Mutational investigation of the specificity determining region of the Src SH2 domain.
Mutational investigation of the specificity determining region of the Src SH2 domain.
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Src SH2 结构域特异性决定区的突变研究。
DOI：
10.1006/jmbi.2000.3765
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
Waksman,G
中科院分区：
文献类型：
--
作者：
Bradshaw,JM;Mitaxov,V;Waksman,G
SH2 结构域是在许多信号传导途径中结合酪氨酸磷酸化序列的蛋白质模块。这些结构域包含两个具有特殊功能的区域：一个区域中的残基形成一个深袋，靶标的磷酸酪氨酸插入其中，而另一个区域包含所谓的“特异性决定残基”，其与靶标中磷酸酪氨酸C端的三个残基相互作用。在这里，滴定量热法和定点诱变已被用来探测 Src 激酶的 SH2 结构域的八个特异性决定残基的重要性，这些残基参与与其酪氨酸磷酸化共有肽靶标（序列 pYEEI，其中 pY 表示磷酸酪氨酸）的接触。将这八个残基中的六个单独突变为丙氨酸，导致结合亲和力损失三倍或更少；因此，特异性决定区中的大多数残基本身对于结合的重要性极小。发现两个残基对结合具有显着影响：Tyr βD5 和 Lys βD3。 Tyr βD5 是最关键的残基，当 Tyr βD5 突变为 Ile 时亲和力损失 30 倍就证明了这一点。然而，虽然该突变消除了 Src SH2 结构域对 pYEEI 肽序列的特异性，但不足以将 Src SH2 结构域的特异性转换为在 βD5 位置具有 Ile 的相关 SH2 结构域的特异性。 Lys βD3 突变为 Ala 残基导致结合亲和力适度降低（七倍）。有趣的是，这种突变导致了影响磷酸酪氨酸 C 端 +1 位残基选择的特异性变化。除了 Lys βD3−+1 Glu 相互作用显着耦合外，如使用双突变体循环评估的那样，在结合界面上仅观察到弱能量耦合。这项研究的结果表明，涉及 SH2 结构域特异性决定区的相互作用本身可能不足以将单个 SH2 结构域靶向特定的磷酸化位点。
SH2 domains are protein modules which bind tyrosine phosphorylated sequences in many signaling pathways. These domains contain two regions with specialized functions: residues in one region form a deep pocket into which the phosphotyrosine of the target inserts, while the other region contains the so-called “specificity determining residues” which interact with the three residues C-terminal to the phosphotyrosine in the target. Here, titration calorimetry and site-directed mutagenesis have been used to probe the importance of eight specificity determining residues of the SH2 domain of the Src kinase involved in contacts with its tyrosine phosphorylated consensus peptide target (sequence pYEEI where pY indicates a phosphotyrosine). Mutating six of these eight residues to Ala individually, resulted in a threefold or less loss in binding affinity; hence the majority of the residues in the specificity determining region are by themselves of minimal importance for binding. Two residues were found to have significant effects on binding: Tyr βD5 and Lys βD3. Tyr βD5 was the most crucial residue as evidenced by the 30-fold loss in affinity when Tyr βD5 is mutated to Ile. However, while this mutation eliminated the specificity of the Src SH2 domain for the pYEEI peptide sequence, it was not sufficient to switch the specificity of the Src SH2 domain to that of a related SH2 domain which has an Ile at the βD5 position. Mutation of Lys βD3 to an Ala residue resulted in a modest reduction in binding affinity (sevenfold). It is interesting that this mutation resulted in a change of specificity affecting the selection of the +1 position residue C-terminal to the phosphotyrosine. Except for the Lys βD3−+1 Glu interaction which is significantly coupled, only weak energetic coupling was observed across the binding interface, as assessed using double mutant cycles. The results of this study suggest that interactions involving the specificity determining region of SH2 domains may be insufficient by themselves to target single SH2 domains to particular phosphorylated sites.
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