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Structural Basis of RasGRP Binding to High-Affinity PKC Ligands

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Drug Discovery Program, Department of Neurology, Georgetown University Medical Center, 3970 Reservoir Road, Washington, D.C. 20007, Molecular Mechanisms of Tumor Promotion Section, National Cancer Institute, Bethesda, Maryland 20892, 123, State Key Laboratory of Bio-organic and Natural Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Fenglin Lu, Shanghai 200032, China, and Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
Cite this: J. Med. Chem. 2002, 45, 4, 853–860
Publication Date (Web):January 22, 2002
Copyright © 2002 American Chemical Society

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    Abstract Image

    The Ras guanyl releasing protein RasGRP belongs to the CDC25 class of guanyl nucleotide exchange factors that regulate Ras-related GTPases. These GTPases serve as switches for the propagation and divergence of signaling pathways. One interesting feature of RasGRP is the presence of a C-terminal C1 domain, which has high homology to the PKC C1 domain and binds to diacylglycerol (DAG) and phorbol esters. RasGRP thus represents a novel, non-kinase phorbol ester receptor. In this paper, we investigate the binding of indolactam(V) (ILV), 7-(n-octyl)-ILV, 8-(1-decynyl)benzolactam(V) (benzolactam), and 7-methoxy-8-(1-decynyl)benzolactam(V) (methoxylated benzolactam) to RasGRP through both experimental binding assays and molecular modeling studies. The binding affinities of these lactams to RasGRP are within the nanomolar range. Homology modeling was used to model the structure of the RasGRP C1 domain (C1-RasGRP), which was subsequently used to model the structures of C1-RasGRP in complex with these ligands and phorbol 13-acetate using a computational docking method. The structural model of C1-RasGRP exhibits a folding pattern that is nearly identical to that of C1b-PKCδ and is comprised of three antiparallel-strand β-sheets capped against a C-terminal α-helix. Two loops A and B comprising residues 8−12 and 21−27 form a binding pocket that has some positive charge character. The ligands phorbol 13-acetate, benzolactam, and ILV are recognized by C1-RasGRP through a number of hydrogen bonds with loops A and B. In the models of C1-RasGRP in complex with phorbol 13-acetate, benzolactam, and ILV, common hydrogen bonds are formed with two residues Thr12 and Leu21, whereas other hydrogen bond interactions are unique for each ligand. Furthermore, our modeling results suggest that the shallower insertion of ligands into the binding pocket of C1-RasGRP compared to C1b-PKCδ may be due to the presence of Phe rather than Leu at position 20 in C1-RasGRP. Taken together, our experimental and modeling studies provide us with a better understanding of the structural basis of the binding of PKC ligands to the novel phorbol ester receptor RasGRP.

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     Georgetown University Medical Center.

     Current address:  NeuroLogic Incorporation, 9700 Great Seneca Highway, Rockville, MD 20850.


     Shanghai Institute of Organic Chemistry.

     National Cancer Institute.

     University of Alberta.


     To whom correspondence should be addressed. For A.P.K.:  phone, 202-687-0686; fax, 202-687-5065; e-mail, [email protected]. For S.W.:  phone, 202-687-2028; fax, 202-687-4032; e-mail, wangs@

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