ABSTRACT
SynTEF1, a prototype synthetic genome reader/regulator (SynGR), was designed to target GAA triplet repeats and restore the expression of frataxin (FXN) in Friedreich's ataxia patients. It achieves this complex task by recruiting BRD4, via a pan-BET ligand (JQ1), to the GAA repeats by using a sequence-selective DNA-binding polyamide. When bound to specific genomic loci in this way, JQ1 functions as a chemical prosthetic for acetyl-lysine residues that are natural targets of the two tandem bromodomains (BD1 and BD2) in bromo- and extra-terminal domain (BET) proteins. As next-generation BET ligands were disclosed, we tested a select set with improved physicochemical, pharmacological, and bromodomain-selective properties as substitutes for JQ1 in the SynGR design. Here, we report two unexpected findings: (1) SynGRs bearing pan-BET or BD2-selective ligands license transcription at the FXN locus, whereas those bearing BD1-selective ligands do not, and (2) rather than being neutral or inhibitory, an untethered BD1-selective ligand (GSK778) substantively enhances the activity of all active SynGRs. The failure of BD1-selective SynGRs to recruit BRD4/BET proteins suggests that rather than functioning as "epigenetic/chromatin mimics," active SynGRs mimic the functions of natural transcription factors in engaging BET proteins through BD2 binding. Moreover, the enhanced activity of SynGRs upon cotreatment with the BD1-selective ligand suggests that natural transcription factors compete for a limited pool of nonchromatin-bound BET proteins, and blocking BD1 directs pan-BET ligands to more effectively engage BD2. Taken together, SynGRs as chemical probes provide unique insights into the molecular recognition principles utilized by natural factors to precisely regulate gene expression, and they guide the design of more sophisticated synthetic gene regulators with greater therapeutic potential.
ABSTRACT
The approximately 300 human cullin-RING ligases (CRLs) are multisubunit E3s in which a RING protein, either RBX1 or RBX2, recruits an E2 to catalyze ubiquitination. RBX1-containing CRLs also can bind Glomulin (GLMN), which binds RBX1's RING domain, regulates the RBX1-CUL1-containing SCF(FBW7) complex, and is disrupted in the disease Glomuvenous Malformation. Here we report the crystal structure of a complex between GLMN, RBX1, and a fragment of CUL1. Structural and biochemical analyses reveal that GLMN adopts a HEAT-like repeat fold that tightly binds the E2-interacting surface of RBX1, inhibiting CRL-mediated chain formation by the E2 CDC34. The structure explains the basis for GLMN's selectivity toward RBX1 over RBX2, and how disease-associated mutations disrupt GLMN-RBX1 interactions. Our study reveals a mechanism for RING E3 ligase regulation, whereby an inhibitor blocks E2 access, and raises the possibility that other E3s are likewise controlled by cellular proteins that mask E2-binding surfaces to mediate inhibition.
Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Carrier Proteins/chemistry , Cullin Proteins/chemistry , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/chemistry , Ubiquitination/physiology , Adaptor Proteins, Signal Transducing/metabolism , Anaphase-Promoting Complex-Cyclosome , Binding Sites/physiology , Carrier Proteins/metabolism , Crystallography, X-Ray , Cullin Proteins/metabolism , Glomus Tumor/metabolism , Humans , Models, Chemical , Mutagenesis/physiology , Paraganglioma, Extra-Adrenal/metabolism , Protein Binding/physiology , Protein Folding , Protein Structure, Tertiary/physiology , Structure-Activity Relationship , Substrate Specificity/physiology , Ubiquitin-Conjugating Enzymes , Ubiquitin-Protein Ligase Complexes/chemistry , Ubiquitin-Protein Ligase Complexes/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
During infection, bacteriophage T4 produces the MotA transcription factor that redirects the host RNA polymerase to the expression of T4 middle genes. The C-terminal 'double-wing' domain of MotA binds specifically to the MotA box motif of middle T4 promoters. We report the crystal structure of this complex, which reveals a new mode of protein-DNA interaction. The domain binds DNA mostly via interactions with the DNA backbone, but the binding is enhanced in the specific cognate structure by additional interactions with the MotA box motif in both the major and minor grooves. The linker connecting the two MotA domains plays a key role in stabilizing the complex via minor groove interactions. The structure is consistent with our previous model derived from chemical cleavage experiments using the entire transcription complex. α- and ß-d-glucosyl-5-hydroxymethyl-deoxycytosine replace cytosine in T4 DNA, and docking simulations indicate that a cavity in the cognate structure can accommodate the modified cytosine. Binding studies confirm that the modification significantly enhances the binding affinity of MotA for the DNA. Consequently, our work reveals how a DNA modification can extend the uniqueness of small DNA motifs to facilitate the specificity of protein-DNA interactions.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , DNA/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolism , Binding Sites , Crystallography, X-Ray , Cytosine/analogs & derivatives , Cytosine/chemistry , Cytosine/metabolism , DNA/chemistry , DNA-Binding Proteins/genetics , Molecular Docking Simulation , Mutagenesis , Protein Conformation , Transcription Factors/genetics , Viral Proteins/geneticsABSTRACT
The position, bonding and dynamics of hydrogen atoms in the catalytic centers of proteins are essential for catalysis. The role of short hydrogen bonds in catalysis has remained highly debated and led to establishment of several distinctive geometrical arrangements of hydrogen atoms vis-à-vis the heavier donor and acceptor counterparts, that is, low-barrier, single-well or short canonical hydrogen bonds. Here we demonstrate how the position of a hydrogen atom in the catalytic triad of an aminoglycoside inactivating enzyme leads to a thirty-fold increase in catalytic turnover. A low-barrier hydrogen bond is present in the enzyme active site for the substrates that are turned over the best, whereas a canonical hydrogen bond is found with the least preferred substrate. This is the first comparison of these hydrogen bonds involving an identical catalytic network, while directly demonstrating how active site electrostatics adapt to the electronic nature of substrates to tune catalysis.
Subject(s)
Acetyltransferases/metabolism , Aminoglycosides/metabolism , Anti-Bacterial Agents/metabolism , Acetyltransferases/chemistry , Aminoglycosides/chemistry , Anti-Bacterial Agents/chemistry , Binding Sites , Catalysis , Catalytic Domain , Crystallography, X-Ray , Humans , Hydrogen Bonding , Molecular Dynamics Simulation , Protein Conformation , Static ElectricityABSTRACT
MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA) and typically down-regulating their stability or translation. Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence (i.e., NMR, FRET, SPR) that purine or pyrimidine-rich microRNAs of appropriate length and sequence form triple-helical structures with purine-rich sequences of duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show that several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 × 10(-16)) for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. This work has thus revealed a new mechanism by which microRNAs could interact with gene promoter regions to modify gene transcription.
Subject(s)
DNA/genetics , Gene Expression Regulation/genetics , MicroRNAs/genetics , Algorithms , Base Composition/genetics , Base Sequence , Binding Sites , Computational Biology , DNA/chemistry , Humans , Leukemia/geneticsABSTRACT
In the largest E3 ligase subfamily, Cul3 binds a BTB domain, and an associated protein-interaction domain such as MATH recruits substrates for ubiquitination. Here, we present biochemical and structural analyses of the MATH-BTB protein, SPOP. We define a SPOP-binding consensus (SBC) and determine structures revealing recognition of SBCs from the phosphatase Puc, the transcriptional regulator Ci, and the chromatin component MacroH2A. We identify a dimeric SPOP-Cul3 assembly involving a conserved helical structure C-terminal of BTB domains, which we call "3-box" due to its facilitating Cul3 binding and its resemblance to F-/SOCS-boxes in other cullin-based E3s. Structural flexibility between the substrate-binding MATH and Cul3-binding BTB/3-box domains potentially allows a SPOP dimer to engage multiple SBCs found within a single substrate, such as Puc. These studies provide a molecular understanding of how MATH-BTB proteins recruit substrates to Cul3 and how their dimerization and conformational variability may facilitate avid interactions with diverse substrates.
