Site-specific replacement of amino acid residues within the CD binding loop of rat oncomodulin.
Relative to the same site in oncomodulin, the CD ion-binding domain of rat parvalbumin exhibits much greater affinity for Ca2+ and Mg2+. As part of an effort to understand the structural basis for these differences, site-specific variants of oncomodulin have been prepared in which the amino acid residues at positions 52, 54, 57, 59, and 60 have been replaced with the residues present at the corresponding positions in rat parvalbumin. The proteins resulting from the single-site substitutions at residues 52, 54, and 57 are indistinguishable from the wild-type protein on the basis Eu3+ luminescence spectroscopy, and none of the three variants displays increased affinity for Ca2+. By contrast, the substitutions at residues 59 and 60 perturb both the Eu3+ luminescence parameters and the Ca2+ and Mg2+ affinities, and these differences are amplified when both replacements are simultaneously incorporated into the protein. The Eu3+ 7F0----5D0 spectrum of the double variant (D59E/G60E) at pH 5.0, with a maximum at 5796 A and pronounced shoulder at 5791 A, strongly resembles that obtained with pike parvalbumin. Consistent with this increased parvalbumin-like character, KCa is decreased from 0.78 microM (for the wild-type protein) to 0.41 microM, and KMg is decreased from 3.5 to 0.74 mM. Nevertheless, the affinity of the CD ion-binding domain in D59E/G60E for Ca2+ remains almost 2 orders of magnitude lower than the corresponding site in rat parvalbumin, strongly suggesting that residues besides those present in the binding loop are involved in dictating the metal ion-binding properties of the oncomodulin CD site.
Palmisano
W A
WA
Department of Chemistry, New Mexico State University, Las Cruces 88003.
Treviño
C L
CL
Henzl
M T
MT
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D002413:Cations, Divalent; D004591:Electrophoresis, Polyacrylamide Gel; D014644:Genetic Variation; D006863:Hydrogen-Ion Concentration; D008163:Luminescent Measurements; D008274:Magnesium / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics*; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
D002135:Calcium-Binding Proteins; D002413:Cations, Divalent; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; C034733:oncomodulin; D008274:Magnesium; D002118:Calcium