Häussinger Daniel, Ahrens Thomas, Sass Hans-Jürgen, Pertz Olivier, Engel Jürgen, Grzesiek Stephan
Division of Structural Biology and Division of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland.
J Mol Biol. 2002 Dec 6;324(4):823-39. doi: 10.1016/s0022-2836(02)01137-3.
Cadherins are calcium-dependent cell surface proteins that mediate homophilic cellular adhesion. The calcium-induced oligomerization of the N-terminal two domains of epithelial cadherin (ECAD12) was followed by NMR spectroscopy in solution over a large range of protein (10 microM-5 mM) and calcium (0-5 mM) concentrations. Several spectrally distinct states could be distinguished that correspond to a calcium-free monomeric form, a calcium-bound monomeric form, and to calcium-bound higher oligomeric forms. Chemical shift changes between these different states define calcium-binding residues as well as oligomerization contacts. Information about the relative orientation and mobility of the ECAD12 domains in the various states was obtained from weak alignment and 15N relaxation experiments. The data indicate that the calcium-free ECAD12 monomer adopts a flexible, kinked conformation that occludes the dimer interface observed in the ECAD12 crystal structure. In contrast, the calcium-bound monomer is already in a straight, non-flexible conformation where this interface is accessible. This mechanism provides a rational for the calcium-induced adhesiveness. Oligomerization induces chemical shift changes in an area of domain CAD1 that is centered at residue Trp-2. These shift changes extend to almost the entire surface of domain CAD1 at high (5 mM) protein concentrations. Smaller additional clusters of shift perturbations are observed around residue A80 in CAD1 and K160 in CAD2. According to weak alignment and relaxation data, the symmetry of a predominantly dimeric solution aggregate at 0.6 mM ECAD12 differs from the approximate C2-symmetry of the crystalline dimer.
钙黏着蛋白是一类依赖钙离子的细胞表面蛋白，介导同种细胞间黏附。利用核磁共振波谱法，在较大的蛋白质浓度范围（10微摩尔/升至5毫摩尔/升）和钙离子浓度范围（0至5毫摩尔/升）内，追踪上皮钙黏着蛋白（ECAD12）N端两个结构域的钙诱导寡聚化过程。可以区分出几种光谱上不同的状态，分别对应无钙单体形式、钙结合单体形式以及钙结合的更高寡聚体形式。这些不同状态之间的化学位移变化确定了钙结合残基以及寡聚化接触位点。通过弱取向和15N弛豫实验，获得了ECAD12结构域在各种状态下的相对取向和流动性信息。数据表明，无钙的ECAD12单体采用灵活的、扭结的构象，该构象封闭了ECAD12晶体结构中观察到的二聚体界面。相比之下，钙结合单体已经处于一种笔直的、不灵活的构象，此时该界面是可及的。这种机制为钙诱导的黏附性提供了一种解释。寡聚化在结构域CAD1中以残基Trp-2为中心的区域诱导化学位移变化。在高蛋白质浓度（5毫摩尔/升）下，这些位移变化几乎扩展到结构域CAD1的整个表面。在CAD1中的残基A80和CAD2中的K160周围观察到较小的额外位移扰动簇。根据弱取向和弛豫数据，0.6毫摩尔/升ECAD12时主要为二聚体的溶液聚集体的对称性与晶体二聚体的近似C2对称性不同。
