BDB -BiopanningDataSet Details

Biopanning Data

BiopanningDataSet ID: 1817

Peptides:

Sequences	Affinity Values
TSFAEYWNLLSP(6) LTFEHYWAQLTS(6) DDWFQRVWSPLM(1) RYEFLDYWSQLH(1) VPRSAPTLWLGT(1)	[3.4][3.3] [NT][20] [NT][NT] [NT][NT] [NT][NT]

Sequences Features:

Motif:

F-X(3)-W-X(2)-L

Unique Sequence Number:

Experimental Method:

Phage display (common panning)

Sequencing Platform:

Sequencing Method:

Sanger sequencing

Rounds of Panning:

Reference:

PMID: 19255450

Target Name:

p53-binding domains of MDM2

Template Name:

Cellular tumor antigen p53

Structure of Target-Template Complex:

1YCR

4HFZ

Structure of Target-Peptide Complex:

3EQS

3LNZ

3G03

3JZR

3JZS

Library Name:

Ph.D.-12 phage display library (X12)

Affinity Measurement Method:

Surface plasmon resonance (SPR),Isothermal titration calorimetry (ITC)

Affinity Measurement Description:

A surface plasmon resonance (SPR)-based competition assay was performed with (15–29)p53 as the competitor. The Kd value (nM) of synthesized peptides was shown. Besides, the direct interaction between p53-binding domains of MDM2 (referred to thereafter as (syn)MDM2) and the peptide (TSFAEYWNLLSP) were quantified at 25°C, using a MicroCal VP-ITC microcalorimeter. The binding affinity (Kd, nM) was shown. NT denotes not tested.

Brief Description:

The p53-binding domains of MDM2 (referred to thereafter as (syn)MDM2) and their site-specifically biotinylated forms were chemically synthesized using native chemical ligation. In fact, biotin-(syn)MDM2 was used as bait against the phage display library. Several MDM2 structures have been determined in complexes with PMI (PDB ID code 3EQS and 3LNZ) and pDI (PDB ID code 3G03, 3JZR and 3JZS). The basic procedures for library screening are as follows: (i) incubate input phage (10 μL) with 400 μL of 10 nM biotin (syn)MDM2 for 60 min before adding phage-target solution to 50 μL of streptavidin-agarose resin (Pierce) for affinity capturing; (ii) wash unbound phage; (iii) elute bound phage with 1 mM (15–29) p53; (iv) amplify the eluate and collect phage for the next round of panning; (v) repeat steps 1– 4; (vi) sequence selected binding clones according to the procedures recommended by the manufacturer.

BiopanningDataSet Comments:

Two consensus sequences emerged for both MDM2 and MDMX: LTFEHYWAQLTS, also termed pDI, and PMI–TSFAEYWNLLSP. The 3 most critical residues involved in p53-MDM2/MDMX recognition, i.e., Phe-19, Trp-23 and Leu-26 (p53 numbering), were all present in the phage-selected consensus sequences. The most potent inhibitor (TSFAEYWNLLSP), termed PMI, bound to MDM2 and MDMX at low nanomolar affinities—approximately 2 orders of magnitude stronger than the wild-type p53 peptide of the same length (ETFSDLWKLLPE). Comparative structural analysis identified an extensive, tightened intramolecular H-bonding network in bound PMI that contributed to its conformational stability, thus enhanced binding to the 2 oncogenic proteins. Importantly, the C-terminal residue Pro of PMI induced formation of a hydrophobic cleft in MDMX previously unseen in the structures of p53-bound MDM2 or MDMX.

Curator:

Curation Date:

0000-00-00

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