Cytoplasmic signal transduction during T cell activation is mediated by a complex of calcineurin and cyclophilin (CyP). CyP is a binding protein for the immunosuppressive drug cyclosporine A (CsA) and also an enzyme catalyzing the cis yields trans isomerization of peptidyl-prolyl bonds. Calcineurin, a seine/threonine phosphatase has been recently identified as a receptor of the CyP-CsA complex in vitro. This proposal is aimed at structural studies of mammalian CyPs and their complexes with substrates and CsA by X-ray protein crystallography, including: (I) determination of three-dimensional structures of human CyP A complexed with CsA and the CsA derivatives of dimethyl-Bmt1-CsA and MeAla6-CsA (II) determination of three-dimensional structures of human CyP A complexed with three proline substrates of Ser-Pro, His-Pro, and Ala-Ala-Pro-Phe, (III) structural comparison between CyP-CsA and CyP-substrate, (IV) determination of the three-dimensional structure of human CyP B, and (V) determination of the three-dimensional structure of murine CyP C. These studies will present a structural basis for cytoplasmic signal transduction in T cell activation, provide insight into the mechanism of peptidyl-prolyl isomerization and its relationship to immunosuppression, and serve as a guideline for the design of new immunosuppressive drugs. Routine methods will be used for crystallization (dialysis or vapor diffusion), preliminary characterization of crystals (precession and still photos), and heavy atom derivative preparation. Diffraction data will be collected on either a Hamlin multiwire detector or a phosphate image plate in our laboratory. Crystal structures will be determined by the molecular replacement or multiple isomorphous replacement method. Models will be built in an ESV10 graphic system and refined by the PROLSQ or XPLOR program.
| Huai, Qing; Kim, Hwa-Young; Liu, Yudong et al. (2002) Crystal structure of calcineurin-cyclophilin-cyclosporin shows common but distinct recognition of immunophilin-drug complexes. Proc Natl Acad Sci U S A 99:12037-42 |
| Xu, R X; Hassell, A M; Vanderwall, D et al. (2000) Atomic structure of PDE4: insights into phosphodiesterase mechanism and specificity. Science 288:1822-5 |
| Chook, Y M; Lipscomb, W N; Ke, H (1998) Detection and use of pseudo-translation in determination of protein structures. Acta Crystallogr D Biol Crystallogr 54:822-7 |
| Zhao, Y; Chen, Y; Schutkowski, M et al. (1997) Cyclophilin A complexed with a fragment of HIV-1 gag protein: insights into HIV-1 infectious activity. Structure 5:139-46 |
| Zhao, Y; Ke, H (1996) Crystal structure implies that cyclophilin predominantly catalyzes the trans to cis isomerization. Biochemistry 35:7356-61 |
| Zhao, Y; Ke, H (1996) Mechanistic implication of crystal structures of the cyclophilin-dipeptide complexes. Biochemistry 35:7362-8 |
