CRYSTALLIZATION OF AN ENGINEERED ICAM-1 DOMAIN 1 WITH THE INSERTED

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 ICAM-1是白细胞整联蛋白LFA-1（淋巴细胞功能相关抗原-1; aLB 2）的反受体，与插入的或I结构域结合，并促进在免疫和炎症中重要的细胞相互作用。ICAM-1由5个胞外IG超家族结构域（D1-D5）、跨膜区和短胞质结构域组成。ICAM-1作为人鼻病毒的受体也被破坏。现有的结构数据（CryoEM和晶体学）表明，只有D1的ICAM-1参与结合整合素I结构域和鼻病毒。到目前为止，可溶性ICAM-1已经以三种不同的形式产生：全长胞外域D1-D5、D1 D2和D3-D5。其中，D1 D2和D3-D5已经通过自身和与其他结合蛋白的复合物结晶。除D1和D5外的所有结构域都含有许多聚糖链，这些聚糖链似乎有助于ICAM-1的稳定性。虽然许多IG结构域似乎是独立折叠的单元，但并非细胞表面受体中的所有IG超家族结构域都保留天然折叠并与相邻结构域隔离地发挥功能。因此，使用定向进化和合理设计的组合来工程化ICAM-1 D1的稳定版本，其保留其与LFA-1 I结构域和鼻病毒的相互作用。 Jin等人（1）关于整联蛋白LFA I结构域的高亲和力突变体的工程化的先前工作也证明了定向进化在发现活化热点中的能力。激活突变的位置在I结构域的内部描绘出变构界面，这反过来将变构性传递给完整整合素中的相邻结构域。这些突变通过稳定活性构象诱导高亲和力结合，并且引入两个活化突变（F265 S/F292 G）导致对ICAM-1的亲和力增加200，000。 突变的高亲和力I结构域与工程化ICAM-1复合物的结晶是一个极其令人兴奋的前景，主要有两个原因。首先，阐明高亲和力结合I结构域的结构将产生关于在与ICAM-1D 1结合时导致该分子活化的实际变构机制的有价值的信息。第二，虽然I结构域与ICAM-1 D1的结构本身是迷人的，未来的目标将是解决ICAM-1 D1与人鼻病毒复合物的结构。因此，从具有I结构域的复合物获得的ICAM-1 D1的结构将是非常有价值的起始工具。ICAM-1 D1的结构还将提供关于突变稳定化的结构基础的有价值的信息和对进一步优化的有价值的见解。 以前曾尝试使ICAM-1 D1结晶（一种较早的不稳定形式，具有整合素I结构域）。筛选I结构域和早期ICAM-1 D1版本的复合物在不同pH、盐、金属离子、沉淀剂和冷冻溶液中的结晶。获得了尺寸为200 x 200 x 50 <$m的平面形状的晶体，其在高级光子源（APS）同步加速器中衍射至3.5-4 <$m。通过使用与ICAM-1 D1 D2（2）结合的中间体L I结构域的晶体结构作为分子置换计算的搜索模型，可以求解二元复合物4处的完整数据集。然而，每个不对称单元被发现含有六聚体二元复合物，并解决它的结构是不平凡的。由于这种大复合物的形成可能是由于D1.v2的聚集倾向，更稳定的D1突变体可能导致更好的衍射质量和更小的晶胞。 参考文献： 1. Jin，M.，例如，定向进化以探测亲和力高20万倍的蛋白质变构和整合素I结构域。Proc Natl Acad Sci U S A，2006. 103（15）：第5758-63页。 2. Shimaoka，M.，Lu，C.，帕尔夫拉曼河T.，von Andrian，U. H、McCormack，A.，Takagi，J. & Springer，T. A.等人（2001）Proc Natl Acad Sci U S A 98，6009-14。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ICAM-1 is the counter receptor for the leukocyte integrin LFA-1 (lymphocyte function-associated antigen-1; aLB2), binding to the ¿inserted` or I domain, and promotes cellular interactions important in immunity and inflammation. ICAM-1 consists of five extracellular Ig superfamily domains (D1-D5), a transmembrane spanning region, and a short cytoplasmic domain. ICAM-1 is additionally subverted as a receptor for human rhinovirus. Existing structural data (CryoEM and crystallography) indicates that only D1 of ICAM-1 is involved in binding both to the integrin I domain and to rhinovirus. Up until now, soluble ICAM-1 has been produced in three different forms: full-length ectodomain D1-D5, D1D2, and D3-D5. Of these, D1D2 and D3-D5 have been crystallized both by themselves and in complex with other binding proteins. All domains except D1 and D5 contain a number of glycan chains, which appear to contribute to ICAM-1 stability. Although many Ig domains appear to be independently folded units, not all Ig superfamily domains in cell surface receptors retain native fold and function in isolation from neighboring domains. Therefore, a combination of directed evolution and rational design was used to engineer a stable version of ICAM-1 D1 that retains its interaction with LFA-1 I domain and rhinovirus. Previous work by Jin et al (1) on the engineering of high affinity mutants of the integrin LFA I domain also demonstrated the power of directed evolution in the discovery of activation hot spots. The location of activating mutations traces out an allosteric interface in the interior of the I domain, which in turn communicates allostery to neighbouring domains in intact integrins. These mutations induce high affinity binding by stabilizing active conformations, and the introduction of two activating mutations (F265S/F292G) leads to an increase of 200,000 affinity to ICAM-1. The crystallization of the mutated high affinity I domain in complex with the engineered ICAM-1 is an extremely exciting prospect for two main reasons. First, elucidation of the structure of the high affinity binding I domain will yield valuable information on the actual allosteric mechanisms that lead to activation of this molecule, upon binding to ICAM-1 D1. Second, although the structure of I domain with ICAM-1 D1 is in itself fascinating, a future goal will be to solve the structure of ICAM-1 D1 in complex with human rhinovirus. Therefore the structure of ICAM-1 D1 obtained from the complex with I domain will be a very valuable starting tool. The structure of the ICAM-1 D1 will also provide valuable information on the structural basis for stabilization by mutations and valuable insight into further optimization. A previous attempt was made to crystallize ICAM-1 D1 (an earlier less stable version with the integrin I domain. The complex of the I domain and the earlier ICAM-1 D1 version was screened for crystallization at different pH, salt, metal ions, precipitants, and freezing solutions. Crystals with a planar shape of 200 x 200 x 50 ¿¿m in size were obtained, which diffracted to 3.5-4 ¿ at the Advanced Photon Source (APS) synchrotron. A complete data set at 4 ¿ of the binary complex could be solved by using the crystal structure of the intermediate ¿¿L I domain bound to ICAM-1 D1D2 (2) as search models for the molecular replacement calculations. However, each asymmetric unit was found to contain hexameric binary complexes, and solving the structure for it was not trivial. As the formation of such large complex may be due to the aggregation tendency of D1.v2, more stable D1 mutants may lead to a better diffraction quality and smaller unit cell. Refs: 1. Jin, M., et al., Directed evolution to probe protein allostery and integrin I domains of 200,000-fold higher affinity. Proc Natl Acad Sci U S A, 2006. 103(15): p. 5758-63. 2. Shimaoka, M., Lu, C., Palframan, R. T., von Andrian, U. H., McCormack, A., Takagi, J. & Springer, T. A. (2001) Proc Natl Acad Sci U S A 98, 6009-14.