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

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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.