The human CD2 molecule, expressed on thymocytes, T lymphocytes and NK cells, binds the ubiquitously expressed counter-receptor, CD58, and defines an important immune recognition/activation/adhesion pathway. During the last funding interval, we elucidated the atomic basis of this CD2-CD58 interaction. A solution structure of the non-glycosylated hCD58 adhesion domain was obtained. NMR titration studies using 15N-labeled CD58 and unlabeled human CD2 identified chemical shifts of those CD58 residues involved in CD2 binding. This interaction was detailed further by the x-ray crystallographic structure of a heterophilic adhesion complex between CD2 and CD58 at 3.2A resolution. A strikingly asymmetric, face-to-face interaction of the major beta-sheets of the two Ig-like domains was observed with poor shape but excellent charge complementarity imparting a high degree of specificity with rapid binding kinetics. The present application will further dissect the molecular components of the CD2-CD58 counter-receptor interface and several CD2-associated proteins that inducibly bind to the CD2 cytoplasmic tail following CD2 ectodomain ligation. First, the functional """"""""hot spot"""""""" of the CD2-CD58 interaction will be identified by isothermal titration calorimetry and by binding analysis with wild-type and mutant CD2 and CD58 proteins including single point mutations of the contacting residues at the intermolecular surface. A molecular assay for inhibitors of CD2-CD58 interaction will be developed and applied to screening of organic compound libraries. Once identified, the effects of such inhibitors on CD2-based cellular functions will be ascertained with the goal of creating new immunosuppressive compounds. Second, the role of the CD2hCD58 (mCD48) interaction in promoting the scanning of antigen-presenting cell surfaces by T cells in concert with the beta2 integrin pathway will be defined in both man and mouse. Such T cell scanning activity provides an important basis for immune surveillance. Third, the molecular mechanism by which the CD2 cytoplasmic tail interacting proteins CD2BP1, CD2BP3 and CD2BP4 affect cell motility, polarity and lipid biogenesis will be investigated.
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