Electrostatic interactions in the reconstitution of an SH2 domain from constituent peptide fragments.
Electrostatic interactions in the reconstitution of an SH2 domain from constituent peptide fragments.
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从组成肽片段重建 SH2 结构域时的静电相互作用。
DOI：
10.1110/ps.0227903
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Wuttke,DeborahS
中科院分区：
文献类型：
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作者：
Ojennus,DeannaDahlke;Lehto,SarahE;Wuttke,DeborahS
片段互补已被用来描绘的基本识别元件的稳定折叠Src同源2（SH 2）域，通过使用NMR光谱，丙氨酸扫描，和表面等离子体共振。通过磷脂酰肌醇3′-激酶p85α亚基的N-末端SH 2结构域的有限蛋白水解消化形成的未折叠的9-kD和5-kD肽片段在相互作用时折叠成活性天然样结构。然而，同源Src蛋白的相应5 kD片段不能与p85 9 kD片段结构互补，表明这些SH 2结构域之间的片段互补对Src和p85结构域之间的序列差异敏感。部分互补和折叠活性可以恢复与这些SH 2结构域的杂交序列。使用5-kD p85片段的完整丙氨酸扫描来鉴定复合物形成所需的序列识别元件。p85 5-kD片段中的丙氨酸取代消除了与同源9-kD片段的结合亲和力，这与SH 2结构域中高度保守的残基密切相关，这些残基整体参与核心包装或在片段之间的界面处发现。然而，令人惊讶的是，发现非保守的表面暴露的天冬氨酸突变为丙氨酸对互补具有显著影响。精氨酸到天冬氨酸的单个额外突变允许天然结构的恢复，并将设计的Src-p85嵌合体的热稳定性提高了18°C。这种修饰似乎减轻了不利的表面静电相互作用，证明了表面电荷相互作用在蛋白质稳定性中的重要性。
Fragment complementation has been used to delineate the essential recognition elements for stable folding in Src homology 2 (SH2) domains by using NMR spectroscopy, alanine scanning, and surface plasmon resonance. The unfolded 9‐kD and 5‐kD peptide fragments formed by limited proteolytic digestion of the N‐terminal SH2 domain from the p85α subunit of phosphatidylinositol 3′‐kinase fold into an active native‐like structure on interaction with one another. The corresponding 5‐kD fragment of the homologous Src protein, however, was not capable of structurally complementing the p85 9‐kD fragment, indicating that fragment complementation among these SH2 domains is sensitive to the sequence differences between the Src and p85 domains. Partial complementation and folding activity could be recovered with hybrid sequences of these SH2 domains. Complete alanine scanning of the 5‐kD p85 fragment was used to identify the sequence recognition elements required for complex formation. The alanine substitutions in the p85 5‐kD fragment that abolished binding affinity with the cognate 9‐kD fragment correlate well with highly conserved residues among SH2 domains that are either integrally involved in core packing or found at the interface between fragments. Surprisingly, however, mutation of a nonconserved surface‐exposed aspartic acid to alanine was found to have a significant effect on complementation. A single additional mutation of arginine to aspartic acid allowed for recovery of native structure and increased the thermal stability of the designed Src‐p85 chimera by 18°C. This modification appears to relieve an unfavorable surface electrostatic interaction, demonstrating the importance of surface charge interactions in protein stability.