STRUCTURAL BASIS OF LIGAND RECOGNITION BY VWF A DOMAINS

“A”结构域，或整合素中通常称为“I”结构域，是一个大约200个残基的蛋白质识别模块，存在于许多参与细胞-细胞和细胞-基质黏附的蛋白质中。在大多数情况下，亲本分子的关键配体结合性质是由重组A结构域概括的，表明该结构域是黏附功能的关键元件。这反过来表明，A结构域是一个有吸引力的靶点，可以作为治疗药物的靶点，从而扰乱异常粘连。我们以前已经确定了这个家族四个成员的晶体结构，并提出了一个涉及结构域上表面的配体识别的一般模型。在整合素中，金属离子位于可能的配体结合界面，我们称之为金属离子依赖的粘附点或MIDAS基序。此外，我们还提出了A结构域的粘附性依赖于A结构域内的三级结构变化(“移形”)，这些变化创建了一个高亲和力的配体结合表面，在这个表面上，结构域从“封闭”构象转换到“开放构象”。现在，我们希望通过确定A结构域与其配体的络合物的晶体结构来检验和扩展这些假设。具体地说，我们将针对整合素α1和α2 I结构域以及von Willebrand因子A3结构域与三螺旋胶原样肽的复合体；整合素αM I结构域与纤维蛋白原和ICAM-1片段；整合素α2 I结构域与层粘连蛋白片段；以及von Willebrand因子A1结构域与糖蛋白1b和蛇毒素Botrocetin的片段。成功地确定了一系列这些靶标的结构将使我们能够解决以下问题：A/I结构域家族识别配体的共同和不同特征是什么？金属桥是整合素-配体接触的一般特征吗？是什么取代了vWF A域中的金属？第三级“变形”是A/I领域的共同特征吗？这是否代表了一种监管机制？最后，我们的结构数据是否表明了设计小分子的策略，这些小分子将模仿配体结合并破坏黏附？

The "A" domain, or "I" domain as it is commonly called in integrins, is a approximately 200 residue protein recognition module that is present in many proteins involved in cell-cell and cell-matrix adhesion. In most cases tested, key ligand binding properties of the parent molecule are recapitulated by recombinant A domains, demonstrating that this domain is a critical element in the adhesion function. This in turn suggests that the A domain is an attractive target for therapeutic agents that would disrupt aberrant adhesion. We have previously determined the crystal structures of four members of this family and proposed a general model for ligand recognition involving the upper surface of the domain. In the integrins, a metal ion is located at the putative ligand binding interface, which we have called the "metal ion-dependent adhesion site" or MIDAS motif. In addition, we have proposed that the adhesiveness of A domains is dependent on tertiary structure changes within the A domain ("shape-shifting") that create a high affinity ligand binding surface, in which the domain switches from a "closed" to an "open conformation. We now wish to test and extend these hypotheses by determining crystal structures of A domains in complex with their ligands. Specifically, we will target complexes of the integrin alpha1 and alpha2 I domains and the von Willebrand Factor A3 domain with triple helical collagen-like peptides; the integrin alphaM I domain with fragments of fibrinogen and ICAM-1; the integrin alpha2 I domain with a fragment of laminin; and the von Willebrand Factor A1 domain with a fragment of glycoprotein lb and the snake toxin botrocetin. Successful structure determination of a range of these targets will enable us to address the following questions: What are the common and distinct features of ligand recognition by the A/I domain family? Is a metal bridge a general feature of integrin-ligand contacts? What replaces the metal in the vWF A domains? Is tertiary "shape- shifting" a common feature of A/I domains, and does this represent a mechanism for regulation? And finally, do our structural data suggest strategies for the design of small molecules that would mimic ligand binding and disrupt adhesion?