Interconversion of the CD and EF sites in oncomodulin. Influence on the Eu3+ 7F0-->5D0 excitation spectrum.

Abstract

The appearance of the parvalbumin Eu3+ 7F0-->5D0 spectrum is markedly pH dependent, the result of a hitherto unidentified deprotonation event in the CD ion-binding domain [Treviño, C.L., et al. (1991) J. Biol. Chem. 265, 9694-9700]. We are studying this phenomenon in the mammalian placental parvalbumin called oncomodulin. As in other parvalbumins, the liganding residues in the CD and EF sites of oncomodulin differ at the +z and -x coordination positions: serine and aspartate, respectively, in the CD site; aspartate and glycine in the EF site. We have prepared a series of oncomodulin variants in which the +z and/or -x residue(s) from one site have been replaced by the corresponding residue(s) from the other. We herein characterize the resulting proteins by Eu3+ luminescence spectroscopy. Simultaneous replacement of serine-55 by aspartate and aspartate-59 by glycine affords the CD site with a coordination sphere superficially equivalent to that of the EF site. As observed previously for the S55D mutation [Henzl, M. T., et al. (1992) FEBS Lett. 314, 130-134], the Eu3+ 7F0-->5D0 spectrum of the 55/59 variant is pH independent. Interestingly, replacement of aspartate-94 by serine at the +z position of the EF site of 55/59 imparts pH dependent behavior to the EF site. The identical mutation in the wild-type background likewise imparts pH dependence to the EF site, affording a protein in which both sites display broad signals near 578.2 nm at pH 8. Significantly, a variant in which threonine replaces serine-55 retains the pH dependent spectroscopic signature. These results indicate that the presence of a hydroxyl group at the +z position is sufficient to confer pH dependence on the 7F0-->5D0 spectrum of a parvalbumin EF-hand domain. Importantly, the data also suggest that the component peaks of the low-pH doublet are not site-specific signals, as previously believed. Rather, they probably represent differences in coordination environment arising from differential hydration or conformational heterogeneity. In wild-type oncomodulin, the CD site signal dominates the low-pH spectrum. Since this dominance persists even when serine-55 and aspartate-59 are replaced by the corresponding EF site residues, it appears that the context of the CD binding site, as dictated by the global polypeptide fold, exerts a major influence on the metal ion-binding properties of the site.