Key motif to gain selectivity at the neuropeptide Y5-receptor: structure and dynamics of micelle-bound [Ala31, Pro32]-NPY.

The structure of [Ala 3 1 , Pro 3 2 ]-NPY, a neuropeptide Y mutant with selectivity for the NPY Y 5 -receptor (Cabrele, C., Wieland, H. A., Stidsen, C., Beck-Sickinger, A. G., (2002) Biochemistry XX, XXXX-XXXX (companion paper)), has been characterized in the presence of the membrane mimetic dodecylphosphocholine (DPC) micelles using high-resolution NMR techniques. The overall topology closely resembles the fold of the previously described Y 5 -receptor-selective agonist [Ala 3 1 , Aib 3 2 ]-NPY (Cabrele, C., Langer, M., Bader, R., Wieland, H. A., Doods, H. N., Zerbe, O., and Beck-Sickinger, A. G. (2000) J. Biol. Chem 275, 36043-36048). Similar to wild-type neuropeptide Y (NPY) and [Ala 3 1 , Aib 3 2 ]-NPY, the N-terminal residues Tyr 1 -Asp 1 6 are disordered in solution. Starting from residue Leu 1 7 , an a helix extends toward the C-terminus. The decreased density of medium-range NOEs for the C-terminal residues resulting in larger RMSD values for the backbone atoms of Ala 3 1 -Tyr 3 6 indicates that the a helix has become interrupted through the [Ala 3 1 , Pro 3 2 ] mutation. This finding is further supported by 1 5 N-relaxation data through which we can demonstrate that the well-defined a helix is restricted to residues 17-31, with the C-terminal tetrapeptide displaying increased flexibility as compared to NPY. Surprisingly, increased generalized order parameter as well as decreased 3 J H N α scalar coupling constants reveal that the central helix is stabilized in comparison to wild-type NPY. Micelle-integrating spin labels were used to probe the mode of association of the helix with the membrane mimetic. The Y5-receptor-selective mutant and NPY share a similar orientation, which is parallel to the lipid surface. However, signal reductions due to efficient electron, nuclear spin relaxation were much less pronounced for the surface-averted residues in [Ala 3 1 , Pro 3 2 ]-NPY when compared to wild-type DPC-bound NPY. Only the signals of residues Asn 2 9 and Leu 3 0 were significantly more reduced in the mutant. The postulation of a different membrane binding mode of [Ala 3 1 , Pro 3 2 ]-NPY is further supported by the faster H/D exchange at the C-terminal amide protons. We conclude that arginine residues 33 and 35, which are believed to be directly involved in forming contacts to acidic receptor residues at the membrane-water interface, are no longer fixed in a well-defined conformation close to the membrane surface in [Ala 3 1 , Pro 3 2 ]-NPY.