title: ENDOR studies of VO2+: probing protein-metal ion interactions in nephrocalcin.
abstract: Nephrocalcin inhibits the growth of calcium oxalate monohydrate crystals in the mammalian kidney. Isoforms A and B contain three equivalents of gamma-carboxyglutamic acid (Gla) residues implicated in Ca2+-binding and exhibit strong inhibitor properties and high Ca2+-binding affinity (Kd approximately 10(-8) M). Isoforms C and D lack these properties and exhibit low Ca2+-binding affinity (Kd approximately 10(-6) M). With VO2+ as a structural probe, electron paramagnetic resonance (EPR) studies of the Ca2+-binding sites of isoforms B and D showed that VO2+ binds competitively with a metal ion:protein stoichiometry of 4:1. EPR spectral parameters of the VO2+ ion were consistent with only equatorial oxygen-donor ligands. EPR and angle-selected electron nuclear double resonance (ENDOR) spectra showed two equatorially positioned, metal coordinating waters in isoform D while in isoform B no ligands undergoing hydrogen exchange were found. Since isoform D showed no evidence for axially coordinated water, similarly to isoform B, it is likely that the protein residues occupying the axial sites are identical in both proteins. ENDOR spectra of VO2+-complexes of isoforms B and D were compared to spectra of the VO2+-complex with 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 the crystal structure of Ca(EMA)2.
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, Illinois 60637, USA.
authors.forename: D
authors.initials: D
authors.lastname: Mustafi
authors.forename: Y
authors.initials: Y
authors.lastname: Nakagawa
authors.forename: M W
authors.initials: MW
authors.lastname: Makinen
mesh_terms: D000818:Animals; D016880:Anisotropy; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D006023:Glycoproteins / Q000737:chemistry* / Q000494:pharmacology*; D007477:Ions; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008280:Magnetics; D008314:Malonates / Q000378:metabolism; D008958:Models, Molecular; D008962:Models, Theoretical; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D011522:Protons; D014466:Ultraviolet Rays; D014638:Vanadates / Q000494:pharmacology*; D017968:Vanadium Compounds / Q000494:pharmacology
chemical_list: D006023:Glycoproteins; D007477:Ions; D008024:Ligands; D008314:Malonates; D020033:Protein Isoforms; D011522:Protons; D017968:Vanadium Compounds; C029490:nephrocalcin; D014638:Vanadates; C038080:ethylmalonic acid; C034028:vanadyl sulfate; D002118:Calcium