Subject(s)
Cullin Proteins/chemistry , Nuclear Proteins/chemistry , Repressor Proteins/chemistry , Ubiquitin-Protein Ligases/chemistry , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Animals , Co-Repressor Proteins , Consensus Sequence/physiology , Crystallography, X-Ray , Cullin Proteins/genetics , Cullin Proteins/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster , Histones/chemistry , Histones/genetics , Histones/metabolism , Humans , Models, Molecular , Molecular Chaperones , Mutation/physiology , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Phosphoprotein Phosphatases/chemistry , Phosphoprotein Phosphatases/genetics , Phosphoprotein Phosphatases/metabolism , Protein Binding/physiology , Protein Interaction Domains and Motifs/physiology , Protein Multimerization/physiology , Protein Structure, Quaternary/physiology , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitination/physiologyABSTRACT
Protein-protein interactions that have large, flat and featureless binding sites are difficult drug targets. In the development of their modulators conventional drug discovery strategies are often unsuccessful. Gaining a detailed understanding of the binding mode of protein-protein interaction inhibitors is therefore of vast importance for their future pharmaceutical use. The MDM2/p53 protein pair is a highly promising target for cancer treatment. Disruption of the protein complex using p53 α-helix mimetics has been shown to be a successful strategy to control p53 activity. To gain further insight into the binding of inhibitors to MDM2, the flexibility of four cyclic ß-hairpins that act as α-helical mimetics and potential MDM2/p53 interaction inhibitors was investigated in relation to their inhibitory activity. MDM2-binding of the mimetics was determined using fluorescence polarization and surface plasmon resonance assays, whereas their conformation and dynamics in solution was described by the combined experimental and computational NAMFIS analysis. Molecular flexibility was shown to be important for the activity of the cyclic ß-hairpin based MDM2 inhibitors.
Subject(s)
Peptidomimetics/chemistry , Peptidomimetics/pharmacology , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/metabolism , Drug Design , Models, Molecular , Protein Binding/drug effects , Protein Conformation, alpha-Helical , Tumor Suppressor Protein p53/chemistryABSTRACT
Gene expression can be regulated through factors that direct RNA polymerase to the correct promoter sequence at the correct time. Bacteriophage T4 controls its development in this way using phage proteins that interact with host RNA polymerase. Using a process called σ appropriation, the T4 co-activator AsiA structurally remodels the σ(70) subunit of host RNA polymerase, while a T4 activator, MotA, engages the C terminus of σ(70) and binds to a DNA promoter element, the MotA box. Structures for the N-terminal (NTD) and C-terminal (CTD) domains of MotA are available, but no structure exists for MotA with or without DNA. We report the first molecular map of the MotA/DNA interaction within the σ-appropriated complex, which we obtained by using the cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE). We conjugated surface-exposed, single cysteines in MotA with FeBABE and performed cleavage reactions in the context of stable transcription complexes. The DNA cleavage sites were analyzed using ICM Molsoft software and three-dimensional physical models of MotA(NTD), MotA(CTD), and the DNA to investigate shape complementarity between the protein and the DNA and to position MotA on the DNA. We found that the unusual "double wing" motif present within MotA(CTD) resides in the major groove of the MotA box. In addition, we have used surface plasmon resonance to show that MotA alone is in a very dynamic equilibrium with the MotA element. Our results demonstrate the utility of fine resolution FeBABE mapping to determine the architecture of protein-DNA complexes that have been recalcitrant to traditional structure analyses.
