Abstract
G protein-coupled receptor (GPCR) structures are of interest as a means to understand biological signal transduction and as tools for therapeutic discovery. The growing number of GPCR crystal structures demonstrates that the extracellular loops (EL) connecting the membrane-spanning helices show tremendous structural variability relative to the more structurally-conserved seven transmembrane α-helical domains. The EL of the LPA1 receptor have not yet been conclusively resolved, and bear limited sequence identity to known structures. This study involved development of a peptide to characterize the intrinsic structure of the LPA1 GPCR second EL. The loop was embedded between two helices that assemble into a coiled-coil, which served as a receptor-mimetic folding constraint (LPA1-CC-EL2 peptide). The ensemble of structures from multi-dimensional NMR experiments demonstrated that a robust coiled-coil formed without noticeable deformation due to the EL2 sequence. In contrast, the EL2 sequence showed well-defined structure only near its C-terminal residues. The NMR ensemble was combined with a computational model of the LPA1 receptor that had previously been validated. The resulting hybrid models were evaluated using docking. Nine different hybrid models interacted with LPA 18:1 as expected, based on prior mutagenesis studies, and one was additionally consistent with antagonist affinity trends.
Highlights
G protein-coupled receptors (GPCRs) are a large family of integral membrane proteins that play critical roles in cellular signaling
This study has demonstrated that the two peptide sequences designed by Oakley et al, to form an antiparallel coiled-coil dimer in an aqueous solution, can self-assemble within a single peptide sequence that included a long linker of 19 amino acids from the LPA1 EL2
The coiled-coil motif provided a receptor-mimetic folding constraint for the longest LPA1 extracellular loop. This folding constraint made possible atomic-resolution structural studies of the LPA1 EL2 in solution, permitting any intrinsic flexibility of the receptor loop sequence to be reflected in the structural data
Summary
G protein-coupled receptors (GPCRs) are a large family of integral membrane proteins that play critical roles in cellular signaling. We report on the structure of the substantially longer LPA1 second extracellular loop sequence in aqueous buffer using a longer optimized antiparallel coiled-coil [32] to promote self-assembly in the absence of both trifluoroethanol and an interhelical disulfide bond. This improved second-generation design was applied to characterize the intrinsic structure of the LPA1 second extracellular loop sequence in aqueous solution, demonstrating that this loop contains no intrinsic secondary structure elements, but does exhibit a strong outward bend at the C-terminal end leading into the C-terminal helix
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