C-Terminal Substitution of MDM2 Interacting Peptides Modulates Binding Affinity by Distinctive Mechanisms

Abstract

The complex between the proteins MDM2 and p53 is a promising drug target for cancer therapy. The residues 19–26 of p53 have been biochemically and structurally demonstrated to be a most critical region to maintain the association of MDM2 and p53. Variation of the amino acid sequence in this range obviously alters the binding affinity. Surprisingly, suitable substitutions contiguous to this region of the p53 peptides can yield tightly binding peptides. The peptide variants may differ by a single residue that vary little in their structural conformations and yet are characterized by large differences in their binding affinities. In this study a systematic analysis into the role of single C-terminal mutations of a 12 residue fragment of the p53 transactivation domain (TD) and an equivalent phage optimized peptide (12/1) were undertaken to elucidate their mechanistic and thermodynamic differences in interacting with the N-terminal of MDM2. The experimental results together with atomistically detailed dynamics simulations provide insight into the principles that govern peptide design protocols with regard to protein-protein interactions and peptidomimetic design.

Recognition Dynamics of p53 and MDM2: Implications for Peptide Design

“…This would incur an additional entropic cost to the binding process, suggesting an enthalpic compensation, in accord with the observation that their binding is more enthalpically driven compared to the p53-based peptide series 31 .…”

“…Based on characterization of the encounter complex -a diffusively bound state that is distinct from the non-diffuse state of the final bound complex -of the peptide-MDM2 interaction, the two series of peptides showed distinct mechanisms of interactions with MDM2.The peptides of the p53-based series were all found to adopt the same mode of interaction as on approach with their C-terminal ends facing the N-terminal region of MDM2. Although, Cterminal substitution (from Pro to Ser, Ala and Asn) does not affect the mode of interaction, it was found to affect the peptide average residence times which increase in the order X=S>A>N>P. This matches the experimentally-observed trend in the binding affinities31 . The phage-derived peptide series, however, showed a distinct mode of interaction, approaching MDM2 in an orientation opposite to that adopted by the p53-based series.…”

“…The peptides are arranged from left to right in order of decreasing experimental binding affinity to MDM2 (-9.54, -9.31, -9.08 and -7.79 kCal/mol) 31 . In a, the residence time profiles were spatially normalized by dividing total residence time at a radius r by the spherical volume slab 4πr 2 and then averaged over the number of BD trajectories.…”

A highly sensitive fluorescent light-up probe for real-time detection of the endogenous protein target and its antagonism in live cells

“…[11] Therefore, for cellular imaging, TPECM dye (Scheme 1) with an excitation wavelength compatible to a 405 nm laser was designed as it displays an orange-red emission color upon aggregation. [12] The peptide, MPRFMDYWEGLS (12.1Pep), a p53derived peptide engineered for high affinity binding to Mdm2 protein through phage display, [9,13] was chosen as a response ligand for Mdm2 detection. The conjugation of 12.1Pep with TPECM fluorogen yielded the target-specific light-up probe, TPECM-12.1Pep, which enables live-cell detection of Mdm2 and screening of Mdm2 antagonisms.…”

Macrostate Identification from Biomolecular Simulations through Time Series Analysis

“…15 MMPBSA_segmentation is written and wrapped in a userfriendly and flexible R package that is available upon request. Our results show that MMPBSA_segmentation is able to identify distinct biologically meaningful subpopulations that appear to have the characteristics of ergodicity.…”