A biweekly scientific journal publishing highquality research in molecular biology and genetics, cancer biology, biochemistry, and related fields. Structure of the mdm2 mdmx ring domain heterodimer. We can predict the density of a material, provided we know the atomic weight, atomic radius, and crystal geometry e. Structurebased design of potent nonpeptide mdm2 inhibitors. Inactivation of p53 is the most prevalent defect in human cancers. The 3d structure of mdm2 with and without p53 derived peptides and small molecules is extensively elaborated with more than 20 high resolution xray and nmr structures available in the protein data bank. Crystal structure databases the following online resources contain files which can be downloaded for interactive viewing either from a standalone visualization software or. As key negative regulator of the p53 tumour suppressor, mdm2 is an attractive therapeutic target. Here, we solved the complex structure of human mdm2rpl11 at 2.
The crystal structure of the binding revealed that three amino acid residues of the p53 peptide green are essential for the binding with mdm2, and they are inserted into a fairly deep cavity on the mdm2 surface yellow. The most potent inhibitor tsfaeywnllsp, termed pmi, bound to mdm2 and mdmx at low nanomolar affinitiesapproximately 2 orders of magnitude stronger than the wildtype p53 peptide of the same length etfsdlwkllpe. Jan 25, 2008 here we report the crystal structure of the mdm2mdmx ring domain heterodimer and provide a molecular basis for understanding how heterodimer formation leads to stabilization of mdm2, and. In recent years, mdm2like protein mdmx, another key downregulator of p53, has gained increasing importance as an additional target for drug development, in order to provide a complementary approach to mdm2 inhibition. Academic resource center illinois institute of technology. Initially, the turn ii peptide was manually docked on the p53 helix binding groove of the crystal structure of mdm2 25109 protein data bank code 1ycr. Material properties generally vary with single crystal orientation i. More importantly, mdm2 functions as the e3 ligase that ubiquitinates p53 for proteasome degradation 14, 18. To understand the structural basis for improved affinity we have solved the crystal structure of this stapled peptide m011 bound to mdm2 residues 6125 at 1.
The central region of mdm2 is critical for p53 activation and tumor suppression. The figure depicts the most important functional domains of p53 and mdm2 proteins. The mdm2 rpl11 complex structure was determined by molecular replacement and refined to a resolution of 2. Mdm2 binds the p53 tumor suppressor protein with high affinity and negatively modulates its transcriptional activity and stability.
How to design a successful p53mdm2x interaction inhibitor. Mdmx, mdm2, p53, structure, drugdesign, cancer the regulation. Structure of human mdm2 complexed with rpl11 reveals the. A novel small molecule inhibitor of mdm2p53 apg115. Computational studies and peptidomimetic design for the. Mdm2 binding to the nh 2 terminal transactivation domain of p53 blocks its transcriptional activity directly 12, 30. Department of medicinal chemistry and institute for structural biology and drug discovery, virginia commonwealth university, richmond, virginia. Mdm2 overexpression, in cooperation with oncogenic ras, promotes transformation of primary rodent fibroblasts, and mdm2 expression led to tumor formation in nude mice. Mdm2 extensively interacts with rpl11 through an acidic domain and two zinc fingers. Tumor protein 53 p53 is a critical regulator of cell cycle and apoptosis that is frequently disabled in human tumors. Hydropathic analysis of inhibitors of the p53 mdm2 interaction.
The crystal structure of this al11co4 approximant with a stoichiometric composition al73. A crystal structure of the rg7112 mdm2 complex revealed that the small molecule binds in the p53 pocket of mdm2, mimicking the interactions of critical p53 amino acid residues. Now let us consider the issue how atoms viewed as hard spheres can be stacked together within a given unit cell. Structural basis for highaffinity peptide inhibition of p53 interactions with mdm2 and mdmx marzena pazgiera,1, min liua,b,1, guozhang zoua, weirong yuana, changqing lia, chong lia, jing lia, juahdi monboa. Throughout this report, these binding pockets on the surface of mdm2 and mdmx are referred to as the phe, trp, and leu pockets. For example in nacl, the basic building block is composed of one na and one cl ion which is repeated in a cubic pattern to make the nacl structure 2. The crystal structure of the 109residue aminoterminal domain of mdm2 bound to a 15residue transactivation domain peptide of p53 revealed that mdm2 has a deep hydrophobic cleft on which the p53 peptide binds as an amphipathic alpha helix. Modulation of the p53mdm2 interplay by hausp inhibitors. Practical suggestions for better crystal structures. The crystal structure of the 109residue aminoterminal domain of mdm2 bound to a 15residue transactivation domain peptide of p53 revealed that mdm2 has a deep hydrophobic cleft on which the p53 peptide binds as an amphipathic. Upon ribosomal stress, this region is bound by ribosomal proteins, particularly ribosomal protein l11 rpl11, leading to mdm2 inactivation and subsequent p53 activation. Mdm2 achieves this repression by binding to and blocking the nterminal transactivation domain of p53. E3 ubiquitinprotein ligase that mediates ubiquitination of p53tp53, leading to its degradation by the proteasome. A complex is formed by binding mdm2 to p53 that plays a regulatory role in adjusting the activity and level of p53 in normal cells.
