Study of MDM2 Binding to p53-Analogues: Affinity, Helicity, and Applicability to Drug Design Ori Kalid* and Nir Ben-Tal Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv 69978, Israel Received September 28, 2008 MDM2 is a key regulator of the p53 tumor-suppressor protein. Here we study the effect of modifications of a p53 N-terminal fragment on its binding to MDM2, using implicit-solvent MD and MM-GB/SA calculations. We provide interpretation of existing experimental data and predict the effect of mutations on binding. Notably 1) We analyze the effect of regulatory phosphorylations at Ser/Thr residues and suggest that a balance between favorable electrostatics and desolvation penalties determines the effect of phosphorylation; 2) We compare the helical stability in solution of p53 alanine mutants and propose a helix stabilizing role for several residues involved in hydrogen bonding and hydrophobic packing; 3) We obtain good correlations between calculated and experimental affinities for a set of peptidomimetic inhibitors, both alone and in combination with p53 analogues, demonstrating potential applicability to drug design. From the technical aspect, protocol optimization and selection of simulation tools are addressed in detail. To the best of our knowledge this is the first published example of MM-GB/SA calculations utilizing a conformational ensemble generated with implicit solvent MD. Our results suggest that this highly efficient variant of classical explicit- solvent MM-GB/SA may be used for studying protein-protein interactions and for the design of peptidomimetic drugs. INTRODUCTION The tumor-suppressor protein p53 leads to cell-cycle arrest and apoptosis in response to cellular damage, acting mostly as a transcription factor. While not required for normal growth and development, p53 is critical for the prevention of tumor development. Defects in the p53 pathway are found in most human cancers, and p53 knockout mice show a dramatic increase in tumor occurrence. 1 Due to its powerful inhibition of cell growth, p53 function must be tightly regulated in normal human cells. This is accomplished by multiple control mechanisms, the most important of which seem to be at the mature protein level, including regulation of protein stability, cellular localization, post-translational modifications, and conformational changes affecting its ability to bind DNA. 1 One of the central components in the regulation of p53 is the p53-interacting protein MDM2. MDM2 is in itself a transcriptional target of p53 and so establishes a negative feedback loop in which p53 promotes the expression of its own down regulator. MDM2 amplifica- tion has been described in certain tumor types, including some sarcomas and brain cancer. In mice, homozygous deletion of MDM2 leads to apoptosis in the embryo and very early embryonic lethality. This effect is countered by a simultaneous knockout of p53. MDM2 interacts with the N-terminal domain of p53 (residues 15-29), containing the major p53 transcription activation domain. This interaction directly interferes with p53’s ability to contact transcriptional coactivator proteins. In addition, MDM2 also functions as a ubiquitin ligase for both p53 and itself and so modulates the half-life of p53. 1 The interaction of p53 with MDM2 has been studied extensively. The following section highlights selected studies from recent years, emphasizing the diversity in acquisition methods, discussing interpretability, and assessing compat- ibility between the studies. One of the first studies, by Picksley et al., tested the effect of alanine mutations on the MDM2 binding capacity of p53(16-25). Using an ELISA binding assay they showed that residues 18-23 (TFSDLW) are all critical for binding. 2 A study by Lin et al. tested the ability of p53 mutants to bind MDM2 using a single concentration immunoprecipi- tation assay. 3 This study showed that mutations Leu22Gln and Trp23Ser reduced MDM2 binding to 56% and 22%, respectively, while the double mutant retained only 2% of the wt activity. However, given only single concentration data, the actual effect on binding affinity is not clear. In a later study, Bottger et al. described a series of MDM2 binding peptides selected from a phage display library. 4 This analysis was later extended to a synthetic library of peptides derived from the initial discovery. 5 Bottger et al. character- ized the ability of these peptides to inhibit p53 binding to MDM2 using three different ELISA formats (peptide-ELISA, MDM2-ELISA, and p53-ELISA, according to the im- mobilized species). A systematic alanine scan of the optimal phage-derived 12-mer peptide revealed that Phe19 and Trp23 are critical for binding, all substitutions leading to complete loss of activity. (The paper also mentions a similar affect on the wt p53, but the data are not shown). Leu26 could only be replaced by Ile, Met, and Val albeit with 3-5-fold reduction in potency. In the crystal structure of p53 with * Corresponding author phone: ++972-3-640-9804; mobile: ++972- 54-2233074; fax: ++972-3-640-6834; e-mail: orikalid@gmail.com. J. Chem. Inf. Model. 2009, 49, 865–876 865 10.1021/ci800352c CCC: $40.75  2009 American Chemical Society Published on Web 03/26/2009 Downloaded by TEL AVIV UNIV (AO) on August 13, 2009 Published on March 26, 2009 on http://pubs.acs.org | doi: 10.1021/ci800352c