Ag. Becksickinger et al., COMPLETE L-ALANINE SCAN OF NEUROPEPTIDE-Y REVEALS LIGANDS BINDING TO Y-1 AND Y-2 RECEPTORS WITH DISTINGUISHED CONFORMATIONS, European journal of biochemistry, 225(3), 1994, pp. 947-958
The synthesis of more than fifty 36-residue oligopeptide analogs of ne
uropeptide Y (NPY) and their affinity to human Y-1 and Y-2 receptors i
s described. Each amino acid of the natural sequence was replaced by L
-alanine, the four alanine residues at position 12, 14, 18 and 23 were
replaced by glycine. Additional residues were exchanged to closely re
lated ones in order to characterize the prerequisites for binding. A c
ombination of automated single and multiple peptide synthesis using fl
uoren-9-ylmethoxycarbonyl/tert-butoxy strategy was applied. The purifi
ed peptides were characterized by electrospray mass spectrometry, anal
ytical HPLC and amino acid analysis. Binding was investigated by displ
acement of I-125-labelled neuropeptide Y from human neuroblastoma cell
lines SK-N-MC and SMS-KAN. Whereas Pro2 and the integrity of the neur
opeptide Y loop is important for the binding to the Y-1 receptor, exch
anges within the C-terminal helix affect the affinity to the Y-2 recep
tor. The C-terminal pentapeptide amide is important for both receptors
and probably represents the binding site. However, Arg33 and Arg35 ma
y not be exchanged by L-alanine in the Y-1 system, whereas Arg35 and T
yr36 are the most susceptible residues in the Y-2 system. In order to
distinguish between conformational effects and direct hormone/receptor
interaction via the side chains of neuropeptide Y, circular dichroic
studies of the alanine-containing peptides were performed and structur
e affinity relationships are discussed. Comparing the affinities of th
e neuropeptide Y analogs to Y-1 and Y-2 receptors significant differen
ces were found for the two binding sites, which suggests a different a
ctive conformation of neuropeptide Y at the two subtypes of receptors.
Using molecular dynamics calculations, two distinct conformations wer
e identified which are in good agreement with the data obtained by str
ucture/ affinity investigations.