title: Improving membrane binding as a design strategy for amphipathic peptide hormones: 2-helix variants of PYY3-36.
abstract: It has been hypothesized that amphipathic peptides might bind to membranes prior to activating their cognate receptors, but this has proven difficult to test. The peptide hormone PYY3-36 is believed to perform its appetite-suppressing actions through binding to hypothalamic Y2 receptors. It has been proposed that PYY3-36 via its amphipathic α-helix binds to the plasma membrane prior to receptor docking. Here, our aim was to study the implication of this hypothesis using new analogs of PYY3-36. We first studied membrane binding of PYY3-36. Next, we designed a series of PYY3-36 analogs to increase membrane-binding affinity by substituting the N-terminal segment with a de novo designed α-helical, amphipathic sequence. These 2-helix variants of PYY3-36 were assembled by solid-phase peptide synthesis. Pharmacological studies demonstrated that even though the native peptide sequence was radically changed, highly active Y2 receptor agonists were generated. A potent analog, with a Kd of 4 nM for membranes, was structurally characterized by NMR in the membrane-bound state, which clearly showed that it formed the expected 2-helix. The topology of the peptide-micelle association was studied by paramagnetic relaxation enhancement using a spin label, which confirmed that the hydrophobic residues bound to the membrane. Our studies further support the hypothesis that PYY3-36 associates with the membrane and indicate that this can be used in the design of novel molecules with high receptor binding potency. These observations are likely to be generally important for peptide hormones and biopharmaceutical drugs derived from them. This new 2-helix variant of PYY3-36 will be useful as a tool compound for studying peptide-membrane interactions.
authors.affiliation: Department of Chemistry, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark.
authors.forename: Søren L
authors.initials: SL
authors.lastname: Pedersen
authors.forename: Vikram K
authors.initials: VK
authors.lastname: Bhatia
authors.forename: Simon
authors.initials: S
authors.lastname: Jurt
authors.forename: Johan F
authors.initials: JF
authors.lastname: Paulsson
authors.forename: Maria H
authors.initials: MH
authors.lastname: Pedersen
authors.forename: Rasmus
authors.initials: R
authors.lastname: Jorgensen
authors.forename: Birgitte
authors.initials: B
authors.lastname: Holst
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Stamou
authors.forename: Niels
authors.initials: N
authors.lastname: Vrang
authors.forename: Oliver
authors.initials: O
authors.lastname: Zerbe
authors.forename: Knud J
authors.initials: KJ
authors.lastname: Jensen
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D036361:Peptide Hormones / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D019894:Peptide YY / Q000737:chemistry*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship
chemical_list: D036361:Peptide Hormones; D019894:Peptide YY