Subject(s)
Bacteriophage T4/chemistry , DNA, Viral/chemistry , DNA-Binding Proteins/chemistry , DNA-Directed RNA Polymerases/chemistry , Escherichia coli Proteins/chemistry , Models, Molecular , Promoter Regions, Genetic , Sigma Factor/chemistry , Transcription Factors/chemistry , Viral Proteins/chemistry , Amino Acid Motifs , Bacteriophage T4/genetics , Bacteriophage T4/metabolism , DNA, Viral/genetics , DNA, Viral/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli/virology , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Protein Structure, Tertiary , Sigma Factor/genetics , Sigma Factor/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is an essential enzyme in the microbial folate biosynthetic pathway. This pathway has proven to be an excellent target for antimicrobial development, but widespread resistance to common therapeutics including the sulfa drugs has stimulated interest in HPPK as an alternative target in the pathway. A screen of a pterin-biased compound set identified several HPPK inhibitors that contain an aryl substituted 8-thioguanine scaffold, and structural analyses showed that these compounds engage the HPPK pterin-binding pocket and an induced cryptic pocket. A preliminary structure activity relationship profile was developed from biophysical and biochemical characterizations of derivative molecules. Also, a similarity search identified additional scaffolds that bind more tightly within the HPPK pterin pocket. These inhibitory scaffolds have the potential for rapid elaboration into novel lead antimicrobial agents.
Subject(s)
Diphosphotransferases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Thioguanine/pharmacology , Crystallography, X-Ray , Diphosphotransferases/metabolism , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Models, Molecular , Molecular Structure , Structure-Activity Relationship , Thioguanine/analogs & derivatives , Thioguanine/chemistryABSTRACT
BCL-2 proteins regulate mitochondrial poration in apoptosis initiation. How the pore-forming BCL-2 Effector BAK is activated remains incompletely understood mechanistically. Here we investigate autoactivation and direct activation by BH3-only proteins, which cooperate to lower BAK threshold in membrane poration and apoptosis initiation. We define in trans BAK autoactivation as the asymmetric "BH3-in-groove" triggering of dormant BAK by active BAK. BAK autoactivation is mechanistically similar to direct activation. The structure of autoactivated BAK BH3-BAK complex reveals the conformational changes leading to helix α1 destabilization, which is a hallmark of BAK activation. Helix α1 is destabilized and restabilized in structures of BAK engaged by rationally designed, high-affinity activating and inactivating BID-like BH3 ligands, respectively. Altogether our data support the long-standing hit-and-run mechanism of BAK activation by transient binding of BH3-only proteins, demonstrating that BH3-induced structural changes are more important in BAK activation than BH3 ligand affinity.
Subject(s)
Apoptosis/physiology , Membrane Proteins/metabolism , Mitochondria/metabolism , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2 Homologous Antagonist-Killer Protein/metabolism , BH3 Interacting Domain Death Agonist Protein/chemistry , BH3 Interacting Domain Death Agonist Protein/genetics , BH3 Interacting Domain Death Agonist Protein/metabolism , Cell Death , Crystallography, X-Ray , Humans , Ligands , Liposomes , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mitochondria/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , bcl-2 Homologous Antagonist-Killer Protein/chemistryABSTRACT
Inhibition of members of the bromodomain and extraterminal (BET) family of proteins has proven a valid strategy for cancer chemotherapy. All BET identified to date contain two bromodomains (BD; BD1 and BD2) that are necessary for recognition of acetylated lysine residues in the N-terminal regions of histones. Chemical matter that targets BET (BETi) also interact via these domains. Molecular and cellular data indicate that BD1 and BD2 have different biological roles depending upon their cellular context, with BD2 particularly associated with cancer. We have therefore pursued the development of BD2-selective molecules both as chemical probes and as potential leads for drug development. Here we report the structure-based generation of a novel series of tetrahydroquinoline analogs that exhibit >50-fold selectivity for BD2 versus BD1. This selective targeting resulted in engagement with BD-containing proteins in cells, resulting in modulation of MYC proteins and downstream