The oncoprotein Mdm2, and the recently intensely studied, homologues protein Mdmx, are principal negative regulators of the p53 tumor suppressor. The mechanisms by which they regulate the stability and activity of p53 are not fully established. We have determined the crystal structure of the N-terminal domain of Mdmx bound to a 15-residue p53 peptide. The structure reveals that although the principle features of the Mdm2-p53 interaction are preserved in the Mdmx-p53 complex, the Mdmx hydrophobic cleft on which the p53 peptide binds is significantly altered: a part of the cleft is blocked by sidechains of Met and Tyr of the p53-binding pocket of Mdmx. Thus specific inhibitors of Mdm2-p53 would not be optimal for binding to Mdmx. Our binding assays show indeed that nutlins, the newly discovered, potent antagonists of the Mdm2-p53 interaction, are not capable to efficiently disrupt the Mdmx-p53 interaction. To achieve full activation of p53 in tumor cells, compounds that are specific for Mdmx are necessary to complement the Mdm2 specific binders.
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.
We present an NMR-based antagonist induced dissociation assay (AIDA) for validation of inhibitor action on protein-protein interactions. As opposed to many standard NMR methods, AIDA directly validates the inhibitor potency in an in vitro NMR competition binding experiment. AIDA requires a large protein fragment (larger than 30 kDa) to bind to a small reporter protein (less than 20 kDa). We show here that a small fragment of a protein fused to glutathione S-transferase (GST) can effectively substitute the large protein component. We successfully used a GST-tagged N-terminal 73-residue p53 domain for binding studies with the human MDM2 protein. Other interactions we studied involved complexes of CDK2, cyclin A, p27, and the retinoblastoma protein. All these proteins play a key role in the cell division cycle, are associated with tumorigenesis, and are thus the subject of anticancer therapy strategies.
Mammalian centromeric cohesin is protected from phosphorylation‐dependent displacement in mitotic prophase by shugoshin‐1 (Sgo1), while shugoshin‐2 (Sgo2) protects cohesin from separase‐dependent cleavage in meiosis I. In higher eukaryotes, progression and faithful execution of both mitosis and meiosis are controlled by the spindle assembly checkpoint, which delays anaphase onset until chromosomes have achieved proper attachment to microtubules. According to the so‐called template model, Mad1–Mad2 complexes at unattached kinetochores instruct conformational change of soluble Mad2, thus catalysing Mad2 binding to its target Cdc20. Here, we show that human Sgo2, but not Sgo1, specifically interacts with Mad2 in a manner that strongly resembles the interactions of Mad2 with Mad1 or Cdc20. Sgo2 contains a Mad1/Cdc20‐like Mad2‐interaction motif and competes with Mad1 and Cdc20 for binding to Mad2. NMR and biochemical analyses show that shugoshin binding induces similar conformational changes in Mad2 as do Mad1 or Cdc20. Mad2 binding regulates fine‐tuning of Sgo2's sub‐centromeric localization. Mad2 binding is conserved in the only known Xenopus laevis shugoshin homologue and, compatible with a putative meiotic function, the interaction occurs in oocytes.
DNA ligases are a structurally diverse class of enzymes which share a common catalytic core and seal breaks in the phosphodiester backbone of double-stranded DNA via an adenylated intermediate. Here, the structure and activity of a recombinantly produced ATP-dependent DNA ligase from the bacterium Psychromonas sp. strain SP041 is described. This minimal-type ligase, like its close homologues, is able to ligate singly nicked double-stranded DNA with high efficiency and to join cohesive-ended and blunt-ended substrates to a more limited extent. The 1.65 Å resolution crystal structure of the enzyme-adenylate complex reveals no unstructured loops or segments, and suggests that this enzyme binds the DNA without requiring full encirclement of the DNA duplex. This is in contrast to previously characterized minimal DNA ligases from viruses, which use flexible loop regions for DNA interaction. The Psychromonas sp. enzyme is the first structure available for the minimal type of bacterial DNA ligases and is the smallest DNA ligase to be crystallized to date.
Chitin, a polymer of β(1–4)-linked N-acetylglucosamine found in e.g. arthropods, is a valuable resource that may be used to produce chitosan and chitooligosaccharides, two compounds with considerable industrial and biomedical potential. Deacetylating enzymes may be used to tailor the properties of chitin and its derived products. Here, we describe a novel CE4 enzyme originating from a marine Arthrobacter species (ArCE4A). Crystal structures of this novel deacetylase were determined, with and without bound chitobiose [(GlcNAc)2], and refined to 2.1 Å and 1.6 Å, respectively. In-depth biochemical characterization showed that ArCE4A has broad substrate specificity, with higher activity against longer oligosaccharides. Mass spectrometry-based sequencing of reaction products generated from a fully acetylated pentamer showed that internal sugars are more prone to deacetylation than the ends. These enzyme properties are discussed in the light of the structure of the enzyme-ligand complex, which adds valuable information to our still rather limited knowledge on enzyme-substrate interactions in the CE4 family.
p53 has been at the centre of attention for drug design since the discovery of its growth-suppressive and pro-apoptotic activity. Herein we report the design and characterisation of a new class of isoquinolinone inhibitors of the MDM2-p53 interaction. Our identification of druglike and selective inhibitors of this protein-protein interaction included a straightforward in silico compound-selection process, a recently reported NMR spectroscopic approach for studying the MDM2-p53 interaction, and selectivity screening assays using cells with the same genetic background. The selected inhibitors were all able to induce apoptosis and the expression of p53-related genes, but only the isoquinolin-1-one-based inhibitors stabilised p53. Our NMR experiments give a persuading explanation for these results, showing that isoquinolin-1-one derivates are able to dissociate the preformed MDM2-p53 complex in vitro, releasing a folded and soluble p53. The joint application of these methods provides a framework for the discovery of protein interaction inhibitors as a promising starting point for further drug design.
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