Calcium-binding properties of wild-type and EF-hand mutants of S100B in the presence and absence of a peptide derived from the C-terminal negative regulatory domain of p53
Calcium-binding properties of wild-type and EF-hand mutants of S100B in the presence and absence of a peptide derived from the C-terminal negative regulatory domain of p53
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DOI：
10.1021/bi050321t
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发表时间：
2005-05-17
期刊：
影响因子：
2.9
通讯作者：
Weber, DJ
中科院分区：
文献类型：
--
作者：
Markowitz, J;Rustandi, RR;Weber, DJ
S100 B是一种二聚体Ca 2+结合蛋白，在加入钙后，其在典型的EF-手结构域（EF 2）中经历螺旋3的90 +/-3度旋转。该螺旋的大的重新定向是S100 B的每个亚基与靶蛋白如肿瘤抑制蛋白p53之间相互作用的先决条件。在这项研究中，Tb 3+被用作探针，以检查如何结合的22个残基的肽衍生自C-末端调节结构域的p53影响钙离子解离的速率。在与Tb 3+的竞争研究中，在不存在和存在p53肽的情况下，Ca 2+（k（off））从S100 B的EF 2结构域的解离速率分别被确定为60和7 s（-1）。这些数据与先前报道的结果一致，该结果显示靶肽与S100 B结合增强了其钙结合亲和力[Rustandi et al.（1998）Biochemistry 37，1951-1960]。在不存在和存在p53肽的情况下，S100 B、EF 2结构域的相应Ca 2+结合速率常数k（on）分别为1.1 x 10（6）和3.5 x 10（5）M-1 s（-1）。这两个结合速率常数显著低于扩散对照（类似于10（9）M-1 s-1），可能涉及Ca 2+离子结合和Ca 2+依赖性结构重排，当靶肽存在时略有不同。在-Z位置对S10013的EF-手型钙结合突变体进行工程改造（EF-手型1，E31 A; EF-手型2，E72 A;两个EF-手型，E31 A + E72 A），并进行检查以进一步了解在不存在和存在p53肽的情况下，特定残基如何促进S100 B中的钙结合。
S100B is a dimeric Ca2+-binding protein that undergoes a 90 +/- 3 degrees rotation of helix 3 in the typical EF-hand domain (EF2) upon the addition of calcium. The large reorientation of this helix is a prerequisite for the interaction between each subunit of S100B and target proteins such as the tumor suppressor protein, p53. In this study, Tb3+ was used as a probe to examine how binding of a 22-residue peptide derived from the C-terminal regulatory domain of p53 affects the rate of Ca2+ ion dissociation. In competition studies with Tb3+, the dissociation rates of Ca2+ (k(off)) from the EF2 domains of S100B in the absence and presence of the p53 peptide was determined to be 60 and 7 s(-1), respectively. These data are consistent with a previously reported result, which showed that that target peptide binding to S100B enhances its calcium-binding affinity [Rustandi et al. (1998) Biochemistry 37, 1951-1960]. The corresponding Ca2+ association rate constants for S100B, k(on), for the EF2 domains in the absence and presence of the p53 peptide are 1.1 x 10(6) and 3.5 x 10(5) M-1 s(-1), respectively. These two association rate constants are significantly below the diffusion control (similar to 10(9) M-1 s-1) and likely involve both Ca2+ ion association and a Ca2+-dependent structural rearrangement, which is slightly different when the target peptide is present. EF-hand calcium-binding mutants of S 10013 were engineered at the -Z position (EF-hand 1, E31A; EF-hand 2, E72A; both EF-hands, E31A + E72A) and examined to further understand how specific residues contribute to calcium binding in S100B in the absence and presence of the p53 peptide.