GroES is an indispensable chaperonin virtually found throughout all life forms. Consequently, mutations of this protein must be critically scrutinized by natural selection. Nevertheless, the homolog from a potentially virulent gastric pathogen, Helicobacter pylori, strikingly features a histidine/cysteine-rich C terminus that shares no significant homology with other family members. Additionally, three more (H45, C51, and C53) are uniquely present in its apical domain. The statistical analyses show that these residues may have originated from negative selection, presumably driven by either dependent or independent amino acid mutations. In the absence of the C-terminal metal-binding domain, the mutant protein still exhibits a substantial capacity for zinc binding in vivo. The biochemical properties of site-directed mutants indicate that H45, C51, and C53 make up an oxidation-sensitive zinc-binding site that may donate the bound metal to a zinc acceptor. Of interest, bismuth antiulcer drugs strongly bind at this site (K(d) of approximately 7 x 10(-26) M), replacing the bound zinc and consequently inducing the disruption of the quaternary structure. Because biological features by negative selection are usually inert to change during evolution, this study sheds light on a promising field whereby medicines can be designed or improved to specifically target the residues that uniquely evolved in pathogenic proteins so as to retard the emergence of drug resistance.