Structure of the stapled p53 peptide bound to mdm2 journal. E3s recruit substrates through degrons or protein domain interactions. Department of biochemistry, university of oxford, south parks road, oxford ox1 3qu, england. The crystal structure of the 109residue aminoterminal domain of mdm2 bound to a 15residue transactivation. Arf binding to mdm2 selectively blocks p53ubiquitination but promotes ubiquitination of mdmx. Pdf small molecule inhibitors of mdm2p53 and mdmxp53.
Crystal structure of human mdm2 with an imidazoline. Atoms are arranged at the corners and center of each cube face of the cell. Mdmx overexpression prevents p53 activation by the mdm2. Molecular mechanism of the interaction between mdm2 and. The xray crystal structure of improved antagonists bound to hdm2 reveals their. Pdf mdm2 smallmolecule antagonist rg7112 activates p53. The crystal structure of the p53mdm2 complex has been solved 29. Structure of the mdm2 oncoprotein bound to the p53 tumor.
Inhibitors that disrupt binding between p53 and mdm2 efficiently activate a p53 response, but their use in the treatment of cancers that retain wildtype p53 may be limited by ontarget toxicities due to p53 activation in normal tissue. Consequently, the mdm2p53 interaction has been extensively targeted for inhibition by small molecules. These optimized molecules increase the transcription of p53 target genes and decrease proliferation of tumor cells expressing wildtype p53. Analysis of crystal structures and molecular dynamics md simulations of complexes of mdm2 and mdmx resulted in the identification of a water molecule with a long residence time that appears to be modulated by the conformation of y10099. Mdm2 smallmolecule antagonist rg7112 activates p53 signaling. The crystal structure of one of nutlins isomers nutlin3a in the first binding site to mdm2 is currently used as a model for creating new. The biochemical basis of mdm2mediated inhibition of p53 function was further elucidated by crystallographic data that showed that the amino terminal domain of mdm2 forms a deep hydrophobic cleft into which the transactivation domain of p53 binds, thereby concealing itself from interaction with the transcriptional. The structure of m06 in complex with mdm2m62a was elucidated using xray crystallography, revealing 2 complexes in the asymmetric unit of the crystal table 1.
A co crystal structure of nutlin3a complex with mdm2 pdb id. Microstructure crystal structure and crystallography crystal defects. Inhibition of mdm2 p53 interaction can stabilize p53 and may offer a novel strategy for cancer therapy. It defines the entire crystal structure with the atom positions within. Temporal activation of p53 by a specific mdm2 inhibitor is. Structural basis for highaffinity peptide inhibition of. The conventional unit cell chosen is usually bigger than the primitive cell in favor of preserving the symmetry of the bravais lattice. In the 1ycrstructure, one way by which the p53peptide contributes to the crystal contacts is through packing oftwo lys24 sidechains. Treatment of cancer cells expressing wildtype p53 with rg7112 activated the. Crystal structure studies of p53 in complex with the mdm proteins have highlighted that three amino acids, phe19, trp23, and leu26, play a key role in the mdm2p53 interaction, differently from the mdmxp53 interaction that must be targeted independently from mdm2, opening the doors to the identification of several classes of antitumor agents. Here, we identify potent and selective smallmolecule antagonists of mdm2 and confirm their mode of action through the crystal structures of complexes.