targets. These compounds were potent cytotoxins toward numerous pediatric cancer cell lines and were minimally toxic to nontumorigenic cells. In addition, unlike the pan BETi (+)-JQ1, these BD2-selective inhibitors demonstrated no rebound expression effects. Finally, we report a pharmacokinetic-optimized, metabolically stable derivative that induced growth delay in a neuroblastoma xenograft model with minimal toxicity. We conclude that BD2-selective agents are valid candidates for antitumor drug design for pediatric malignancies driven by the MYC oncogene. SIGNIFICANCE: This study presents bromodomain-selective BET inhibitors that act as antitumor agents and demonstrates that these molecules have in vivo activity towards neuroblastoma, with essentially no toxicity.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Design , Neoplasms , Transcription Factors/antagonists & inhibitors , Animals , Cell Line, Tumor , Child , Female , Humans , Mice , Mice, SCID , Neoplasms/genetics , Neoplasms/metabolism , Protein Domains , Proto-Oncogene Proteins c-myc/genetics , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
p27Kip1 is an intrinsically disordered protein (IDP) that inhibits cyclin-dependent kinase (Cdk)/cyclin complexes (e.g., Cdk2/cyclin A), causing cell cycle arrest. Cell division progresses when stably Cdk2/cyclin A-bound p27 is phosphorylated on one or two structurally occluded tyrosine residues and a distal threonine residue (T187), triggering degradation of p27. Here, using an integrated biophysical approach, we show that Cdk2/cyclin A-bound p27 samples lowly-populated conformations that provide access to the non-receptor tyrosine kinases, BCR-ABL and Src, which phosphorylate Y88 or Y88 and Y74, respectively, thereby promoting intra-assembly phosphorylation (of p27) on distal T187. Even when tightly bound to Cdk2/cyclin A, intrinsic flexibility enables p27 to integrate and process signaling inputs, and generate outputs including altered Cdk2 activity, p27 stability, and, ultimately, cell cycle progression. Intrinsic dynamics within multi-component assemblies may be a general mechanism of signaling by regulatory IDPs, which can be subverted in human disease.
Subject(s)
Cell Division/physiology , Cyclin A/metabolism , Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Crystallography, X-Ray , Cyclin A/isolation & purification , Cyclin-Dependent Kinase 2/isolation & purification , Cyclin-Dependent Kinase Inhibitor p27/genetics , Cyclin-Dependent Kinase Inhibitor p27/isolation & purification , Fusion Proteins, bcr-abl/metabolism , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Phosphorylation/physiology , Protein Binding/physiology , Protein Processing, Post-Translational/physiology , Protein Structure, Tertiary/physiology , Proteolysis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Signal Transduction/physiology , Threonine/metabolism , Tyrosine/metabolism , src-Family Kinases/isolation & purification , src-Family Kinases/metabolismABSTRACT
Post-translational covalent modification by ubiquitin and ubiquitin-like proteins (UBLs) is a major eukaryotic mechanism for regulating protein function. In general, each UBL has its own E1 that serves as the entry point for a cascade. The E1 first binds the UBL and catalyzes adenylation of the UBL's C-terminus, prior to promoting UBL transfer to a downstream E2. Ubiquitin's Arg 72, which corresponds to Ala72 in the UBL NEDD8, is a key E1 selectivity determinant: swapping ubiquitin and NEDD8 residue 72 identity was shown previously to swap their E1 specificity. Correspondingly, Arg190 in the UBA3 subunit of NEDD8's heterodimeric E1 (the APPBP1-UBA3 complex), which corresponds to a Gln in ubiquitin's E1 UBA1, is a key UBL selectivity determinant. Here, we dissect this specificity with biochemical and X-ray crystallographic analysis of APPBP1-UBA3-NEDD8 complexes in which NEDD8's residue 72 and UBA3's residue 190 are substituted with different combinations of Ala, Arg, or Gln. APPBP1-UBA3's preference for NEDD8's Ala72 appears to be indirect, due to proper positioning of UBA3's Arg190. By contrast, our data are consistent with direct positive interactions between ubiquitin's Arg72 and an E1's Gln. However, APPBP1-UBA3's failure to interact with a UBL having Arg72 is not due to a lack of this favorable interaction, but rather arises from UBA3's Arg190 acting as a negative gate. Thus, parallel residues from different UBL pathways can utilize distinct mechanisms to dictate interaction selectivity, and specificity can be amplified by barriers that prevent binding to components of different conjugation cascades.
