Exploring the systematic effect of N‐substituted PxxP motifs on peptoid affinity to ARHGEF5/TIM SH3 domain and its relationship with ARHGEF5/TIM activation
Exploring the systematic effect of N‐substituted PxxP motifs on peptoid affinity to ARHGEF5/TIM SH3 domain and its relationship with ARHGEF5/TIM activation
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DOI：
10.1002/prot.25760
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发表时间：
2019-06
期刊：
影响因子：
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通讯作者：
Yong Fu;P. He;Yu Zhou-;Shengyuan Huang;Lin Liang;Shengchun Liu
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文献类型：
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作者：
Yong Fu;P. He;Yu Zhou-;Shengyuan Huang;Lin Liang;Shengchun Liu
TIM蛋白是全长Rho鸟嘌呤核苷酸交换因子5(ARHGEF5)的一个短亚型，它通过激活Rho家族的GTP酶来调节Rho依赖的信号通路。TIM的DH域的激活被假定的螺旋N端自动抑制，而TIM的DH域则通过C端SH3结构域与假定螺旋和DH域之间的富含Pro的区域47SSPRQPRKAL56(称为SSP肽)的分子内相互作用而稳定。以前，我们证明了通过合理设计的多肽配体靶向TIM蛋白的SH3结构域，可以缓解TIM蛋白的自我抑制状态。然而，与同源的SH3-SSP相互作用相比，所设计的天然多肽只有适度增加的亲和力(~2倍)，并且容易被蛋白酶降解。这里，考虑到Pro是唯一在SH3-肽识别中起关键作用的内源性N-取代氨基酸，本文将SSP肽核心49PxxP52基序中的两个关键的Pro残基Pro49和Pro52系统地替换为19种N-取代氨基酸类型，从而衍生出一系列针对Tim SH3结构域的非天然类肽配体。动力学和能量学分析表明，这种取代会破坏SSP多肽的活性多肽II(PPII)的螺旋构象，这是因为天然Pro侧链的五元环没有引入结构约束，从而增加了多肽的灵活性，从而增加了与结构域结合时可能产生的较大的熵惩罚。然而，如果衍生多肽配体的N-取代基序能有效地与Tim SH3结构域的PxxP结合位点相互作用，这种损伤并不是很明显，多肽亲和力也可能得到恢复和提高。因此，通过荧光光谱的确认，成功地设计了一些有效的类肽，其中三个(即SSP[N-Ile49，N-Asn52]，SSP[N-Phe49，N-Gln52]和SSP[N-Tyr49，N-Asn52])的亲和力显著高于天然SSP(Kd=0.87μM)(Kd=0.09，0.07和0.04μM)。此外，鸟嘌呤核苷酸交换实验还证实，所设计的SH3靶向多肽能够有效地增强Tim催化的RhoA交换活性(EA)，这与所测量的SH3-肽结合亲和力(PKD)呈指数关系。
The TIM protein is a short isoform of full‐length Rho guanine nucleotide exchange factor 5 (ARHGEF5), which acts as a functional regulator of Rho‐dependent signaling pathways by activating the Rho family of GTPases. The activation is auto‐inhibited by a putative helix N‐terminal to the DH domain of TIM, which is stabilized by the intramolecular interaction of C‐terminal SH3 domain with a proline‐rich region 47SSPRQPRKAL56 (termed as SSP peptide) between the putative helix and the DH domain. Previously, we demonstrate that the auto‐inhibitory state of TIM protein can be relieved by targeting its SH3 domain with rationally designed peptide ligands. However, the designed natural peptides have only a moderately increased affinity (~2‐fold) as compared to the cognate SH3‐SSP interaction and are susceptible to protease degradation. Here, considering that proline is the only endogenous N‐substituted amino acid that plays a critical role in SH3‐peptide recognition, the two key proline residues Pro49 and Pro52 in the core 49PxxP52 motif of SSP peptide are systematically replaced by 19 N‐substituted amino acid types to derive a variety of nonnatural peptoid ligands for TIM SH3 domain. Dynamics and energetics analyses reveal that the replacement would impair the active polyproline II (PPII) helical conformation of SSP peptide due to lack of structural constraint introduced by the five‐membered ring of native proline side‐chains, thus increasing the peptide flexibility that could incur a large entropy penalty upon binding to the domain. However, the impairment is not very significant and the peptide affinity may also be restored and improved if the N‐substituted motif of derived peptiod ligands can effectively interact with the PxxP‐binding site of TIM SH3 domain. Consequently, a number of potent peptoids are successfully designed by fluorescence spectroscopy confirmation, in which three (ie, SSP[N‐Ile49, N‐Asn52], SSP[N‐Phe49, N‐Gln52], and SSP[N‐Tyr49, N‐Asn52]) exhibit considerably increased affinity (Kd = 0.09, 0.07, and 0.04 μM, respectively) relative to the native SSP peptide (Kd = 0.87 μM). In addition, guanine nucleotide exchange assays also substantiate that the designed SH3‐targeted peptiods can effectively enhance TIM‐catalyzed RhoA exchange activity (EA), which is observed to present an exponential relationship with the measured SH3‐peptoid binding affinity (pKd).