Remodeling of the AB site of rat parvalbumin and oncomodulin into a canonical EF-hand

Parvalbumin (PV) and the homologous protein oncomodulin (OM) contain three EF-hand motifs, but the first site (AE) cannot bind Ca2+. Were we aimed to recreate the putative ancestral proteins [D19-28E]PV and [D19-28E]OM by rep lacing the 10-residue-long nonfunctional loop in the AB site by a 12-residu e canonical loop. To create an optical conformational probe we also express ed the homologs with a F102W replacement. Unexpectedly, in none of the prot eins did the mutation reactivate the AB site. The AB-remodeled parvalbumins bind two Ca2+ ions with strong positive cooperativity (nx = 2) and moderat e affinity ([Ca2+](0.5) = 2 mu M ) compared with [Ca2+](0.5) = 37 nM and n( H) = 1 for the wild-type protein. Increasing Mg2+ concentrations changed nx from 2 to 0.65, but without modification of the [Ca2+](0.5)-value. CD reve aled that the Ca2+ and Mg2+ forms of the remodeled parvalbumins lost one-th ird of their or helix content compared with the Ca2+ form of wild-type parv albumin. However, the microenvironment of single Trp residues in the hydrop hobic cores, monitored using intrinsic fluorescence and difference optical density, is the same. The metal-free remodeled parvalbumins possess unfolde d conformations. The AB-remodeled oncomodulins also bind two Ca2+ with [Ca2 +](0.5) = 43 mu M and nH = 1.45. Mg2+ does not affect Ca2+ binding. Again t he Ca2+ forms display two-thirds of the alpha-helical content in the wild-t ype, while their fore is still strongly hydrophobic as monitored by Trp and Tyr fluorescence. The metal-free oncomodulins are partially unfolded and s eem not to possess a hydrophobic core. Our data indicate that AB-remodeled parvalbumin has the potential to regulate cell functions, whereas it is unl ikely that [D19-28E]OM can play a regulatory role in vivo. The predicted ev olution of the AB site from a canonical to an abortive EF-hand may have bee n dictated by the need for stronger interaction with Mg2+ and Ca2+, and a h igh conformational stability of the metal-free forms.