CRYSTAL STRUCTURE OF ANTI-P-GLYCOPROTEIN FAB MRK-16 IN COMPLEX WITH ITS PEPTIDE EPITOPE. S. Vasudevan, K. Johns and D.R. Rose, Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, M5G 2M9 Canada

Cancer cells undergoing chemotherapy can develop multidrug resistance, one form of which is associated with the overexpression of a membrane protein, P-glycoprotein (pgp). Pgp is an ATP-binding cassette (ABC) transporter that consists of two putative membrane-spanning domains and two cytoplasmic ATPase domains. Pgp has been shown to participate in energy-dependent efflux of a wide range of common anti-cancer drugs as well as other substrates. Inhibition of pgp function can improve the effectiveness of chemotherapy.

Monoclonal antibody MRK-16 binds to a discontinuous epitope consisting of two extracellular loops distant in the amino-acid sequence of pgp. It has been used as an adjuvant in anti-cancer treatments. We are studying MRK-16 firstly to understand its mode of interaction with pgp with the possible goal of improved pgp inhibitors, and secondly as part of a broad strategy to use antibodies as tools towards the structure of pgp itself. We report here the crystal structure of the MRK-16 Fab. Crystals were grown in the presence of a synthetic peptide representing one of the epitope loops. The space group is P2(1) with unit cell dimensions(a,b,c) of 54.5, 67.8, 117.2Å, (=97.6deg., and there are two Fab's per asymmetric unit. The structure was determined by standard molecular replacement techniques and refined to 2.8Å resolution with x-plor. Due to crystal packing, only one of the Fab's is complexed with peptide, permitting a comparison of liganded and unliganded structures in the same crystal. The elbow angles of the two copies of the Fab differ by about 7 degrees and there are some intriguing differences in the conformations of some of the complementarity-determining loops that make up the binding site. Conclusions based on the structure reported here, in the light of information on other pgp inhibitors, will be discussed.

MRK-16 antibody was provided by Prof. T. Tsuruo, University of Tokyo. This work was supported by an NCI(Canada) grant to DRR.