Molecular characterization of a ghrelin-l-aptamer complex

Abstract

The orexigenic peptide ghrelin is involved in various and vital physiological processes such as food uptake, regulation of body weight and glucose metabolism. At Ser3 ghrelin is modified with a fatty acid. This unique posttranslational modification renders ghrelin active to bind to the growth hormone secretagogue receptor (GHSR)1a whereas deacylated ghrelin is inactive. The L-aptamer NOX-B11 was identified to specifically bind and neutralize octanoylated, i.e. active ghrelin. L-aptamers (also termed Spiegelmers) are oligonucleotide binders built from non-natural L-nucleotides that confer resistance to nucleases. Dynamic light and X-ray scattering measurements were applied to learn more about the specific recognition of a fatty acid modification by an oligonucleotide. First data revealed that upon ghrelin binding NOX-B11 undergoes a conformational change resulting in a 1:1 Ghrelin•NOX-B11 complex. Furthermore, crystals of Ghrelin•NOX-B11 were obtained and optimized by multiple cycles of micro seeding. X-ray diffraction data were collected from a single crystal to a resolution of 2.65 Å and the space group was determined to be C2. The Matthews coefficient was calculated to be 2.75 Å3Da−1, corresponding to a solvent content of approx. 60%.