Durussel Isabelle, Méhul Bruno, Bernard Dominique, Schmidt Rainer, Cox Jos A
Department of Biochemistry, University of Geneva, 1211 Genève, Switzerland.
Biochemistry. 2002 Apr 30;41(17):5439-48. doi: 10.1021/bi016062z.
Human CLSP, a new Ca(2+)-binding protein specifically expressed in differentiated keratinocytes, is a 15.9 kDa, four EF-hand containing protein with 52% sequence identity to calmodulin (CaM). The protein binds four Ca(2+) ions at two pairs of sites with Ca(2+) values of 1.2 and 150 microM, respectively. Mg(2+) at millimolar concentrations strongly decreases the affinity for Ca(2+) of the two high-affinity sites, but has no effect on the low-affinity sites. The protein can also bind two Mg(2+) (Mg(2+) = 57 microM) at the sites of high Ca(2+) affinity. Thus, as fast skeletal muscle troponin C (TnC), CLSP possesses two high-affinity Ca(2+)-Mg(2+) mixed sites and two low-affinity Ca(2+)-specific sites. Studies on the isolated recombinant N- (N-CLSP) and C-terminal half domains of CLSP (C-CLSP) revealed that, in contrast to the case of TNC, the high-affinity Ca(2+)-Mg(2+) mixed sites reside in the N-terminal half. The binding of cations modifies the intrinsic fluorescence of the two Tyr residues. Upon Ca(2+) binding, hydrophobicity is exposed at the protein surface that can be monitored with a fluorescent probe. The Ca(2+)-dependency of the two conformational changes is biphasic in the absence of Mg(2+), but monophasic in the presence of 2 mM Mg(2+), both corresponding closely to direct binding of Ca(2+) to CLSP. In the presence of Ca(2+), human CLSP forms a high-affinity 1:1 complex with melittin, a natural peptide considered to be a model for the interaction of CaM with its targets. In the complex, CLSP binds Ca(2+) with high affinity to all four binding sites. Isolated N- and C-CLSP show only a weak interaction with melittin, which is enhanced when both halves are simultaneously presented to the model peptide.
人角质形成细胞特异性蛋白（CLSP）是一种新的钙结合蛋白，在分化的角质形成细胞中特异性表达，分子量为15.9 kDa，含有四个EF手结构域，与钙调蛋白（CaM）的序列同源性为52%。该蛋白在两对位点结合四个钙离子，其半数结合浓度（Ca(2+)）分别为1.2微摩尔和150微摩尔。毫摩尔浓度的镁离子会强烈降低两个高亲和力位点对钙离子的亲和力，但对低亲和力位点没有影响。该蛋白还能在高钙离子亲和力位点结合两个镁离子（Mg(2+) = 57微摩尔）。因此，与人快速骨骼肌肌钙蛋白C（TnC）一样，CLSP拥有两个高亲和力的钙-镁混合位点和两个低亲和力的钙离子特异性位点。对CLSP分离的重组N端（N-CLSP）和C端半结构域（C-CLSP）的研究表明，与TnC不同，高亲和力的钙-镁混合位点位于N端半结构域。阳离子的结合会改变两个酪氨酸残基的固有荧光。钙离子结合后，蛋白质表面会暴露疏水性，可用荧光探针进行监测。在没有镁离子的情况下，两种构象变化的钙依赖性呈双相性，但在存在2 mM镁离子时呈单相性，两者都与钙离子直接结合到CLSP密切相关。在有钙离子存在的情况下，人CLSP与蜂毒素形成高亲和力的1:1复合物，蜂毒素是一种天然肽，被认为是CaM与其靶标相互作用的模型。在复合物中，CLSP以高亲和力结合钙离子到所有四个结合位点。分离的N-CLSP和C-CLSP与蜂毒素仅表现出微弱的相互作用，当两个半结构域同时与模型肽结合时，这种相互作用会增强。
