NMR solution structure and SRP54M predicted interaction of the N-terminal sequence (1-30) of the ovine Doppel protein

Abstract

Prion protein (PrP(C)) biosynthesis involves a multi-step process that includes translation and post-translational modifications. While PrP has been widely investigated, for the homolog Doppel (Dpl), limited knowledge is available. In this study, we focused on a vital step of eukaryotic protein biosynthesis: targeting by the signal recognition particle (SRP). Taking the ovine Dpl (OvDpl(1-30)) peptide as a template, we studied its behavior in two different hydrophobic environments using CD and NMR spectroscopy. In both trifluoroethanol (TFE) and dihexanoyl-sn-glycero-3-phosphatidylcholine (DHPC), the OvDpl(1-30) peptide revealed to fold in an alpha-helical conformation with a well-defined central region extending from residue Cys8 until Ser22. The NMR structure was subsequently included in a computational docking complex with the conserved M-domain of SRP54 protein (SRP54M), and further compared with the N-terminal structures of mouse Dpl and bovine PrP(C) proteins. This allowed the determination of (i) common predicted N-terminal/SRP54M polar contacts (Asp331, Gln335, Glu365 and Lys432) and (ii) different N-C orientations between prion and Dpl peptides at the SRP54M hydrophobic groove, that are in agreement with each peptide electrostatic potential. Together, these findings provide new insights into the biosynthesis of prion-like proteins. Besides they also show the role of protein conformational switches in signalization toward the endoplasmic membrane, a key event of major significance in the cell cycle. They are thus of general applicability to the study of the biological function of prion-like as well as other proteins.

Keywords: (Adaptive Poisson-Boltzmann Solver); 54-kDa subunit of SRP S domain; APBS; CD; DHPC; Doppel protein; Dpl; E. coli; ER; Escherichia coli; N-terminal signal peptide; NMR; Nuclear Magnetic Resonance; OvDpl; PrP; PrP(C); Prion-like; RMSD; SP; SRP; SRP54; SRP54 C-terminal methionine-rich M-domain; SRP54M; Signal peptide; bPrP; bPrP(1-30) peptide; bPrPp; bovine PrP(C); cDNA; cellular isoform of PrP; circular dichroism; complementary deoxyribonucleic acid; dihexanoyl-sn-glycero-3-phosphatidylcholine; endoplasmic reticulum; hSRP54M; human SRP54M; mPrP; mouse PrP(C); ovine Dpl; prion protein; prion-like protein Doppel gene; prnd; root mean square deviation matrix; signal recognition particle.