Our findings deciphered the structural basis for highaffinity peptide inhibition of p53 interactions with mdm2 and mdmx, shedding new light on structure based rational design of different classes of p53 activators for potential. The crystal structures of the hausp traflike domain in complex with p53 and mdm2 peptides, determined at 2. P53mdm2 crystal structure of the p53binding domain of mdm2. Mdm2 or hdm2 in humans is an oncoprotein that can inactivate p53 tumor suppressor. Overlay of nmr and xray structure, mdm2 6125pip2 dimer observed in the crystal structure, chemical shift indices of apo and holo mdm2 pip1, sequence homology between human, dog, mouse and rat mdm2 1188 and p53 183, secondary structure predictions within nterminal region of mdm2, 3,2 c, 15 n filtered noesyhsqc pulse sequence and nmr experimental details as well as. Crystal structures reveal tight mdmx dimer formation. Upon ribosomal stress, this region is bound by ribosomal. The biochemical basis of mdm2mediated inhibition of p53 function was further elucidated by crystallographic data that showed that the amino terminal domain of mdm2 forms a deep hydrophobic cleft into which the transactivation domain of p53 binds, thereby concealing itself from interaction with the transcriptional machinery 29. This model suggests that small molecules may be able to. A detailed understanding of how stapled peptides are recalcitrant to mdm2 mutations conferring nutlinresistance will aid in the further development of potent mdm2 antagonists. In certain cancers, mdm2 amplification is a common event and contributes to the inactivation of p53. Crystal structures have been formed so far only of the.
Structural basis for highaffinity peptide inhibition of p53. As mdm2 could be easily degraded, we coexpressed a series of mdm2 and rpl11 fragments in the escherichia coli cells and then copurified them through a series of procedures see the materials and methods. The murine double minute mdm2 oncogene, which codes for the mdm2 protein, was originally cloned, along with two other genes mdm1 and mdm3 from the transformed mouse cell line 3t3dm. In vivo activation of the p53 pathway by smallmolecule. How p53 concentration is maintained at low level in normal cells. There are two complementary charges in the crystal structure of mdm2, k51 and h73. A miniaturized thermal denaturation assay was used to screen chemical libraries, resulting in the discovery of a novel series of benzodiazepinedione antagonists of the hdm2. The crystal structure of mdm2 nutlin complexes revealed that nutlins project functional groups into the binding pocket that mimic to a high degree the interaction of the three p53 amino acids. These pockets were defined by the original structure of a p53 peptide bound to mdm2 32 which showed that there were three key residues from the p53 peptide, phe 19, trp 23, and leu 26. Ordering of the nterminus of human mdm2 by small molecule. Mdm2 ring is shown in orange and mdmx ring in yellow, with the zinc ions and coordinating residues shown as spheres and sticks, respectively. Structurebased design of novel inhibitors of the mdm2.
The crystal structure of the 109residue aminoterminal domain of mdm2 bound to a 15residue transactivation domain peptide of p53 revealed that mdm2 has three hydrophobic clefts that bind three critical residues of p53 phe19, trp23, and leu26. Crystals of mdmx bound to ro2443 were grown that diffracted to relatively high resolution 1. Cocrystal structure of mdm2 green in complex with p53 peptide cyan pdb id. The structure of an mdm2 nutlin3a complex solved by the use of a. Using this mutant, the cocrystal structure of mdm2 with the benchmark inhibitor nutlin3a has been determined, revealing subtle differences from the previously described cocomplex of mdm2 with nutlin2. The structure gives an insight into the remarkable plasticity of mdm2 that facilitates binding of small molecule and peptide antagonists. Synthetic access to these compounds as well as their derivatives are described in detail. Discovery and cocrystal structure of benzodiazepinedione hdm2. Introduction to materials science, chapter, structure and properties of ceramics university of tennessee, dept. Activation of the p53 pathway by smallmoleculeinduced. Rational design of mi219 as a potent, specific, and orally available smallmolecule inhibitor of the mdm2 p53 interaction. Discovery and cocrystal structure of benzodiazepinedione. Inhibits p53tp53 and p73tp73mediated cell cycle arrest and apoptosis by binding its transcriptional activation domain.
Structureactivity studies of mdm2mdm4binding stapled. Surprisingly, near identity to the structure of a related peptide m06 without the 6chloro modification is observed. Genetic and biochemical studies mapped p53mdm2 binding sites to the nterminal domain of mdm2 and the nterminal part of the transactivation domain of p53 5. Mar 02, 2016 spirocycles and the development of a mdm2 antagonist 1. Home search guide structure home 3d macromolecular structures conserved domains pubchem biosystems help. Facecentered cubic crystal structure iii introduction to materials science, chapter 3, the structure of crystalline solids university of tennessee, dept. The biochemical basis of mdm2mediated inhibition of p53 function was further elucidated by crystallographic data that showed that the amino terminal domain of mdm2 forms a.
