The electrostatic interactions of relaxin‐3 with receptor RXFP4 and the influence of its B‐chain C‐terminal conformation
The electrostatic interactions of relaxin‐3 with receptor RXFP4 and the influence of its B‐chain C‐terminal conformation
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DOI：
10.1111/febs.12830
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发表时间：
2014-07
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影响因子：
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通讯作者：
Xin-Yi Wang;Yu-Qi Guo;Wei-jie Zhang;X. Shao;Ya‐Li Liu;Zeng-guang Xu;Zhan-Yun Guo
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作者：
Xin-Yi Wang;Yu-Qi Guo;Wei-jie Zhang;X. Shao;Ya‐Li Liu;Zeng-guang Xu;Zhan-Yun Guo
松弛素-3（也称为胰岛素样肽7）是一种胰岛素/松弛素超家族肽激素，可以结合并激活三种松弛素家族肽受体：RXFP 3，RXFP 4和RXFP 1。最近，我们通过使用电荷交换诱变方法确定了松弛素-3与其同源受体RXFP 3之间的关键静电相互作用。在本研究中，松弛素-3和RXFP 4之间的静电相互作用用相同的方法进行了研究。人RXFP 4的带负电荷的细胞外残基的突变鉴定了一个保守的EXXXD（100-104）基序，该基序对于松弛素-3激活RXFP 4是必需的。对松弛素-3保守的带正电荷的Arg残基的突变表明，B12 Arg、B16 Arg和B26 Arg均参与RXFP 4的结合和激活，尤其是B26 Arg。突变配体和突变受体之间的活性互补表明了两种可能的静电相互作用对：RXFP 4的Glu 100与松弛素-3的B26 Arg，以及RXFP 4的Asp 104与松弛素-3的B12 Arg和B16 Arg。为了与RXFP 3和RXFP 4的基本EXXXD基序相互作用，松弛素-3 B-链C-末端的折叠构象似乎是关键的，因为它使B26 Arg足够接近B12 Arg和B16 Arg。为了验证这一假设，我们将松弛素-3的保守B23 Gly-B24 Gly二肽替换为占据胰岛素样肽5相应位置的Ala-Ser二肽，并导致延长的螺旋构象。突变体松弛素-3对RXFP 3和RXFP 4的受体激活效力显着降低，表明B-链C-末端的折叠构象对于松弛素-3与两种受体的EXXXD基序有效相互作用是重要的。
Relaxin‐3 (also known as insulin‐like peptide 7) is an insulin/relaxin‐superfamily peptide hormone that can bind and activate three relaxin‐family peptide receptors: RXFP3, RXFP4, and RXFP1. Recently, we identified key electrostatic interactions between relaxin‐3 and its cognate receptor RXFP3 by using a charge‐exchange mutagenesis approach. In the present study, the electrostatic interactions between relaxin‐3 and RXFP4 were investigated with the same approach. Mutagenesis of the negatively charged extracellular residues of human RXFP4 identified a conserved EXXXD(100–104) motif that is essential for RXFP4 activation by relaxin‐3. Mutagenesis of the conserved positively charged Arg residues of relaxin‐3 demonstrated that B12Arg, B16Arg and B26Arg were all involved in the binding and activation of RXFP4, especially B26Arg. The activity complementation between the mutant ligands and the mutant receptors suggested two probable electrostatic interaction pairs: Glu100 of RXFP4 versus B26Arg of relaxin‐3, and Asp104 of RXFP4 versus both B12Arg and B16Arg of relaxin‐3. For interaction with the essential EXXXD motifs of both RXFP3 and RXFP4, a folding‐back conformation of the relaxin‐3 B‐chain C‐terminus seems to be critical, because it brings B26Arg sufficiently close to B12Arg and B16Arg. To test this hypothesis, we replaced the conserved B23Gly‐B24Gly dipeptide of relaxin‐3 with an Ala‐Ser dipeptide that occupied the corresponding position of insulin‐like peptide 5 and resulted in an extended helical conformation. The mutant relaxin‐3 showed a significant decrease in receptor‐activation potency towards both RXFP3 and RXFP4, suggesting that a folding‐back conformation of the B‐chain C‐terminus was important for relaxin‐3 to efficiently interact with the EXXXD motifs of both receptors.