PRELIMINARY X-RAY DIFFRACTION ANALYSIS OF AN ANTIBODY FRAGMENT AGAINST P-GLYCOPROTEIN. Valerie Monem, Sona Vasudevan, Kathy L. Johns and David R. Rose, Ontario Cancer Institute, Department of Medical Biophysics, University of Toronto, Canada.

P-glycoprotein (P-gp) has been shown to be involved in the development of drug resistance in cancer cells, thus impeding successful chemotherapy treatment. P-gp is predicted to consist of twelve membrane-spanning domains and is thought to act as an ATP-dependent drug pump with specificity for a broad range of structurally unrelated drugs¹. The structure of P-gp is as yet unknown. We are using monoclonal antibodies as one approach to obtaining structural information about P-gp. The Fab portion of the antibody C219 that recognizes both cytoplasmic ATP-binding domains of all known P-gp molecules² was crystallized with and without its peptide epitope. The unliganded Fab crystallized in the monoclinic space group P2₁ with 4 molecules in the asymmetric unit and data were collected to 3.2 Å resolution. Previous self-rotation analysis suggested the presence of a two-fold non-crystallographic symmetry (NCS)³. Subsequent analysis was done using molecular replacement methods. The super-imposed structures of several Fabs with a wide range of elbow-bends were used as starting probes. The program AMORE showed the same four strong rotation and translation peaks with two of the probes against data from 15 to 4 Å resolution. Rigid-body refinement was executed by X-PLOR, allowing for adjustment of the elbow-bend and of the heavy and light chains independently for each molecule. Only two of the four molecules in the asymmetric unit were found to be non-crystallographically related by 180[[ring]]. The unrefined electron density maps support the molecular replacement solutions. Further analysis will include the exploitation of the two-fold NCS with averaging techniques. The crystals of C219 with bound peptide are orthorhombic and belong to the space group P2₁2₁2₁, most likely with 4 molecules in the asymmetric unit (42% solvent content). However, these crystals did not diffract well enough for thorough crystallographic analysis. Better crystals are being grown using seeding techniques in preparation for synchrotron data collection. Supported by NSERC and NCI (Canada).

¹ Childs, S. J. and Ling V. (1994) In Important Advances In Oncology, DeVita, V.T. et al. Ed., Philadelphia, pp. 21-36.

² Georges, E., Bradley, G., Gariepy, J. and V. Ling (1989) P.N.A.S.USA 87, 152.

³ Vasudevan, S., Johns, K. L. and Rose, D. R. (1994) J. Mol. Biol. 241, 736.