Elucidation of the Molecular Basis of Cholecystokinin Peptide Docking to Its Receptor Using Site-Specific Intrinsic Photoaffinity Labeling and Molecular Modeling

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• 作者：Maoqing Dong ; Polo C.-H. Lam ; Delia I. Pinon ; Ruben Abagyan ; Laurence J. Miller
• 刊名：Biochemistry
• 出版年：2009
• 出版时间：June 16, 2009
• 年：2009
• 卷：48
• 期：23
• 页码：5303-5312
• 全文大小：995K
• 年卷期：v.48,no.23(June 16, 2009)
• ISSN：1520-4995

文摘

G protein-coupled receptors represent the largest family of receptors and the major target of current drug development efforts. Understanding of the mechanisms of ligand binding and activation of these receptors remains limited, despite recent advances in structural determination of family members. This work focuses on the use of photoaffinity labeling and molecular modeling to elucidate the structural basis of binding a natural peptide ligand to a family A G protein-coupled receptor, the type 1 cholecystokinin receptor. Two photolabile cholecystokinin analogues were developed and characterized as representing high-affinity, fully biologically active probes with sites of covalent attachment at positions 28 and 31. The sites of receptor labeling were identified by purification, proteolytic peptide mapping, and radiochemical sequencing of labeled wild-type and mutant cholecystokinin receptors. The position 28 probe labeled second extracellular loop residue Leu¹⁹⁹, while the position 31 probe labeled first extracellular loop residue Phe¹⁰⁷. Along with five additional spatial approximation constraints coming from previous photoaffinity labeling studies and 12 distance restraints from fluorescence resonance energy transfer studies, these were built into two homology models of the cholecystokinin receptor, based on the recent crystal structures of the β2-adrenergic receptor and A2a-adenosine receptor. The resultant agonist ligand-occupied receptor models fully accommodate all existing experimental data and represent the best refined models of a peptide hormone receptor in this important family.