Peptide design and structural characterization of a GPCR loop mimetic

Abstract

G protein-coupled receptors (GPCRs) control fundamental aspects of human physiology and behaviors. Knowledge of their structures, especially for the loop regions, is limited and has principally been obtained from homology models, mutagenesis data, low resolution structural studies, and high resolution studies of peptide models of receptor segments. We developed an alternate methodology for structurally characterizing GPCR loops, using the human S1P(4) first extracellular loop (E1) as a model system. This methodology uses computational peptide designs based on transmembrane domain (TM) model structures in combination with CD and NMR spectroscopy. The characterized peptides contain segments that mimic the self-assembling extracellular ends of TM 2 and TM 3 separated by E1, including residues R3.28(121) and E3.29(122) that are required for sphingosine 1-phosphate (S1P) binding and receptor activation in the S1P(4) receptor. The S1P(4) loop mimetic peptide interacted specifically with an S1P headgroup analog, O-phosphoethanolamine (PEA), as evidenced by PEA-induced perturbation of disulfide cross-linked coiled-coil first extracellular loop mimetic (CCE1a) (1)H and (15)N backbone amide chemical shifts. CCE1a was capable of weakly binding PEA near biologically relevant residues R29 and E30, which correspond to R3.28 and E3.29 in the full-length S1P(4) receptor, confirming that it has adopted a biologically relevant conformation. We propose that the combination of coiled-coil TM replacement and conformational stabilization with an interhelical disulfide bond is a general design strategy that promotes native-like structure for loops derived from GPCRs.

Unraveling the Structure and Function of G Protein-Coupled Receptors Through NMR Spectroscopy

“…Lastly, Pham and coworkers developed an alternative methodology for studying GPCR loops based on the computational design of a peptide containing segments that mimic the self assembling of the ends of two TMs connected by the loop under examination [61]. When applying their strategy to the analyses of EL1 of the sphingosine 1-phosphate receptor S 1 P 4 , the authors obtained the best results with the coiled-coil strategy, i.e.…”

Lysophospholipid interactions with protein targets

“…S1P headgroup recognition by the S1P 4 first extracellular loop and the extracellular end of TM3 has been examined using NMR spectroscopy [83]. This study provided additional evidence of a direct interaction between R3.28 and the phosphate group and between E3.29 and the ammonium group first proposed based on modeling studies described in section 3.3.…”

GPCR Conformations: Implications for Rational Drug Design

“…This is in part due to the likely flexibility of the loops within the context of the full-length receptor, as well as due to the lack of a well-folded transmembrane domain to provide a conformational restraint. NMR-based structural characterization of peptide segments from the β-adrenoceptor [121], parathyroid hormone receptor [122], angiotensin II AT 1A receptor [123], neurokinin-1 receptor [124,125], thromboxane A 2 receptor [126-128], V 1A vasopressin receptor [129], corticotrophin releasing factor receptor 2β [130], Ste2p [131,132], the muscarinic acetylcholine M2 receptor [133], the fourth sphingosine 1-phosphate receptor, S1P 4 [134], and the CCR5 receptor [135] then followed. Unique features of these subsequent studies include the use of conformational restraints such as an octamethylene linker between the peptide termini [122], disulfide linkages between the termini [126,127,132], or between helical loop extensions [134], use of bacterial expression systems to produce isotopically-labelled peptides to simplify chemical shift assignments [130,133,134], inclusion of coiled-coil motifs at the termini to promote self-association of the loop termini [134] and use of saturation transfer NMR to investigate interactions with binding partners [135].…”

“…The third cytoplasmic loop of the parathyroid hormone receptor has been shown to activate G proteins [122]. Fluorescence intensity changes on antagonist treatment [126,127] as well as chemical shift perturbation in response to specific agonists or agonist headgroups [125,130,134] have been used to show that extracellular segments are capable of specific ligand recognition. Table 4 summarizes segment characterization studies and the experiments utilized to validate the structures obtained as representative of the corresponding segment in the full-length GPCR.…”