Subject(s)
Ubiquitins/metabolism , Alanine/chemistry , Alanine/genetics , Alanine/metabolism , Amino Acid Sequence , Arginine/chemistry , Arginine/genetics , Arginine/metabolism , Binding Sites/genetics , Crystallography, X-Ray , Glucosamine/chemistry , Glucosamine/metabolism , Humans , Kinetics , Molecular Sequence Data , Multienzyme Complexes/chemistry , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , NEDD8 Protein , Polymerase Chain Reaction , Protein Binding , Protein Structure, Secondary , Sequence Homology, Amino Acid , Substrate Specificity , Surface Plasmon Resonance , Ubiquitin-Activating Enzymes/chemistry , Ubiquitin-Activating Enzymes/genetics , Ubiquitin-Activating Enzymes/metabolism , Ubiquitins/chemistry , Ubiquitins/geneticsABSTRACT
The SUMO ubiquitin-like proteins play regulatory roles in cell division, transcription, DNA repair, and protein subcellular localization. Paralleling other ubiquitin-like proteins, SUMO proteins are proteolytically processed to maturity, conjugated to targets by E1-E2-E3 cascades, and subsequently recognized by specific downstream effectors containing a SUMO-binding motif (SBM). SUMO and its E2 from the budding yeast Saccharomyces cerevisiae, Smt3p and Ubc9p, are encoded by essential genes. Here we describe the 1.9 A resolution crystal structure of a non-covalent Smt3p-Ubc9p complex. Unexpectedly, a heterologous portion of the crystallized complex derived from the expression construct mimics an SBM, and binds Smt3p in a manner resembling SBM binding to human SUMO family members. In the complex, Smt3p binds a surface distal from Ubc9's catalytic cysteine. The structure implies that a single molecule of Smt3p cannot bind concurrently to both the non-covalent binding site and the catalytic cysteine of a single Ubc9p molecule. However, formation of higher-order complexes can occur, where a single Smt3p covalently linked to one Ubc9p's catalytic cysteine also binds non-covalently to another molecule of Ubc9p. Comparison with other structures from the SUMO pathway suggests that formation of the non-covalent Smt3p-Ubc9p complex occurs mutually exclusively with many other Smt3p and Ubc9p interactions in the conjugation cascade. By contrast, high-resolution insights into how Smt3p-Ubc9p can also interact with downstream recognition machineries come from contacts with the SBM mimic. Interestingly, the overall architecture of the Smt3p-Ubc9p complex is strikingly similar to recent structures from the ubiquitin pathway. The results imply that non-covalent ubiquitin-like protein-E2 complexes are conserved platforms, which function as parts of larger assemblies involved in many protein post-translational regulatory pathways.
