Davila Samantha, Liu Peilu, Smith Alexis, Marshall Alan G, Pedigo Susan
Department of Chemistry and Biochemistry , University of Mississippi , University , Mississippi 38677 , United States.
Department of Chemistry & Biochemistry , Florida State University , Tallahassee , Florida 32306 , United States.
Biochemistry. 2018 Nov 13;57(45):6404-6415. doi: 10.1021/acs.biochem.8b00733. Epub 2018 Nov 2.
Cadherins are calcium-dependent, transmembrane adhesion molecules that assemble through direct noncovalent association of their N-terminal extracellular modular domains. As the transmembrane component of adherens junctions, they indirectly link adherent cells' actin cytoskeletons. Here, we investigate the most distal extracellular domain of neural cadherin (N-cadherin), a protein required at excitatory synapses, the site of long-term potentiation. This domain is the site of the adhesive interface, and it forms a dimer spontaneously without binding calcium, a surprising finding given that calcium binding is required for proper physiological function. A critical tryptophan at position 2, W2, provides a spectroscopic probe for the "closed" monomer and strand-swapped dimer. Spectroscopic studies show that W2 remains docked in the two forms but has a different apparent interaction with the hydrophobic pocket. Size-exclusion chromatography was used to measure the levels of the monomer and dimer over time to study the kinetics and equilibria of the unexpected spontaneous dimer formation ( K = 130 μM; τ = 2 days at 4 °C). Our results support the idea that NCAD1 is missing critical contacts that facilitate the rapid exchange of the βA-strand. Furthermore, the monomer and dimer have equivalent and exceptionally high intrinsic stability for a 99-residue Ig-like domain with no internal disulfides ( T = 77 °C; Δ H = 85 kcal/mol). Ultimately, a complete analysis of synapse dynamics requires characterization of the kinetics and equilibria of N-cadherin. The studies reported here take a reductionist approach to understanding the essential biophysics of an atypical Ig-like domain that is the site of the adhesive interface of N-cadherin.
钙黏着蛋白是钙依赖性跨膜黏附分子，通过其N端细胞外模块化结构域的直接非共价结合进行组装。作为黏着连接的跨膜成分，它们间接连接黏附细胞的肌动蛋白细胞骨架。在这里，我们研究神经钙黏着蛋白（N-钙黏着蛋白）最远端的细胞外结构域，该蛋白在兴奋性突触（长期增强的部位）是必需的。这个结构域是黏附界面的位点，它在不结合钙的情况下自发形成二聚体，鉴于正常生理功能需要钙结合，这一发现令人惊讶。位置2的关键色氨酸W2为“封闭”单体和链交换二聚体提供了一个光谱探针。光谱研究表明，W2在两种形式中都保持对接，但与疏水口袋有不同的表观相互作用。使用尺寸排阻色谱法测量单体和二聚体随时间的水平，以研究意外自发二聚体形成的动力学和平衡（K = 130 μM；4°C下τ = 2天）。我们的结果支持这样一种观点，即NCAD1缺少促进βA链快速交换的关键接触。此外，对于一个没有内部二硫键的99个残基的免疫球蛋白样结构域，单体和二聚体具有同等且异常高的内在稳定性（T = 77°C；ΔH = 85 kcal/mol）。最终，对突触动力学的完整分析需要对N-钙黏着蛋白的动力学和平衡进行表征。这里报道的研究采用简化方法来理解一个非典型免疫球蛋白样结构域的基本生物物理学，该结构域是N-钙黏着蛋白黏附界面的位点。
