NICKEL BINDING AND IMMUNOLOGICAL PROPERTIES OF THE C-TERMINAL DOMAIN OF THE HELICOBACTER-PYLORI GROES HOMOLOG (HSPA)

Helicobacter pylori synthesizes a heat-shock protein of the GroES clas s, The gene encoding this protein (heat-shock protein A, HspA) was rec ently cloned and it was shown to be unique in structure. H. pylori Hsp A consists of two domains: the N-terminal domain (domain A) homologous with other GroES proteins, and a C-terminal domain (domain B) corresp onding to 27 additional residues resembling a metal-binding domain, Va rious 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 cat ion binding properties of the various MBP and MBP-fused proteins allow ed us to conclude that HspA binds nickel ions by means of its C-termin al domain, HspA exhibited a high and specific affinity for nickel ions in comparison with its affinity for other divalent cations (copper, z inc, cobalt). Equilibrium dialysis experiments revealed that MBP-HspA binds nickel ions with an apparent dissociation constant (K-d) of 1.8 mu M 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, r espectively. The two variants differed from each other by the simultan eous 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 t he two HspA variants, When co-expressed together with the H. pylori ur ease gene cluster in Escherichia coil cells, the two HspA variant-enco ding genes led to a fourfold increase in urease activity, demonstratin g that HspA in H. pylori has a specialized function with regard to the nickel metalloenzyme urease.