A detailed understanding of the molecular basis of ligand binding and activation of a receptor provides key information that can be useful in the development and refinement of receptor-active drugs. The recent solution of the high-resolution crystal structures of the β2-adrenergic receptor and A2a-adenosine receptor has provided two very useful templates for GPCRs in family A (1
). Both structures were as different from the previous best structure for a receptor in this family, rhodopsin, as the structure of rhodopsin had been different from the structure of bacteriorhodopsin, the previous best template (6
).While these models are clearly improving for the prediction of structures of other classical GPCRs in this family, a detailed molecular understanding of the basis of ligand binding to them is largely limited to the GPCRs that are naturally activated by small molecules that act within the relatively constrained helical bundle domain. In contrast, peptides are believed to bind predominantly to extracellular loop and tail domains of their receptors (35
). These regions of the receptors are much less well conserved and are likely quite varied in structure from one receptor to another. This creates major challenges for meaningful insights into the molecular basis of binding and activation of such receptors by peptide ligands.
The purpose of this work was to enhance our understanding of the molecular basis of binding and activation of a peptide hormone receptor in this family, the type 1 (or type A) CCK receptor. This is a receptor that plays important roles in nutrient homeostasis. It is present in the pancreas, gallbladder, gastrointestinal smooth muscle and nerves, and specific brain nuclei, where it acts to stimulate pancreatic exocrine secretion, gallbladder emptying, enteric motility, and postcibal satiety (36
). To achieve these insights, we have developed two homology models for this receptor, one based on the β2-adrenergic receptor structure and another based on the A2a-adeonsine receptor structure, and we have utilized a broad variety of experimentally derived constraints to refine these models. This includes the development, characterization, and application of two new intrinsic photoaffinity labeling probes, increasing the number of spatial approximation constraints to seven. It is note-worthy that these experimentally derived constraints were all generated with full agonist analogues of the natural peptide ligand, and that this included sites of covalent attachment in six of seven residues within its highly focused pharmacophoric domain.
On the basis of these data, the site of docking of CCK in molecular modeling approaches was highly consistent and converged quite nicely from the two distinct templates. CCK was found to bind at the extracellular surface and above the lipid bilayer to a pocket defined by the top of transmembrane segment 2, extracellular loops 2 and 3, and the top of transmembrane segment 7.The carboxyl terminus of CCK in this model is located above the helical bundle. This is generally consistent with the previously described model that was based on a smaller number of photoaffinity labeling constraints and the rhodopsin homology template (8
). It is, however, quite distinct from the alternative model that was based largely on mutagenesis data (12
). As in both previous molecular models, the functionally important acidic tyrosine sulfate moiety within CCK in the current model is found to form a complex nicely with the basic Arg197
in extracellular loop 2.
This site of peptide docking with the CCK receptor in the current refined molecular model was also consistent with 12 measurements coming from FRET experiments with distances from each of three positions distributed throughout the CCK peptide to each of four positions in the ectodomain of the CCK receptor (30
). These data nicely support the docked pose of CCK with its carboxyl terminus close to Asn102
near the top of transmembrane segment 2, with its midregion closest to Ala204
in extracellular loop 2 and Thr341
near the top of transmembrane segment 6. In contrast, the alternative model that came largely from indirect mutational studies in which the amino terminus of CCK resides closest to the top of transmembrane segment 1 and the carboxyl terminus resides closest to the top of transmembrane segment 6 is inconsistent with these FRET data.
The models currently being proposed are also consistent with everything we know from existing peptide and receptor structure–activity studies. Key is the ability of this model to accommodate the various extended molecular forms of CCK, with the amino terminus of CCK-26–33 residing in a position that would easily allow peptide extension without interference with any important structures. Similarly, the sites of glycosylation are fully accommodated by this model, all situated directly out and away from any site of peptide docking or any other important structure. The functionally important salt bridge postulated to exist between acidic tyrosine sulfate at position 27 of CCK and the basic Arg197
residue within receptor extracellular loop 2 (37
) is partially shielded from solvation by the amino terminus of the receptor, as this is positioned in the current models.
Indeed, the current models provide substantial enhancements from that previously proposed that was based on the rhodopsin structure. Both models originating from the homology structure based on the β2-adrenergic receptor and the A2a-adenosine receptor positioned the docked peptide above the second extracellular loop, forming the essential salt bridge in that region. In contrast, in the previous model based on the rhodopsin structure, the peptide was situated closer to the second and third helices, forming the salt bridge behind the second extracellular loop on top of the third helix. The convergence from quite distinct starting structures to yield a consistent mode of peptide docking in the two current models provides strong support for the validity of this new insight. This provides the highest level of refinement yet available to demonstrate the docking of a peptide hormone to a family A GPCR.