Monitoring the effects of antagonists on protein-protein interactions with NMR spectroscopy.
We describe an NMR method that directly monitors the influence of ligands on protein-protein interactions. For a two-protein interaction complex, the size of one component should be small enough (less than ca. 15 kDa) to provide a good quality (15)N((13)C) HSQC spectrum after (15)N((13)C) labeling. The size of the second unlabeled component should be large enough so that the molecular weight of the preformed complex is larger than ca. 40 kDa. When the smaller protein binds to a larger one, broadening of NMR resonances results in the disappearance of most of its cross-peaks in the HSQC spectrum. Addition of an antagonist that can dissociate the complex would restore the HSQC spectrum of the smaller component. The method directly shows whether an antagonist releases proteins in their wild-type folded states or whether it induces their denaturation, partial unfolding, or precipitation. We illustrate the method by studying lead compounds that have recently been reported to block the MDM2-p53 interaction. Activation of p53 in tumor cells by inhibiting its interaction with MDM2 offers new strategy for cancer therapy.
D'Silva
Loyola
L
Max Planck Institute for Biochemistry, D-82152 Martinsried, Germany.
Ozdowy
Przemyslaw
P
Krajewski
Marcin
M
Rothweiler
Ulli
U
Singh
Mahavir
M
Holak
Tad A
TA
D001896:Boron Compounds / Q000494:pharmacology*; D002247:Carbon Isotopes; D045744:Cell Line, Tumor; D002599:Chalcone / Q000031:analogs & derivatives / Q000494:pharmacology*; D005786:Gene Expression Regulation; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009587:Nitrogen Isotopes; D010879:Piperazines / Q000494:pharmacology*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship; D013449:Sulfonamides / Q000494:pharmacology*; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics
D001896:Boron Compounds; D002247:Carbon Isotopes; D007093:Imidazoles; D008024:Ligands; D009587:Nitrogen Isotopes; D010879:Piperazines; D011994:Recombinant Proteins; D013449:Sulfonamides; D016159:Tumor Suppressor Protein p53; C482205:nutlin 3; D002599:Chalcone; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2