Endor studies of VO2+: Probing protein-metal ion interactions in nephrocalcin

Nephrocalcin inhibits the growth of calcium oxalate monohydrate crystals in the mammalian kidney. Isoforms A and B contain three equivalents bf gamma -carboxyglutamic acid (Gla) residues implicated in Ca2+-binding and exhibit strong inhibitor properties and high Ca2+-binding affinity (Kd similar to 10(-8) M). Isoforms C and D lack these properties and exhibit low Ca2+-bind ing affinity (Kd similar to 10(-6) M). With VO2+ as a structural probe, ele ctron paramagnetic resonance (EPR) studies of the Ca2+-binding sites of iso forms B and D showed that VO2+ binds competitively with a metal ion : prote in stoichiometry of 4:1. EPR spectral parameters of the VO2+ ion were consi stent with only equatorial oxygen-donor ligands. EPR and angle-selected ele ctron nuclear double resonance (ENDOR) spectra showed two equatorially posi tioned, metal coordinating waters in isoform D while in isoform B no ligand s undergoing hydrogen exchange were found. Since isoform D showed no eviden ce for axially coordinated water, similarly to isoform B, it is likely that the protein residues occupying the axial sites are identical in both prote ins. ENDOR spectra of VO2+-complexes of isoforms B and D were compared to s pectra of the VO2+-complexwith alpha -ethylmalonic acid (EMA), a molecular mimic of Gla. Spectra of the VO2+-complex of EMA showed axial water located trans to the V=O bond and outer shell water hydrogen-bonded to the vanadyl oxygen, consistent with the X-ray structure of Ca(EMA)(2). We, therefore, conclude that the spatial disposition of carboxylate groups of Gla residues coordinating Ca2+ in isoforms A and B must differ from that observed in th e crystal structure of Ca(ENLA)(2).