IDENTIFICATION OF A MODEL CARDIAC GLYCOSIDE RECEPTOR - COMPARISONS WITH NA-ATPASE(,K+)

The availability of high-affinity anti-digoxin monoclonal antibodies ( mAbs) offers the potential for their use as models for the characteriz ation of the relationship between receptor structure and cardiac glyco side binding. We have characterized the binding of anthroylouabain (AO ), a fluorescent derivative of the cardiac glycoside ouabain, to mAbs 26-10, 45-20, and 40-50 [Mudgett-Hunter, M., et al. (1995) Mol. Immuno l. 22, 477] and lamb kidney Na+, K+-ATPase by monitoring the resultant AO fluorescence emission spectra, anisotropy, lifetime values, and Fo rster resonance energy transfer (FRET) from protein tryptophan(s) (Trp ) to AO. These data suggest that the structural environment in the vic inity of the AO-binding site of Na+,K+-ATPase is similar to that of mA b 26-10 but not mAbs 45-20 and 40-50. A model of AO complexed to the a ntigen binding fragment (Fab) of mAb 26-10 which was generated using k nown X-ray crystal structural data [Jeffrey, P. D., et al. (1993) Proc . Natl. Acad. Sci. U.S.A. 90, 10310] shows a heavy chain Trp residue ( Trp-H100) that is close (similar to 3 Angstrom) to the anthroyl moiety . This is consistent with the energy transfer seen upon AO binding to mAb 26-10 and suggests that Trp-H100, which is part of the antibody's cardiac glycoside binding site, is a major determinant of the fluoresc ence properties of bound AO. In contrast, the generated model of AO co mplexed to Fab 40-50 [Jeffrey, P. D., et al. (1995) J. Mel. Biol. 248, 344] shows a heavy chain Tyr residue (Tyr-H100) which is part of the cardiac glycoside binding site, located similar to 10 Angstrom from th e anthroyl moiety. The closest Trp residues (H52 and L35) are located similar to 17 Angstrom from the anthroyl moiety, and no FRET is observ ed despite the fact that these Trp residues are close enough for signi ficant FRET to occur. The energy transfer seen upon AO binding to Na+, K+-ATPase suggests the presence of one completely quenched or two hig hly quenched enzyme Trp residues similar to 10 and similar to 17 Angst rom, respectively, from the anthroyl moiety. These data suggest that t he Na+, K+-ATPase Trp residue(s) involved in fluorescence energy trans fer to AO is likely to be part of the cardiac glycoside binding site.