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42859-AC4
Determination of Ligand Binding Epitopes Using NMR Diffusion Measurements

Cynthia K. Larive, University of California (Riverside)

During the second year of this grant we continued to make good progress on this project. We have completed a study on the binding of R- and S-enantiomers of propranolol to the serum protein alpha1-acid glycoprotein (AGP) and hope to be finished soon with a study of the binding of the smaller and achiral drug lidocaine at the same binding site. About 45% of the mass of AGP is made up by carbohydrate and no high resolution structure of AGP is available. The accepted picture of the binding pocket is that it is flexible, conically shaped, and hydrophobic with a patch of negatively charged/hydrophilic residues near the base. From our NMR results, it appears that the protons near the positively charged amine group of the R enantiomer have greater contact with the protein than in the S enantiomer. The stronger interactions for the hydrophilic protons of the weaker binding R-enantiomer, could reflect steric interactions that distance the naphthyl ring from the accommodating hydrophobic part of the AGP binding pocket. Our preliminary results suggest that the smaller but achiral lidocaine molecule appears to have a similar binding motif to propranolol. The propranolol results are currently being prepared for publication in Chemistry and Biology. I gave an invited talk at the Experimental NMR Conference (ENC) in April and my students gave posters on this work at AAPS and at the fall ACS meeting.

An exciting recent development is that we have established a collaboration with Prof. Zhenbio Yang's group in Plant Cell Biology at UCR. Prof. Yang's group studies intracellular signaling pathways linking cell surface receptors to nuclear components in the model plant Arabidopsis thaliana. The Arabidopsis genome sequence reveals that plants lack many of the signaling G proteins used by animals and yeast; and instead contain a unique family of small GTPases, termed ROP. ROP acts as a versatile switch in signal transduction in plants. In addition to knock-out studies of the 11 ROP genes identified in Arabidopsis to probe the pathways controlled by ROP, Prof. Yang has been active in studying the interactions of ROP with a regulatory protein, GAP. Binding of GAP switches off the GTPase activity of ROP. In a chemical genomics screen of the Chem Bridge Library, the Yang group has identified a series of small organic ligands that appear to block the protein-protein contacts of ROP and GAP. We plan to use NMR diffusion and saturation-transfer difference experiments to probe the binding of these ligands. These experiments provide a nice transition from method development to using these techniques to address a complex and important biological problem.

This grant assisted in the support and development of two students, Bridget Becker and Jennifer Cruz. Bridget received her Ph.D last fall and has been working at Procter and Gamble. Jennifer is a third year graduate student. She has passed her qualifying examinations and advanced to candidacy.

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