In the current absence of a crystal or NMR structure of a specific agonist-docked GPCR, many divergent and less definitive experimental approaches have been taken to gain insights into the conformation of the complex and into the molecular basis of ligand binding and activation. In the case of the CCK receptor, these approaches have yielded working models of the hormone-bound receptor that share many similarities, yet differ particularly in regard to the mode of docking the carboxyl-terminal portion of CCK (10
). In the current work, we have utilized multidimensional FRET to establish distances between residues scattered throughout the docked CCK ligand and residues in distinct extracellular regions of this receptor to try to gain new, independently derived insights, and to possibly distinguish between the molecular models that have been proposed. Multidimensional FRET becomes extremely powerful, considering that this approach in the current work, using three ligands and four receptor constructs, generates twelve distance constraints.
Both of the proposed models of CCK docked at its receptor share substantial similarities in their helical bundle domains (10
). These structures are also quite similar to this region of the high resolution crystal structures of rhodopsin and the β
2-adrenergic receptor (1
), consistent with the close primary structural relationships of the predicted transmembrane segments of those receptors with the CCK receptor. It is notable that the distances measured in the FRET studies from a residue fixed high in the intramembranous region of the central core of the helical bundle of the CCK receptor, at the level of Cys94
in transmembrane segment two, are not different to the amino terminus, midregion, and carboxyl terminus of CCK. These distances of approximately 22 Å are fully consistent with the peptide lying at the surface of the membrane, as has been proposed in the model based on photoaffinity labeling (10
). In contrast, this experimentally derived distance from the carboxyl terminus of CCK makes the model in which this region of CCK is proposed to reside immediately adjacent to Cys94
much less likely.
The microenvironment of the fluorophore at the carboxyl terminus of CCK as docked at its receptor was recently demonstrated to be exposed to hydrophilic solvent, based on potassium iodide quenching, and highly mobile, based on fluorescence anisotropy (23
). Of particular note, the quenching of the carboxyl-terminal fluorescence indicator was even more pronounced when the receptor was in its active conformation than when in its inactive conformation (23
). This is the opposite of what might be expected if activation involved moving the carboxyl terminus of CCK more deeply into the helical bundle. These fluorescence data are fully consistent with the current FRET data and with the molecular model derived from photoaffinity labeling data (10
), again distinguishing it from the less-compatible model that places the peptide carboxyl terminus within the helical bundle (11
The FRET distances to the extracellular loop regions are more difficult to interpret than those to the helical bundle region. This relates to the less well refined structures of the loops and the absence of experimentally generated constraints for those regions. In fact, the current FRET distances to the residues within each of these loops will represent important new constraints for building more meaningful models of the extracellular regions of this receptor. Evaluating compatibility of these distances with the existing models, it becomes clear that, while many of the distances measured are compatible with both proposed molecular models of the hormone-receptor complex, only the working model derived largely based on the photoaffinity labeling data (10
) is fully compatible with all of the FRET distance constraints.
It is interesting that the FRET studies identified that the longest distances from all three of the CCK fluorophores to any of the extracellular loops were those to residue 204 within loop two. The second extracellular loop is the longest of the loops, constrained only by the presence of the architecturally important disulfide bond between the first and second loops that has been shown to be present in many GPCRs (30
). That bond (involving Cys196
) is present nine residues away from residue 204. It is also interesting that the Arg197
residue adjacent to this disulfide bond has been proposed to interact with the sulfate moiety within tyrosine 27 of CCK in both working models (10
). This was determined using multiple different experimental approaches, including direct photoaffinity labeling. Here, too, this residue is eight residues away from residue 204 that was utilized as a site of one of the fluorescence acceptors in the current study. The long distance to this residue might suggest that part of the second extracellular loop might move away from the docked peptide.
The short distances to residues within the first and third extracellular loops is consistent with the peptide-binding pocket situated nestled between these loops. This is consistent with both models that have been proposed (10
), although detailed, experimentally based resolution of these loops has not yet been achieved.
The current report has utilized a unique and complementary technique to provide insights into the mode of docking the CCK natural peptide ligand to its Family A GPCR. The distance constraints are fully consistent with one of the models proposed in the current literature (10
) and are less compatible with some features of the other model currently proposed (11
). In addition to distinguishing between these models, the current data provide the first comprehensive set of experimentally derived constraints for the extracellular loops of this receptor. This should contribute to further refinement of our understanding of the conformation of this physiologically important receptor and to the molecular basis of its binding its natural peptide ligand.