A multipronged footprinting map validates the predicted structure of stress biomarker Neuropeptide Y (NPY) and its binding with novel peptide binders

Neuropeptide Y (NPY) signals important stress and mood phenotypes in the brain. Peptide biological recognition elements (BREs) based on phage display can be used in hormone biosensors for stress and weight management. We employed protein footprinting mass spectrometry (PF-MS) to probe NPY structure and provide readout of NPY residues responsive to BRE binding. Three complementary PF-MS approaches using hydroxyl radical, trifluoromethyl radical, and carboxyl chemistries were applied independently. This multipronged approach mapped changes in NPY solvent accessibility with 42% labeling coverage.The location and mechanism of binding of two BREs (N2, N3) with NPY was different. Deprotection of NPY residues due to N2 binding suggest an extended NPY conformation, whereas the absence of deprotection in the NPY-N3 complex points to a hairpin conformation. Protection of sites in the NPY-N3 complex agree with reported MD results, confirming a C-terminal binding site. A published NMR structure of NPY shows multiple distinct conformations, including an extended structure with unfolded N-terminus and a poly-proline (PP) α-helix that is back folded onto the NPY C-terminus. AlphaFold2 predicted a PP α-helix structure for NPY. Knowing the true NPY conformation is essential to optimize sensors based upon the NPY-N2 and NPY-N3 complexes. To this end, we reversed the order of PF-MS experiments via a “digest and label” strategy. We found the baseline structure of NPY has a higher order structure, validating the AlphaFold2 prediction. Residue level PF-MS results from this approach are compared to those from footprinting of the intact NPY-BRE complexes to decipher the structural factors impacting NPY solvent accessibility.