Peptide-Based Drug Design

NMR of Peptides 107
protein prevents �-phage superinfection by blocking gene expression. They
found residues Ser24 to K42 formed an �-helix when in complex with the RNA
construct, as seen from secondary shifts, while the free Nun protein was unstructured.
The N-terminal residues did not show secondary shifts or homonuclear
NOEs to indicate the presence of a stable secondary structure on the �s-ms
time scale. However, small heteronuclear 1 H- 15 N NOEs and 15 N relaxation rates
ranging from 3.5 to 5.3 s −1 for these residues indicated the presence of a limited
set of backbone conformations in the ps-ns time scale. From increased relaxation
rates of R2 > 11 s −1 for residues Ser24, Arg25, Asp26, Ala31, Trp33, Ile37,
and Ala38, all part of the �-helix, the presence of chemical exchange in the
�s-ms time scale was deduced. The dissociation of the complex is slow on the
chemical shift time scale, and therefore these relaxation rates must be a result of
intramolecular conformational changes in the complex. Stuart et al. assumed that
restricted flexibility of the N-terminus may play a role in the formation of larger
molecular assemblies involved in transcription termination. These results show
that NMR can reveal the presence of preferred conformations, even if secondary
structural motifs are not present in the peptide.
5.5. Chemical Shifts
5.5.1. Description of a Peptide–Protein Complex
Using isotopically labeled peptides, Stamos et al. determined the structure of
the high-affinity IgE receptor FC�RI in complex with a �-hairpin IGE32 peptide
and helical zeta peptide e117. The study is a nice example of the use of NMR
techniques for analyzing peptide–receptor complexes (81). For the bound state
Stamos et al. found dramatic changes in the 1H chemical shifts of Pro16 in the
zeta peptide, ranging from 1.7 to 3.1 ppm. Shift changes of this magnitude are
a result of close vicinity to aromatic side chains in the receptor, in a sandwichlike
packing. This finding was in agreement with an x-ray structure of FC�RI�
in complex with an Fc fragment of IgE (82) where Pro426 of IgE is placed
between Trp87 and Trp110 of FC�RI�. Combining mutagenesis data and intermolecular
NOEs, it was confirmed that the zeta peptide bound in site 2 of
FC�RI. These data were used to model the complex of FC�RI and e117 zeta
peptide on the basis of the crystal structure of FC�RI� and zeta peptide e131
(83), revealing high similarity between the structures. Using the same NMR
experimental approach, the structure of the hairpin peptide–receptor complex
was determined and shown to retain the �-hairpin structure upon binding. Intermolecular
NOEs were used to orient the ligand in binding site 2 of the receptor
with Pro9 of the peptide packed between Trp87 and Trp110, indicated as before
by dramatic shift changes of the proline residue.