Solution Structure of a β-Peptide Ligand for hDM2

Abstract

Recently, we described a β-peptide foldamer, β53-1 ( Figure 1A), that assembles into a 14-helix in aqueous solution, binds the oncoprotein hDM2 with submicromolar affinity, and inhibits the interaction of hDM2 with a peptide derived from the activation domain of p53 (p53AD). 1 The intact recognition epitope of β53-1, including a high degree of helical structure, is required for selective inhibition of the p53AD·hDM2 interaction. Here, we present the solution structure of β53-1 in methanol. The structure reveals details of a helixstabilizing salt bridge on one helical face, novel "wedge into cleft" packing along another, and how distortions in the β53-1 14-helix maximize presentation of the p53AD recognition epitope. These details deepen our understanding of how β 3 -peptides fold and how they can be designed to form higher order structures and bind macromolecules. 2,3 Two-dimensional NMR spectroscopy was performed using 5 mM β53-1 in CD 3 OH at 10°C. Previous circular dichroism and analytical ultracentrifugation experiments 1 and the NMR line widths observed herein are consistent with a monomeric, 14-helical structure for β53-1 under these conditions. The proton resonances of β53-1 were assigned unambiguously using TOCSY and natural abundance 1 H-13 C HSQC spectra. 4 ROESY experiments were then performed using mixing times of 200, 350, and 500 ms. 5 The observed series of NH-C α H ROEs confirmed the sequential assignment by providing a backbone "ROE walk". Three classes of medium-range ROEs characterize a 14-helical conformation: those between H N (i) and H β (i+2), H N (i) and H β (i+3), and H α (i) and H β (i+3). 6,7 All 20 potential medium-range interactions of this type were observed in the ROESY spectra of β53-1; in addition, 27 additional medium-range ROEs between side chains three positions apart were also observed. 4 The large number of medium-range ROEs observed by NMR provides clear evidence for a high level of 14-helix structure in β53-1; 449 ROEs quantified using a 350 ms mixing time were subsequently assigned and integrated using SPARKY. 8 Peak volumes were converted to 151 upper-limit distance constraints 4 and used to perform simulated annealing torsional dynamics on 100 random starting configurations of β53-1 using DYANA. 4,9 No constraint violations were reported among the resulting 20 lowest-energy structures, which are shown in Figure 1B.The ensemble of calculated structures of β53-1 ( Figure 1B) shows a 14-helix with an average backbone atom RMSD from the mean structure of 0.17 ± 0.07 Å. The backbone torsions of individual structures deviate little from the mean, even at the termini ( Figure 1C), illustrating the robustness of the β53-1 14-helix in methanol. .0 residues per turn for residues 1-6, with a slight unwinding to approximately 1.49 Å rise per residue and 3.3 residues per turn for residues 7-10. This unwinding appears to be unique to β53-1, as it was not observed in NMR structures of unrelated β 3 -peptides with and without side chain ion pairing. 6,7,10 Side chains are also well-de...

Targeting protein–protein interactions: Lessons from p53/MDM2

“…124 b-Peptides were designed to mimic the projection of the three hydrophobic side chains (Phe19, Trp23, Leu26) from the a-helical segment of p53. 125 Since there are three residues per turn of 14-helix, b -hVal residues. The overall design is unusual from a structure-based design standpoint, because a left-handed b-peptide helix is being used to mimic a righthanded a-helical structure.…”

“…Investigation by 2D 1H NMR spectroscopy suggested that b-peptide 55 adopts a slightly distorted 14-helical conformation in methanol. 125 Schepartz and coworkers reasoned that b-peptides composed entirely of b…”

Biophysical and Structural Characterization of a Robust Octameric β-Peptide Bundle

“…22 Importantly, the inherent conformational flexibility of β 3 -peptides, when compared to cyclic analogues such as ACHC, 23 may be essential to the general success of this approach, as all β 3 -peptides of known structure that effectively inhibit protein interactions possess geometries that deviate from the ideal 3 14 -helix imposed by ACHC. 21,22,24 Larger mixedsequence peptides containing both α-and β-amino acids have also shown success as protein ligands for the BH3 recognition site of Bcl-x L . 25,26 Several strategies have been pursued to identify β-peptides that assemble into discrete, wellfolded quaternary structures whose thermodynamic properties resemble those of natural proteins.…”

Foldameric β‐H18/20P Mixed Helix Stabilized by Head‐to‐Tail Contacts: A Way to Higher‐Order Structures

“…Previously unreported NOE interactions were observed in CD 3 OH: long-range NOEs arising from C β H i -NH i+4 (i = odd) and NH i -C β H i+4 (i = even) interactions could be clearly identified (Figure 2 and Figure S5 in the Supporting Information). Furthermore, C α H 1 -C β H 6 , C β H 3 -NH 7 and NH 4 -C β H 6 NOE contacts could be observed (Figure 2). The initial structure refinement with a single chain and the full set of NOE-derived distance restraints revealed that the regular i-i+4 interactions are mutually exclusive with the last three NOEs.…”

“…[2,[5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] Although the formation of secondary structures of peptidic foldamers has been thoroughly studied, their higher-order self-organization (tertiary and quaternary structures) and its effects on the folding propensities are less well understood. It has been shown that peptidic foldamers are able to fold cooperatively into helix bundles; [20] β-and α,β-peptide sequences that were disordered at low concentrations have recently been shown to form quaternary structures through self-assembling helical building blocks.…”