Tuning Inner-Ear Tip-Link Affinity Through Alternatively Spliced Variants of Protocadherin-15.
Tuning Inner-Ear Tip-Link Affinity Through Alternatively Spliced Variants of Protocadherin-15.
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DOI：
10.1021/acs.biochem.7b01075
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发表时间：
2018-03-20
期刊：
影响因子：
2.9
通讯作者：
Sotomayor M
中科院分区：
文献类型：
--
作者：
Narui Y;Sotomayor M
人类的听觉依赖于两种非经典钙粘蛋白--原钙粘蛋白-15（PCDH 15）和钙粘蛋白-23（CDH 23）--的尖端-尖端相互作用。这些蛋白质一起形成称为尖端连接的细丝，其连接机械敏感毛细胞的相邻静纤毛。当声波进入耳蜗时，静纤毛偏转并且张力被施加到尖端连接件，从而打开附近的换能通道。通过响亮的声音或钙螯合剂破坏尖端链接消除了转导电流，并说明尖端链接的完整性对于机械传感至关重要。破坏后的尖端连接重塑是一个动态过程，这可能导致形成非典型复合物，其中包含PCDH 15的可变剪接变体。根据前两个细胞外钙粘蛋白（EC）重复序列的差异，将这些变体分为6组（N1-N6）。在此，我们表征了所有PCDH 15变体（pcdh 15（N1）至pcdh 15（N6））的两个N-末端EC重复，并将这些变体组合以测试复合物形成。我们在2.3 nm分辨率下测定了一个新的复合物的晶体结构，并与经典的cdh 23-pcdh 15（N1）复合物进行了比较。虽然存在细微的结构差异，但如通过表面等离子体共振分析所确定的，cdh 23和pcdh 15（N2）之间的结合亲和力比cdh 23和pcdh 15（N1）弱约6倍。导向分子动力学模拟预测，cdh 23-pcdh 15（N2）复合物的解束缚力可以低于典型的尖端链接。我们的研究结果表明，替代异嗜性尖端链接结构形成稳定的蛋白质-蛋白质相互作用在体外，并表明，嗜同性PCDH 15-PCDH 15尖端链接通过额外的EC重复序列的相互作用形成。
Human hearing relies upon the tip-to-tip interaction of two non-classical cadherins, protocadherin-15 (PCDH15) and cadherin-23 (CDH23). Together, these proteins form a filament called the tip link that connects neighboring stereocilia of mechanosensitive hair cells. As sound waves enter the cochlea, the stereocilia deflect and tension is applied to the tip link opening nearby transduction channels. Disruption of the tip link by loud sound or calcium chelators eliminates transduction currents and illustrates that tip-link integrity is critical for mechanosensing. Tip-link remodeling after disruption is a dynamic process, which can lead to the formation of atypical complexes that incorporate alternatively spliced variants of PCDH15. These variants are categorized into 6 groups (N1-N6) based upon differences in the first two extracellular cadherin (EC) repeats. Here, we characterized the two N-terminal EC repeats of all PCDH15 variants (pcdh15(N1) to pcdh15(N6)), and combined these variants to test complex formation. We solved the crystal structure of a new complex composed of CDH23 EC1-2 (cdh23) and pcdh15(N2) at 2.3 Å resolution and compared it to the canonical cdh23-pcdh15(N1) complex. While there were subtle structural differences, the binding affinity between cdh23 and pcdh15(N2) is ~6 times weaker than cdh23 and pcdh15(N1) as determined by surface plasmon resonance analysis. Steered molecular dynamics simulations predict that the unbinding force of the cdh23-pcdh15(N2) complex can be lower than the canonical tip link. Our results demonstrate that alternative heterophilic tip-link structures form stable protein-protein interactions in vitro and suggest that homophilic PCDH15-PCDH15 tip links form through the interaction of additional EC repeats.
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DOI：
10.1073/pnas.90.4.1330
发表时间：
1993-02-15
影响因子：
11.1
作者：
JARAMILLO, F;HUDSPETH, AJ
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影响因子：
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作者：
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影响因子：
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
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Cowtan, K