Subject(s)
Proteins/chemistry , Repressor Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitin/chemistry , Amino Acid Sequence , Catalytic Domain , Cloning, Molecular , Crystallography, X-Ray , Cysteine/chemistry , Humans , Molecular Conformation , Molecular Sequence Data , Protein Binding , Saccharomyces cerevisiae/metabolism , Sequence Homology, Amino Acid , Small Ubiquitin-Related Modifier ProteinsABSTRACT
The transcription factor p53 is the main tumour suppressor in cells and many cancer types have p53 mutations resulting in a loss of its function. In tumours that retain wild-type p53 function, p53 activity is down-regulated by MDM2 (human murine double minute 2) via a direct protein-protein interaction. We have designed and synthesised two series of 2,5-diketopiperazines as inhibitors of the MDM2-p53 interaction. The first set was designed to directly mimic the α-helical region of the p53 peptide, containing key residues in the i, i+4 and i+7 positions of a natural α-helix. Conformational analysis indicated that 1,3,6-trisubstituted 2,5-diketopiperazines were able to place substituents in the same spatial orientation as an α-helix template. The key step of the synthesis involved the cyclisation of substituted dipeptides. The other set of tetrasubstituted 2,5-diketopiperazines were designed based on structure-based docking studies and the Ugi multicomponent reaction was used for the synthesis. This latter set comprised the most potent inhibitors which displayed micromolar IC50-values in a biochemical fluorescence polarisation assay.
Subject(s)
Diketopiperazines/chemical synthesis , Diketopiperazines/pharmacology , Drug Design , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/metabolism , Binding Sites , Chemistry Techniques, Synthetic , Diketopiperazines/chemistry , Humans , Models, Molecular , Protein Binding/drug effects , Protein Structure, Secondary , Proto-Oncogene Proteins c-mdm2/chemistry , Tumor Suppressor Protein p53/chemistryABSTRACT
p27(Kip1) (p27), a prototypical intrinsically disordered protein (IDP), regulates eukaryotic cell division through interactions with cyclin-dependent kinase (Cdk)/cyclin complexes. The activity, stability, and subcellular localization of p27 are regulated by phosphorylation. We illustrate how p27 integrates regulatory signals from several non-receptor tyrosine kinases (NRTKs) to activate Cdk4 and initiate cell cycle entry. Unmodified p27 potently inhibits Cdk/cyclin complexes, including Cdk4/cyclin D (IC(50), 1 nM). Some NRTKs (e.g., Abl) phosphorylate p27 on Tyr 88, which facilitates a second modification on Tyr 74 by another NRTK (e.g., Src). Importantly, this second modification causes partial reactivation of Cdk4 within ternary complexes containing doubly Tyr phosphorylated p27. Partial activation of Cdk4 initiates entry into the cell division cycle. Therefore, p27's disordered features enable NRTKs to sequentially promote a phosphorylation cascade that controls cell fate. Beyond cell cycle control, these results illustrate general concepts regarding why IDPs are well-suited for roles in signaling and regulation in biological systems.
Subject(s)
Cell Cycle Checkpoints , Cyclin-Dependent Kinase Inhibitor p27/physiology , Animals , Cell Cycle , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinases , Cyclins , Humans , PhosphorylationABSTRACT
Elimination of defective mitochondria is essential for the health of long-lived, postmitotic cells. To gain insight into this process, we examined programmed mitochondrial clearance in reticulocytes. BNIP3L is a mitochondrial outer membrane protein that is required for clearance. It has been suggested that BNIP3L functions by causing mitochondrial depolarization, activating autophagy, or engaging the autophagy machinery. Here we showed in mice that BNIP3L activity localizes to a small region in its cytoplasmic domain, the minimal essential region (MER). The MER is a novel sequence, which comprises three contiguous hydrophobic amino acid residues, and flanking charged residues. Mutation of the central leucine residue causes complete loss of BNIP3L activity, and prevents rescue of mitochondrial clearance. Structural bioinformatics analysis predicts that the BNIP3L cytoplasmic domain lacks stable tertiary structure, but that the MER forms an α-helix upon binding to another protein. These findings support an adaptor model of BNIP3L, centered on the MER.
