title: Revisiting the structure of the Vps10 domain of human sortilin and its interaction with neurotensin.
abstract: Sortilin is a multifunctional receptor involved in sorting and apoptosis. We have previously reported a 2.0-Å structure of the Vps10 ectodomain in complex with one of its ligands, the tridecapeptide neurotensin. Here we set out to further characterize the structural properties of sortilin and its interaction with neurotensin. To this end, we have determined a new 2.7 Å structure using a crystal grown with a 10-fold increased concentration of neurotensin. Here a second peptide fragment was observed within the Vps10 β-propeller, which may in principle either represent a second molecule of neurotensin or the N-terminal part of the molecule bound at the previously identified binding site. However, in vitro binding experiments strongly favor the latter hypothesis. Neurotensin thus appears to bind with a 1:1 stoichiometry, and whereas the N-terminus does not bind on its own, it enhances the affinity in context of full-length neurotensin. We conclude that the N-terminus of neurotensin probably functions as an affinity enhancer for binding to sortilin by engaging the second binding site. Crystal packing differs partly from the previous structure, which may be due to variations in the degree and pattern of glycosylations. Consequently, a notable hydrophobic loop, not modeled previously, could now be traced. A computational analysis suggests that this and a neighboring loop may insert into the membrane and thus restrain movement of the Vps10 domain. We have, furthermore, mapped all N-linked glycosylations of CHO-expressed human sortilin by mass spectrometry and find that their locations are compatible with membrane insertion of the hydrophobic loops.
authors.affiliation: Department of Molecular Biology and Genetics, MIND Centre, Aarhus University, Gustav Wieds Vej 10C, DK 8000, Aarhus C, Denmark; Department of Medical Biochemistry and Biophysics, Karolinska Institute, 17177, Stockholm, Sweden.
authors.forename: Esben M
authors.initials: EM
authors.lastname: Quistgaard
authors.forename: Morten K
authors.initials: MK
authors.lastname: Grøftehauge
authors.forename: Peder
authors.initials: P
authors.lastname: Madsen
authors.forename: Lone T
authors.initials: LT
authors.lastname: Pallesen
authors.forename: Brian
authors.initials: B
authors.lastname: Christensen
authors.forename: Esben S
authors.initials: ES
authors.lastname: Sørensen
authors.forename: Poul
authors.initials: P
authors.lastname: Nissen
authors.forename: Claus M
authors.initials: CM
authors.lastname: Petersen
authors.forename: Søren S
authors.initials: SS
authors.lastname: Thirup
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D009496:Neurotensin / Q000737:chemistry*; D011487:Protein Conformation; D033921:Vesicular Transport Proteins / Q000737:chemistry*; D000099183:Sortilin
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D009496:Neurotensin; D033921:Vesicular Transport Proteins; D000099183:Sortilin
keywords: N-linked glycosylations; Vps10 domain (Vps10p domain); crystal structure; hydrophobic loop; membrane imbedded loop; neurotensin; neurotensin receptor 3; sortilin; β-propeller