C-terminal domain (domain B) corresponding to 27 additional residues resembling a metal-binding domain. Various recombinant proteins consisting of the entire HspA polypeptide, the A domain, or the B domain were produced independently as proteins fused to maltose-binding protein (MBP). Comparison of the divalent cation binding properties of the various MBP and MBP-fused proteins allowed us to conclude that HspA binds nickel ions by means of its C-terminal domain. HspA exhibited a high and specific affinity for nickel ions in comparison with its affinity for other divalent cations (copper, zinc, cobalt). Equilibrium dialysis experiments revealed that MBP-HspA binds nickel ions with an apparent dissociation constant (Kd) of 1.8 microM and a stoichiometry of 1.9 ions per molecule. The analysis of the deduced HspA amino acid sequences encoded by 35 independent clinical isolates demonstrated the existence of two molecular variants of HspA, i.e. a major and a minor variant present in 89% and 11% of strains, respectively. The two variants differed from each other by the simultaneous substitution of seven amino acids within the B domain, whilst the A domain was highly conserved amongst all the HspA proteins (99-100% identity). On the basis of serological studies, the highly conserved A domain of HspA was found to be the immunodominant domain. Functional complementation experiments were performed to test the properties of the two HspA variants. When co-expressed together with the H. pylori urease gene cluster in Escherichia coli cells, the two HspA variant-encoding genes led to a fourfold increase in urease activity, demonstrating that HspA in H. pylori has a specialized function with regard to the nickel metalloenzyme urease.
Kansau
I
I
Unité de Pathogénie Bactérienne des Muqueuses, Institut Pasteur, INSERM U389, Paris, France.
Guillain
F
F
Thiberge
J M
JM
Labigne
A
A
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology*; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics; D018835:Chaperonin 10 / Q000235:genetics / Q000276:immunology*; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins / Q000235:genetics / Q000276:immunology*; D016480:Helicobacter pylori / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007477:Ions; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009532:Nickel / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology
D018528:ATP-Binding Cassette Transporters; D000942:Antigens, Bacterial; D002352:Carrier Proteins; D018835:Chaperonin 10; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D007477:Ions; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D009532:Nickel