HYPB PROTEIN OF BRADYRHIZOBIUM-JAPONICUM IS A METAL-BINDING GTPASE CAPABLE OF BINDING 18 DIVALENT NICKEL IONS PER DIMER

Bradyrhizobium japonicum hypB encodes a protein containing an extremely histidine-rich region (24 histidine residues within a 39-amino-acid stretch) and guanine nucleotide-binding domains. The product of the hypB gene was overexpressed in Escherichia coli and purified by Ni2+-charged metal chelate affinity chromatography (MCAC) in a single step. In SDS/PAGE, HypB migrated at 38 kDa-slightly larger than the calculated molecular mass (32.8 kDa). Purified HypB has GTPase activity with a k(cat) of 0.18 min(-1) and a K-m for GTP of 7 mu M, and it has dGTPase activity as well. HypB exists as a dimer of molecular mass 78 kDa in native solution as determined by fast protein liquid chromatography on Superose 12. It binds 9.0 +/- 0.14 divalent nickel ions per monomer (18 Ni2+ per dimer) with a K-d of 2.3 mu M; it also binds Zn2+, CU2+, Co2+, Cd2+) and Mn2+. In-frame deletion of the histidine rich region (deletion of 38 amino acids including 23 histidine residues) resulted in a truncated HypB that did not bind to the MCAC column, whereas in-frame deletion of 14 amino acids including 8 histidine residues within HypB resulted in a truncated HypB that still bound to the column, The results indicate that the histidine residues within the histidine-rich region of HypB are involved in metal binding.