Use of multidimensional fluorescence resonance energy transfer to establish the orientation of cholecystokinin docked at the type A cholecystokinin receptor

Abstract

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.

Figure 1

Control spectra. Shown are representative emission spectra of a fluorescence donor (Aladan²⁴-CCK) bound to a monoreactive CCK receptor construct (N102C) after excitation with 362 nm light, and that of the same receptor derivatized with the fluorescence acceptor, Alexa⁵⁴⁶, after excitation with light at either 362 nm or at 546 nm (left panel). This clearly establishes that the wavelength of light used to excite the donor has no ability to excite the acceptor in the absence of energy transfer from donor to acceptor. Also shown is the elimination of the FRET signal when nonfluorescent CCK (1 μM) competed for occupation of this monoreactive receptor by the fluorescence donor (right panel). This establishes the critical nature of spatial approximation between donor and acceptor provided by receptor occupation of donor in the experimental paradigm.

Figure 2

Experimental spectra. Shown are representative data generated with cells stably expressing the null-cysteine-reactive CCK receptor construct (top panel) and a representative pseudowild type, monocysteine-reactive CCK receptor construct (N102C) (middle panel). The cells were derivatized with the Alexa⁵⁴⁶-MTS reagent and subsequently allowed to bind fluorescent Aladan ligand, as described in Methods. Cells were then excited by light at 362 nm and the illustrated emission spectra were collected. A peak at 572 nm in the monoreactive receptor-bearing cells indicates significant fluorescence resonance energy transfer, while the null-cysteine-reactive CCK receptor-bearing cells did not have such a peak. Also shown in the middle panel is the ability to deconvolute the FRET emission spectrum into its component donor emission and acceptor emission spectra. The bottom panel shows the calibration of Alexa fluorescence acceptor excitation and acceptor emission spectra, permitting the determination of the acceptor excitation associated with any observed acceptor emission. The Pearson correlation coefficient, “r”, for the relationship between values of acceptor excitation and acceptor emission was 0.99.

Figure 3

Molecular model of the CCK-occupied CCK receptor. Shown are views from the side (left panel) and top (right panel) of the molecular model of the CCK-occupied CCK receptor that is based on photoaffinity labeling data (14). This model is fully compatible with all twelve of the current experimentally derived FRET distance constraints. Highlighted in dashed white lines are the four distances between the C_β positions of the fluorescence donor at the critical residue at the carboxyl terminus of CCK (Phe³³) and the sites of the fluorescence acceptors within the CCK receptor. These distances best distinguish the two divergent working models of the CCK-occupied receptor, with the carboxyl terminus of the peptide situated in substantially different places in the two models. The backbone of the CCK ligand is illustrated in blue-to-red from amino terminus to carboxyl terminus. The sites of fluorescence donors are expanded and labeled. Each of the extracellular loop (ECL) regions of the CCK receptor is labeled. The disulfide bond that links extracellular loops one and two is illustrated as well.