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+.


CONCLUSIONS
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+.
Ueki
Tatsuya
T
Molecular Physiology Laboratory, Department of Biological Science, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan. ueki@hiroshima-u.ac.jp
Kawakami
Norifumi
N
Toshishige
Masaaki
M
Matsuo
Koichi
K
Gekko
Kunihiko
K
Michibata
Hitoshi
H
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D002942:Circular Dichroism; D003035:Cobalt / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014561:Urochordata / Q000235:genetics / Q000378:metabolism*; D014639:Vanadium / Q000737:chemistry / Q000378:metabolism*; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
D002352:Carrier Proteins; D014639:Vanadium; D003035:Cobalt; D015032:Zinc