INTERCONVERSION OF THE CD AND EF SITES IN ONCOMODULIN - INFLUENCE ON THE EU(3-]D-5(O) EXCITATION SPECTRUM() (7)FO)

The appearance of the parvalbumin Eu3+ F-7(0)-->D-5(0) spectrum is mar kedly pH dependent, the result of a hitherto unidentified deprotonatio n event in the CD ion-binding domain [Trevino, C. L., et al. (1991) J. Biol. Chem. 265, 9694-9700]. We are studying this phenomenon in the m ammalian placental parvalbumin called oncomodulin. As in other parvalb umins, the liganding residues in the CD and EF sites of oncomodulin di ffer at the +z and -x coordination positions: serine and aspartate, re spectively, in the CD site; aspartate and glycine in the EF site. We h ave prepared a series of oncomodulin variants in which the +z and/or - x residue(s) from one site have been replaced by the corresponding res idue(s) from the other.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. (19 92) FEES Lett. 314, 130-134], the Eu3+ F-7(0)-->D-5(0) spectrum of the 55/59 variant is pH independent. Interestingly, replacement of aspart ate-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, afford ing a protein in which both sites display broad signals near 578.2 nm at pH 8. Significantly, a variant in which threonine replaces serine-5 5 retains the pH dependent spectroscopic signature. These results indi cate that the presence of a hydroxyl group at the +z position is suffi cient to confer pH dependence on the F-7(0)-->D-5(0) spectrum of a par valbumin EF-hand domain. Importantly, the data also suggest that the c omponent peaks of the low-pH doublet are not site-specific signals, as previously believed. Rather, they probably represent differences in c oordination environment arising from differential hydration or conform ational heterogeneity. In wildtype oncomodulin, the CD site signal dom inates the low-pH spectrum. Since this dominance persists even when se rine-55 and aspartate-59 are replaced by the corresponding EF site res idues, 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.