Structural Insight into G-Protein Coupled Receptor Binding by Apelin

Apelin peptides are the cognate ligands for the G-protein coupled receptor APJ, with functions in the cardiovascular and central nervous systems, in glucose metabolism and as a human immunodeficiency virus (HIV-1) coreceptor. Apelin is found in 13−36 residue forms in vivo. The structures of five isoforms of apelin at physiological versus low (5−6 °C) temperature are compared here using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, demonstrating increased structure at low temperature. Far-ultraviolet (UV) CD spectra are predominantly random coil for apelin isoforms, but are convoluted by unusual bands from the C-terminal phenylalanine side chain. These bands, assigned using F13A-apelin-13, are accentuated at 5 °C and imply conformational restriction. At 35 °C, the R6−L9 region of apelin-17 is well structured, consistent with previous mutagenesis results showing necessity of this segment for apelin−APJ binding and activation. At 5 °C, R6−L9 retains its structuring while the functionally critical C-terminal G13−F17 region also becomes highly structured. Type IV β-turns and some polyproline-II structure alongside F17 side chain motional reduction correlate well with CD spectral properties. Cis−trans peptide bond isomerization at P14 and P16 produces two sequentially assignable conformers (both trans:both cis ∼4:1) alongside less populated conformers. Chemical shift assignment of apelin-12, -13 and pyroglutamate-apelin-13 implies highly similar structuring and the same isomerization at the C-terminus. Based on the apelin-17 structure, a two-step binding and activation mechanism is hypothesized.