Characterization of vanadium-binding sites of the vanadium-binding protein Vanabin2 by site-directed mutagenesis

Background: Vanabins are a unique protein family of vanadium-binding proteins with nine disulfide bonds. Possible binding sites for VO²⁺ in Vanabin2 from a vanadium-rich ascidian Ascidia sydneiensis samea have been detected by nuclear magnetic resonance study, but the metal selectivity and metal-binding ability of each site was not examined. Methods: In order to reveal functional contribution of each binding site, we prepared several mutants of Vanabin2 by in vitro site-directed mutagenesis and analyzed their metal selectivity and affinity by immobilized metal-ion affinity chromatography and Hummel Dreyer method. Results: Mutation at K10/R60 (site 1) markedly reduced the affinity for VO²⁺. Mutation at K24/K38/R41/R42 (site 2) decreased the maximum binding number, but only slightly increased the overall affinity for VO²⁺. Secondary structure of both mutants was the same as that of the wild type as assessed by circular dichroism spectroscopy. Mutation in disulfide bonds near the site 1 did not affect its high affinity binding capacity, while those near the site 2 decreased the overall affinity for VO²⁺. General significance: These results suggested that the site 1 is a high affinity binding site for VO²⁺, while the site 2 composes a moderate affinity site for multiple VO²⁺.