Linse S, Voorhies M, Norström E, Schultz D A
Lund University, Chemical Centre, Physical Chemistry 2, Lund, S-221 00, Sweden.
J Mol Biol. 2000 Feb 18;296(2):473-86. doi: 10.1006/jmbi.1999.3452.
The interaction between the two EF-hands, EF3 and EF4, in the C-terminal domain of vertebrate calmodulin is addressed using an EF-hand phage display library. Significant specificity is observed in the presence of Ca(2+), as EF3-EF4 heterodimers are favored over EF3-EF3 and EF4-EF4 homodimers. Primarily EF4-type (and not EF3-type) amino acids are selected when an EF3 peptide is used as the target and vice versa. The results show that this specificity is promoted by several factors. There are three positions, corresponding to Phe89, Ala102, and Leu105, that are strongly selected as EF3-type hydrophobic residues with an EF4 target. When EF3 is the target peptide, EF4-type residues, Ile125, Tyr138 and Phe141, are selected. Remarkably, this subset consists of the same three residue positions in EF3 or EF4 and seems to be involved in specifying the heterodimer preference in both cases. In addition, electrostatic repulsion between the acidic monomers in an EF4 homodimer may further influence the preferred stability of heterodimers. This hypothesis is based on the observation that positively charged residues are strongly selected at four positions when EF4 is the target. A survey of EF-hand pairs suggests that charge separation is a common way to achieve efficient attraction of Ca(2+) without causing electrostatic repulsion between the subdomains. No significant specificity of binding is observed in the ion free state or in the presence of magnesium as no sequence is preferentially selected. The residues at the interface between the two EF-hands are thus highly optimized for the Ca(2+) bound state. At some residue positions, EF3-type amino acids are chosen with EF3-target in the presence of Ca(2+). These residues are not involved in the preference for heterodimer over homodimer formation, but represent key positions to mutate in the intact domain to stabilize its Ca(2+)-bound state.
利用一个EF手型噬菌体展示文库，研究了脊椎动物钙调蛋白C端结构域中两个EF手型结构EF3和EF4之间的相互作用。在Ca(2+)存在的情况下观察到显著的特异性，因为EF3 - EF4异二聚体比EF3 - EF3和EF4 - EF4同二聚体更受青睐。当以EF3肽作为靶标时，主要选择EF4型（而非EF3型）氨基酸，反之亦然。结果表明，这种特异性受到多种因素的促进。有三个位置，对应于Phe89、Ala102和Leu105，在以EF4为靶标的情况下被强烈选择为EF3型疏水残基。当EF3是靶标肽时，选择EF4型残基Ile125、Tyr138和Phe141。值得注意的是，该子集由EF3或EF4中相同的三个残基位置组成，似乎在两种情况下都参与确定异二聚体偏好。此外，EF4同二聚体中酸性单体之间的静电排斥可能进一步影响异二聚体的优先稳定性。这一假设基于以下观察结果：当以EF4为靶标时，在四个位置强烈选择带正电荷的残基。对EF手型对的调查表明，电荷分离是在不引起亚结构域之间静电排斥的情况下实现Ca(2+)有效吸引的常见方式。在离子自由状态或镁存在的情况下未观察到显著的结合特异性，因为没有序列被优先选择。因此，两个EF手型之间界面处的残基针对Ca(2+)结合状态进行了高度优化。在某些残基位置，在Ca(2+)存在的情况下以EF3为靶标时选择EF3型氨基酸。这些残基不参与异二聚体相对于同二聚体形成的偏好，但代表了在完整结构域中进行突变以稳定其Ca(2+)结合状态的关键位置。
