Use of multidimensional fluorescence resonance energy transfer to establish the orientation of cholecystokinin docked at the type A cholecystokinin receptor.
Fluorescence resonance energy transfer (FRET) represents a powerful tool to establish relative distances between donor and acceptor fluorophores. By utilizing several donors situated in distinct positions within a docked full agonist ligand and several acceptors distributed at distinct sites within its receptor, multiple interdependent dimensions can be determined. These can provide a unique method to establish or confirm three-dimensional structure of the molecular complex. In this work, we have utilized full agonist analogues of cholecystokinin (CCK) with Aladan distributed throughout the pharmacophore in positions 24, 29, and 33, along with receptor constructs derivatized with Alexa (546) at positions 94, 102, 204, and 341 in the helical bundle and first, second, and third extracellular loops, respectively. These provided 12 FRET distances to overlay on working models of the CCK-occupied receptor. These established that the carboxyl terminus of CCK resides at the external surface of the lipid bilayer, adjacent to the receptor amino-terminal tail, rather than being inserted into the helical bundle. They also provide important experimentally derived constraints for understanding spatial relationships between the docked ligand and the flexible extracellular loop regions. Multidimensional FRET provides a new independent method to establish and refine structural insights into ligand-receptor complexes.
Harikumar
Kaleeckal G
KG
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, 13400 East Shea Boulevard, Scottsdale, Arizona 85259, USA.
Gao
Fan
F
Pinon
Delia I
DI
Miller
Laurence J
LJ
D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000737:chemistry; D000409:Alanine / Q000031:analogs & derivatives* / Q000737:chemistry; D000818:Animals; D016466:CHO Cells; D002766:Cholecystokinin / Q000737:chemistry* / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D008024:Ligands; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D011806:Quinolinium Compounds / Q000737:chemistry*; D043886:Receptor, Cholecystokinin A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
C481052:Alexa fluor 546; D008024:Ligands; D008051:Lipid Bilayers; D011806:Quinolinium Compounds; D043886:Receptor, Cholecystokinin A; C459091:aladan; D002766:Cholecystokinin; D015081:2-Naphthylamine; D000409:Alanine