Interactions between residues in the oncomodulin CD domain influence Ca2+ ion-binding affinity.
Despite striking sequence homology with rat parvalbumin, oncomodulin exhibits much lower affinity for Ca2+ ion. We are attempting to identify the structural basis for this difference by systematically substituting the parvalbumin residue for the oncomodulin residue at points of nonidentity. In this paper, we examine two mutations in the helical segments flanking the CD ion-binding loop. Replacement of Asp-45 in the C helix by lysine, to produce D45K, reduces the dissociation constant for Ca2+ at the CD site from 0.81 to 0.53 microM. Replacement of Lys-69 in the D helix by glycine, to afford K69G, similarly reduces KCa to 0.59 microM. Both mutations perturb the Eu3+ 7Fo----5Do spectral parameters. We also examine the consequences of simultaneous mutations involving positions 57, 59, 60, and 69. Ca(2+)-binding assays and Eu3+ luminescence measurements indicate that there is a conformational interaction between residues 57 and 69 and that this interaction is modulated by residues 59 and 60. When the mutations at positions 57, 59, 60, and 69 are combined, the resulting variant exhibits a KCa value for the CD site of 0.25 microM, reflecting a 3-fold increase in affinity relative to the wild-type protein. Moreover, the pK alpha governing the interconversion of low and high pH forms of the Eu3+ 7Fo----5Do spectrum is increased to 8.1, very close to the value of 8.25 determined previously for rat parvalbumin. In this paper, we also complete our survey of single mutations in the CD loop by examining L58I. Replacement of Leu-58 by isoleucine reduces the affinity of the CD site for Ca2+, raising KCa to 2.2 microM. Finally, we revise our previous estimate of the KCa value for Y57F downward, from 0.80 to 0.64 microM. The earlier result is believed to have been inflated by heterogeneity in the preparation, a consequence of proteolysis.
Treviño
C L
CL
Department of Chemistry, New Mexico State University, Las Cruces 88003.
Boschi
J M
JM
Henzl
M T
MT
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D014644:Genetic Variation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000378:metabolism; D010320:Parvalbumins / Q000235:genetics; D011487:Protein Conformation; D051381:Rats; D012689:Sequence Homology, Nucleic Acid
D002135:Calcium-Binding Proteins; D009363:Neoplasm Proteins; D010320:Parvalbumins; C034733:oncomodulin; D002118:Calcium