The solved structure contained rpl11 and part of mdm2 fig. Structure of the mdm2mdmx ring domain heterodimer reveals. Reports from initial clinical trials showed that blocking this interaction with a smallmolecule inhibitor can have great value in the treatment of cancer for patients with p53 wildtype tumors. Nov 08, 1996 in certain cancers, mdm2 amplification is a common event and contributes to the inactivation of p53. Structural analysis of mdm2 ring separates degradation from. The crystal structure of a p53derived peptide bound to the p53. However, the crystal structure of mdm2 bound to a peptide from the transacti vation domain of p53 has revealed that mdm2 possesses. Structures of mdm2 inhibitors with spirooxindole core structure. Sep 20, 2011 crystal contacts from mdm2 crystal structure 1ycr, 1t4e and 1rv1. Crystal structure of human mdm2 with an imidazoline inhibitor. Several classes of small molecules have been identified as potent, selective, and efficient inhibitors of the p53mdm2x interaction, and many co. Activation of the p53 pathway by smallmolecule induced mdm2.
The crystal structure of mdm2 in complex with p53 residues 1529 shows that the interaction is largely mediated by three key residues of p53, namely phe19, trp23 and leu26, and the binding surface on mdm2 is well defined kussie et al. Structures of low molecular weight inhibitors bound to mdmx and. A unit cell is a volume, when translated through some subset of the vectors of a bravais lattice, can fill up the whole space without voids or overlapping with itself. In fact, p53 and mdm2 protein are in a balanced situation in normal cell. The crystal structure of the p53mdm2 complex has been solved. Mdm2 has been identified as a p53 interacting protein that represses p53 transcriptional activity. Small molecule inhibitors of mdm2p53 and mdmxp53 interactions as new cancer therapeutics. Here, we present the three dimensional crystal structure of a stapled peptide antagonist bound to nutlinresistant mutant mdm2. Structure of a stapled peptide antagonist bound to nutlinresistant. The mdm2 oncoprotein is a cellular inhibitor of the p53 tumor suppressor in that it can bind the transactivation domain of p53 and downregulate its ability to activate transcription. Pdf structure of human mdm2 complexed with rpl11 reveals.
Recent smallmolecule inhibitors of the p53mdm2 protein. However, we found no ionicstrength dependency of the association kinetics of the wildtype p53 peptide p53 1529. Jul 15, 2015 crystal structure of the mdm2rpl11 complex. We solved the crystal structures of synthetic mdm2 and mdmx, both in complex with pmi, at 1.
Crystal structure 3 unit cell and lattice constants. Computational studies and peptidomimetic design for the human p53 mdm2 complex. The modeled complex structure was based on the contact points from the nmr chemical shift perturbations. Mdm2 is a p53 responsive genethat is, its transcription can be activated by p53. Development of novel protac smallmolecule degraders of mdm2. May 29, 2017 guided by a novel crystal structure of the mdm2mdmxe2ubch5bubiquitin complex, we designed mdm2 mutants that prevent e2ubiquitin binding without altering the ringdomain structure. Crystal structure analyses demonstrate that although mdm2 interacts with hausp at a much higher affinity than p53, they both bind to the same shallow groove in the traflike domain of hausp in a mutually exclusive manner hu et al. Spirocycles and the development of a mdm2 antagonist.
Mdm2mdmx complexes bind the p53 tumorsuppressor protein, inhibiting p53s transcriptional activity and targeting p53 for proteasomal degradation. Structural basis of competitive recognition of p53 and. We previously reported the discovery of potent and selective morpholinone and piperidinone inhibitors of the mdm2p53 interaction. Structure of a stapled peptide antagonist bound to nutlin. Inhibitors of the mdm2p53 interaction that restore the functional p53 constitute potential nongenotoxic anticancer agents with a novel mode of action. Also acts as a ubiquitin ligase e3 toward itself and arrb1. The structural nature and the critical role of the mdm2p53 interaction in regulating p53 levels. Employing a structure based approach and based on the crystal structure of the mdm2 p53 complex, we have designed spirooxindoles fig.
Moreover, a preliminary water analysis of the current crystal structures is presented. In many tumor types, p53 is deleted or mutated, but in others p53 is inactivated by overexpression or amplification of its negative regulator mouse double minute 2 mdm2. In the 1t4e structure, an mdm2bound benzodiazepinedione forms ahydrogen bond with an adjacent mdm2 molecule. The crystal structure of mdm2 in complex with an nterminal peptide of p53 showed that the p53 peptide forms an amphipathic. The structural basis of the p53mdm2 interaction has been established by xray crystallography. Structural effects and competition mechanisms targeting the. Lattice points lattice points are theoretical points arranged periodically in 3d space. Iucr the structure of an mdm2nutlin3a complex solved by.
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