Solution Structure and Internal Motion of a Bioactive Peptide Derived from Nerve Growth Factor

Abstract

The conformation and internal dynamics of a bioactive cyclic peptide, N-acetyl-YCTDEKQCY, derived from the C-D loop of ␤-nerve growth factor (␤-NGF) were analyzed by solution NMR spectroscopy. NMR experimental data were used to calculate an ensemble of peptide structures. All of the structures had a ␤-turn at residues Asp 4 -Gln 7 but could be divided into two families according the presence or absence of a hydrogen bond at Gln 7 . Comparison of the calculated structures with the corresponding C-D loops from the x-ray structures of the NGF revealed striking similarity. The orientation of Glu 5 , Lys 6 , and Gln 7 side chains in the NGF mimetic was very similar to the C-D loop of NGF. These residues are known to participate in interactions with the TrkA receptor. Relaxation measurements of the peptidomimetic ␣-carbons at 13 C natural abundance and calculated dynamic parameters suggest that the loop region of peptide is well structured but that residues Thr 3 , Asp 4 , Glu 5 , and Lys 6 undergo slow conformational exchange. These results suggest that conformational similarity and possibly peptide dynamics are responsible for the bioactivity of the peptide. Nerve growth factor (NGF)1 is a member of the neurotrophin family of polypeptide growth factors. NGF promotes the growth and survival of sympathetic, trigeminal, dorsal root ganglia neurons and cholinergic neurons of the basal forebrain. Two cell surface neurotrophin receptors have been characterized: the low affinity p75 receptor (K D ϳ10 Ϫ9 M), which is common to all members of the neurotrophin family (1-3), and the intermediate affinity receptors (K D ϳ10 Ϫ11 M), TrkA, TrkB, and TrkC, which afford the binding specificity of the different neurotrophins (4 -7). TrkA is selective for NGF and has tyrosine kinase enzymatic activity that mediates most of the downstream signaling for this growth factor. Highest affinity binding of NGF (K D ϳ10 Ϫ12 M) occurs in cells coexpressing TrkA and p75 (4, 8).NGF, like other neurotrophins, is a dimer of two identical polypeptides of 118 residues (9). X-ray structures of NGF (10, 11), a brain-derived neurotrophic factor-neurotrophin-3 heterodimer (12), and the 7 S NGF complex (13) have been determined. The neurotrophin protomers have the same overall topology of seven ␤-strands connected by three disulfide bridges arranged in a cysteine knot motif. Comparison of the primary sequence of different neurotrophins shows clusters of high homology (Ͼ50% identity) and high diversity. The hypervariable domains occur in the exposed ␤-loops (residues 29 -35, 43-48, and 92-98), in an exposed reverse turn (residues 59 -66), in a solvent-exposed ␤-strand (residues 79 -89), and at both N and C termini. By analogy with findings from other growth factors, the hypervariable domains are believed to be responsible for Trk receptor selectivity

Genuine Monovalent Ligands of TrkA Nerve Growth Factor Receptors Reveal a Novel Pharmacological Mechanism of Action

“…correlation spectroscopy spectra were consistent with a monomeric, nonaggregated state and a five-residue pharmacophore within a ␤-turn (27)…”

“…Purification, quality control, and characterization of the peptides were done as described (21) and by NMR diffusion studies (this report). More convincingly, the full NMR spectra of N-Ac-C(92-96) were analyzed (27). Assignment of all resonances and distances and resolution of the structure showed the peptide to be monomeric.…”

“…97) dimer is more efficient. Monomeric C(92-96) is monovalent, because it has a 5-residue pharmacophore (27) and could not interact with two receptors simultaneously. The intriguing possibility that C(92-96) could dimerize after docking is unlikely (see below), but it remains unexplored and would require structural analysis of receptor-ligand complexes.…”

“…The C(92-96) and C(92-97) peptides are cyclized by intrachain disulfide bonds (indicated by the underlines) (21). These peptides are structural mimics of the C-D ␤-turn of NGF (27). Linear peptides with the same sequences do not bind TrkA and were prepared as controls by substituting Cys with Met (primary sequence YMTDEKQMY).…”

A synthetic peptide containing loop 4 of nerve growth factor for targeted gene delivery

“…The design of the peptide vector was inspired by the successful development of NGF small mimetics [18,20,[37][38][39] and based on the structural analysis of NGF/TrkA complexes. NGF loop 4 is a hydrophilic hairpin loop exposed on the NGF surface, serving as one of the two primary NGF loop domains interacting with TrkA receptors [40,41].…”