Mapping spatial approximations between the amino terminus of secretin and each of the extracellular loops of its receptor using cysteine trapping

Abstract

While it is evident that the carboxyl-terminal region of natural peptide ligands bind to the amino-terminal domain of class B GPCRs, how their biologically critical amino-terminal regions dock to the receptor is unclear. We utilize cysteine trapping to systematically explore spatial approximations among residues in the first five positions of secretin and in every position within the receptor extracellular loops (ECLs). Only Cys(2) and Cys(5) secretin analogues exhibited full activity and retained moderate binding affinity (IC(50): 92±4 and 83±1 nM, respectively). When these peptides probed 61 human secretin receptor cysteine-replacement mutants, a broad network of receptor residues could form disulfide bonds consistent with a dynamic ligand-receptor interface. Two distinct patterns of disulfide bond formation were observed: Cys(2) predominantly labeled residues in the amino terminus of ECL2 and ECL3 (relative labeling intensity: Ser(340), 94±7%; Pro(341), 84±9%; Phe(258), 73±5%; Trp(274) 62±8%), and Cys(5) labeled those in the carboxyl terminus of ECL2 and ECL3 (Gln(348), 100%; Ile(347), 73±12%; Glu(342), 59±10%; Phe(351), 58±11%). These constraints were utilized in molecular modeling, providing improved understanding of the structure of the transmembrane bundle and interconnecting loops, the orientation between receptor domains, and the molecular basis of ligand docking. Key spatial approximations between peptide and receptor predicted by this model (H(1)-W(274), D(3)-N(268), G(4)-F(258)) were supported by mutagenesis and residue-residue complementation studies.

Use of Cysteine Trapping to Map Spatial Approximations between Residues Contributing to the Helix N-capping Motif of Secretin and Distinct Residues within Each of the Extracellular Loops of Its Receptor

“…The current project utilized cysteine trapping to systematically explore spatial approximations for each of the helix N-capping motif residues within secretin, representing residues Phe 6 , Thr 7 , and Leu 10 , and those residues within the core of the secretin receptor that could potentially contribute to this important binding pocket. We recently reported a similar approach to define spatial approximations with the more distal amino-terminal residues two and five within secretin (5). That effort supported the presence of this pocket, but the establishment of multiple disulfide bonds for each of the probes suggested that there was a dynamic component to the docking, and definition of more approximations would be necessary to refine our understanding.…”

“…Each of these analogues was a full agonist for cAMP, although each probe bound with lower affinity than that of the natural hormone. We applied these probes to the same extensive series of secretin receptor mutants in which the natural residues in each of 61 positions throughout the tops of transmembrane segments (TM) and extracellular loop (ECL) regions were replaced with cysteines that had been probed previously (5). These receptor constructs were expressed in COS cells, and the ligand probes were allowed to bind under conditions permitting the spontaneous formation of disulfide bonds.…”

“…The position 10 probe did not efficiently label any of the residues in any of these regions. Receptor residues contributing to disulfide bond formation with the helix N-capping motif residues were distinct from the predominant sites of labeling previously observed with the position two and five cysteine probes (5). These sets of spatial approximation data were added to all previous experimentally derived constraints to refine our previous working model of the secretin-occupied secretin receptor (5).…”

“…Receptor residues contributing to disulfide bond formation with the helix N-capping motif residues were distinct from the predominant sites of labeling previously observed with the position two and five cysteine probes (5). These sets of spatial approximation data were added to all previous experimentally derived constraints to refine our previous working model of the secretin-occupied secretin receptor (5). Predictions for spatial approximations made based on this model were further tested by receptor mutagenesis and complementary changes in residues within ligand and receptor.…”

“…Cysteine Trapping-COS-1 cells grown in 24-well plates (ϳ80% confluence) were transfected with wild type and all the 61 cysteine secretin mutants using the polyethyleneimine method as we have previously described (5). After washing once with PBS, cells were incubated for 1.5 h at room temperature with 200 l of DMEM containing 5% fetal clone II and 125 ICys 6 -sec or 125 I-Cys 7 -sec or 125 I-Cys 10 -sec (ϳ100,000 cpm per well) in the absence or presence of 0.1 M secretin.…”