Abstract：
Ca2+-binding proteins are present in almost all living organisms and different types display different levels of binding affinities for the cation. Here, we report two new scoring schemes enabling the user to estimate and manipulate the calcium binding affinities in EF hand containing proteins. To validate this, we designed a unique EF-hand loop capable of binding calcium with high affinity by altering five residues. The N-terminal domain of Entamoeba histolytica calcium-binding protein1 (NtEhCaBP1) is used for site-directed mutagenesis to incorporate the designed loop sequence into the second EF-hand motif of this protein, referred as Nt-EhCaBP1-EF2 mutant. The binding isotherms calculated using ITC calorimetry showed that Nt-EhCaBP1-EF2 mutant site binds Ca2+ with higher affinity than Wt-Nt-EhCaBP1, by ∼600 times. The crystal structure of the mutant displayed more compact Ca2+-coordination spheres in both of its EF loops than the structure of the wildtype protein. The compact coordination sphere of EF-2 causes the bend in the helix-3, which leads to the formation of unexpected hexamer of NtEhCaBP1-EF2 mutant structure. Further dynamic correlation analysis revealed that the mutation in the second EF loop changed the entire residue network of the monomer, resulting in stronger coordination of Ca2+ even in another EF-hand loop.
摘要：
Ca2+结合蛋白存在于几乎所有的活生物体中,并且不同类型对阳离子显示不同水平的结合亲和力。这里,我们报告了两种新的评分方案,使用户能够评估和操纵EF手含蛋白的钙结合亲和力.为了验证这一点，我们设计了一个独特的EF手环，能够通过改变五个残基以高亲和力结合钙。溶组织内阿米巴钙结合蛋白1（NtEhCaBP1）的N末端结构域用于定点诱变，以将设计的环序列掺入该蛋白的第二EF手基序中，称为Nt-EhCaBP1-EF2突变体。使用ITC量热法计算的结合等温线显示,Nt-EhCaBP1-EF2突变位点以高于Wt-Nt-EhCaBP1的亲和力结合Ca2+约600倍。突变体的晶体结构在其两个EF环中显示出比野生型蛋白的结构更紧密的Ca2配位球。EF-2的紧密配位球会导致螺旋3弯曲，从而形成NtEhCaBP1-EF2突变体结构的意外六聚体。进一步的动态相关分析显示,第二EF环中的突变改变了单体的整个残基网络,即使在另一个EF手环中，也能产生更强的Ca2+配位。
