Leucine 85 is an important determinant of divalent ion affinity in rat beta-parvalbumin (Oncomodulin).
Despite 69% sequence identity with chicken parvalbumin 3 (CPV3), rat beta-parvalbumin (beta-PV) exhibits a substantially lower Ca(2+) affinity (DeltaDeltaG degrees ' = 2.0 kcal/mol). This difference largely reflects the disparate behavior of the respective CD sites. Replacement of the rat beta-PV codon with the CPV3 codon at positions 49, 50, and 57-60 produces virtual sequence identity with the CPV3 CD site. However, the resulting protein exhibits a modest (0.5 kcal/mol) improvement in Ca(2+) affinity, implying that sequence differences beyond the binding site modulate divalent ion binding behavior. The solution structure of Ca(2+)-free rat beta-PV suggested that Leu-85, phenylalanine in CPV3, might be an important determinant. Therefore, the impact of the L85F mutation on divalent ion affinity was examined in rat beta-PV, in the variant harboring all six of the aforementioned CD site mutations, and in the intermediate CD site variants. We find that the identity of residue 85, located within the E helix, strongly influences divalent ion affinity in the mammalian beta-PV isoform and that its impact is mediated by interactions with residues in the CD site. In the wild-type protein, L85F primarily affects the EF site. By contrast, in the presence of the six CD site mutations, L85F also improves the CD site performance, yielding a protein with Ca(2+) affinity rivaling that of CPV3 and markedly enhanced Mg(2+) affinity as well. The impact of L85F on CD site Ca(2+) affinity is particularly sensitive to the identities of residues 59 and 60. Interestingly, however, significant improvement in CD site Mg(2+) affinity also requires mutation of additional CD site residues.
Henzl
Michael T
MT
Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, USA. henzlm@missouri.edu
Davis
Meredith E
ME
Tan
Anmin
A
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000737:chemistry; D003062:Codon; D007477:Ions; D007930:Leucine / Q000737:chemistry*; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010320:Parvalbumins / Q000737:chemistry* / Q000235:genetics*; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D017386:Sequence Homology, Amino Acid
D003062:Codon; D007477:Ions; D010320:Parvalbumins; D007930:Leucine; D002118:Calcium