Pauls T L, Durussel I, Clark I D, Szabo A G, Berchtold M W, Cox J A
Institute of Histology, University of Fribourg, Switzerland.
Eur J Biochem. 1996 Dec 1;242(2):249-55. doi: 10.1111/j.1432-1033.1996.0249r.x.
Rat parvalbumin (PV) and oncomodulin (OM) display considerable sequence similarity and structural similarity, but differ in the affinity and selectivity of metal binding to their CD site, a Ca2+/Mg(2+)-mixed site in PV and a Ca(2+)-specific site in OM. In an attempt to identify the structural basis for these differences, mutations were introduced in the previously generated [W102]PV mutant, which contains a unique tryptophan as a conformational-sensitive fluorescent probe inside the hydrophobic core. In the present report, we substituted selected amino acid residues in the CD site of PV by those present at identical positions in OM. One mutant protein, named [F66, W102]PV, has one new substitution in which isoleucine at position 66 was exchanged by phenylalanine. The second mutant protein, [I46, I50, L58, F66, W102]PV, has four new substitutions, namely V46-->I, L50-->I, I58-->L and I66-->F. Tryptophan fluorescence and difference spectrophotometry indicated that the mutations do not alter significantly the hydrophobic core. Both mutant proteins display two metal-binding sites of identical affinities with intrinsic affinity constants K'Ca2+ of 2.9 x 10(7) M-1 for [F66, W102]PV and 1.7 x 10(7) M-1 for [I46, I50, L58, F66, W102]PV and K'Mg2+ of 3.1 x 10(4) M-1 for [F66, W102]PV and 1.9 x 10(4) M-1 for [I46, I50, L58, F66, W102]PV. Thus, the five-residue substitution, but not the two-residue one, leads to a small decrease of affinity compared to [W102]PV (K'Ca2+ = 2.7 x 10(7) M-1, K'Mg2+ = 4.4 x 10(4) M-1). Despite these similarities, the Mg2+ effect on Ca2+ binding is different for the two mutant parvalbumins: the Ca(2+)-binding isotherms of [F66, W102]PV undergo a parallel shift upon increasing Mg2+ concentrations, which indicates that the Mg2+ effect on the two Ca(2+)-binding sites is the same and quantitatively very similar to that described for [W102]PV. In [I46, I50, L58, F66, W102]PV, Mg2+ antagonizes the binding of the second Ca2+ (likely at the EF site) much more than that of the first Ca2+ (likely the CD site). According to the competition equation, the two sites display KMg2+.compet values of 390 M-1 and 3.9 x 10(3) M-1, respectively. These data indicate that (a) the single I66-->F mutation does not modify the cation binding parameters. (b) Multiple modifications in the hydrophobic core still do not change the affinity for Ca2+ and Mg2+, but strongly affect the Mg2+ antagonism and probably the selectivity of the CD site.
大鼠小白蛋白（PV）和癌调蛋白（OM）在序列和结构上有相当大的相似性，但在金属离子与它们的CD位点结合的亲和力和选择性上有所不同，PV的CD位点是一个Ca2+/Mg(2+)混合位点，而OM的是一个Ca(2+)特异性位点。为了确定这些差异的结构基础，我们在之前构建的[W102]PV突变体中引入了突变，该突变体在疏水核心内部含有一个独特的色氨酸作为构象敏感荧光探针。在本报告中，我们用OM中相同位置的氨基酸残基替换了PV的CD位点中的特定氨基酸残基。一种突变蛋白，命名为[F66, W102]PV，有一个新的替换，即66位的异亮氨酸被苯丙氨酸取代。第二种突变蛋白，[I46, I50, L58, F66, W102]PV，有四个新的替换，即V46→I、L50→I、I58→L和I66→F。色氨酸荧光和差示分光光度法表明，这些突变并没有显著改变疏水核心。两种突变蛋白都显示出两个具有相同亲和力的金属结合位点，[F66, W102]PV的固有亲和常数K'Ca2+为2.9×10(7) M-1，[I46, I50, L58, F66, W102]PV的为1.7×10(7) M-1；[F66, W102]PV的K'Mg2+为3.1×10(4) M-1，[I46, I50, L58, F66, W102]PV的为1.9×(4) M-1。因此，与[W102]PV（K'Ca2+ = 2.7×10(7) M-1，K'Mg2+ = 4.4×10(4) M-1）相比，五个残基的替换而非两个残基的替换导致亲和力略有下降。尽管有这些相似之处，但两种突变小白蛋白中Mg2+对Ca2+结合的影响不同：[F66, W102]PV的Ca(2+)结合等温线在Mg2+浓度增加时发生平行移动，这表明Mg对两个Ca(2+)结合位点的影响相同，并且在数量上与[W1]PV中描述的非常相似。在[I46, I50, L58, F66, W102]PV中，Mg2+对第二个Ca2+（可能在EF位点）结合的拮抗作用比第一个Ca2+（可能是CD位点）大得多。根据竞争方程，两个位点的KMg2+.compet值分别为390 M-1和3.9×10(3) M-1。这些数据表明：（a）单个I66→F突变不会改变阳离子结合参数。（b）疏水核心中的多个修饰仍然不会改变对Ca2+和Mg2+的亲和力，但会强烈影响Mg2+的拮抗作用，可能还会影响CD位点的选择性。