Subject(s)
Membrane Proteins/chemistry , Membrane Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/metabolism , Reticulocytes/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Computational Biology , Hydrophobic and Hydrophilic Interactions , Leucine/metabolism , Membrane Proteins/deficiency , Mice , Mitochondrial Proteins/deficiency , Molecular Sequence Data , Protein Structure, Tertiary , Structure-Activity Relationship , bcl-X Protein/metabolismABSTRACT
The sulfonamide antibiotics inhibit dihydropteroate synthase (DHPS), a key enzyme in the folate pathway of bacteria and primitive eukaryotes. However, resistance mutations have severely compromised the usefulness of these drugs. We report structural, computational, and mutagenesis studies on the catalytic and resistance mechanisms of DHPS. By performing the enzyme-catalyzed reaction in crystalline DHPS, we have structurally characterized key intermediates along the reaction pathway. Results support an S(N)1 reaction mechanism via formation of a novel cationic pterin intermediate. We also show that two conserved loops generate a substructure during catalysis that creates a specific binding pocket for p-aminobenzoic acid, one of the two DHPS substrates. This substructure, together with the pterin-binding pocket, explains the roles of the conserved active-site residues and reveals how sulfonamide resistance arises.
Subject(s)
Anti-Bacterial Agents/pharmacology , Dihydropteroate Synthase/chemistry , Dihydropteroate Synthase/metabolism , Drug Resistance, Bacterial , Sulfamethoxazole/pharmacology , Sulfathiazoles/pharmacology , 4-Aminobenzoic Acid/chemistry , 4-Aminobenzoic Acid/metabolism , Amino Acid Sequence , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Bacillus anthracis/drug effects , Bacillus anthracis/enzymology , Biocatalysis , Catalytic Domain , Crystallization , Crystallography, X-Ray , Dihydropteroate Synthase/genetics , Diphosphates/chemistry , Diphosphates/metabolism , Magnesium/chemistry , Models, Chemical , Models, Molecular , Molecular Sequence Data , Mutagenesis , Parabens/chemistry , Parabens/metabolism , Protein Conformation , Sulfamethoxazole/chemistry , Sulfamethoxazole/metabolism , Sulfathiazole , Sulfathiazoles/chemistry , Sulfathiazoles/metabolism , Yersinia pestis/drug effects , Yersinia pestis/enzymologyABSTRACT
p27Kip1 controls cell proliferation by binding to and regulating the activity of cyclin-dependent kinases (Cdks). Here we show that Cdk inhibition and p27 stability are regulated through direct phosphorylation by tyrosine kinases. A conserved tyrosine residue (Y88) in the Cdk-binding domain of p27 can be phosphorylated by the Src-family kinase Lyn and the oncogene product BCR-ABL. Y88 phosphorylation does not prevent p27 binding to cyclin A/Cdk2. Instead, it causes phosphorylated Y88 and the entire inhibitory 3(10)-helix of p27 to be ejected from the Cdk2 active site, thus restoring partial Cdk activity. Importantly, this allows Y88-phosphorylated p27 to be efficiently phosphorylated on threonine 187 by Cdk2 which in turn promotes its SCF-Skp2-dependent degradation. This direct link between transforming tyrosine kinases and p27 may provide an explanation for Cdk kinase activities observed in p27 complexes and for premature p27 elimination in cells that have been transformed by activated tyrosine kinases.
Subject(s)
Cell Transformation, Neoplastic/genetics , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Cyclin-Dependent Kinases/metabolism , Gene Expression Regulation, Neoplastic/genetics , Oncogenes/genetics , src-Family Kinases/metabolism , Animals , Binding Sites/genetics , Cyclin-Dependent Kinase 2/genetics , Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinase Inhibitor p27/genetics , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/genetics , Fibroblasts , Fusion Proteins, bcr-abl , HeLa Cells , Humans , Mice , Phosphorylation , Protein Binding/genetics , Protein Structure, Tertiary/genetics , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , S-Phase Kinase-Associated Proteins/genetics , S-Phase Kinase-Associated Proteins/metabolism , SKP Cullin F-Box Protein Ligases/genetics , SKP Cullin F-Box Protein Ligases/metabolism , Signal Transduction/physiology , src-Family Kinases/geneticsABSTRACT
A total of 22 individuals participated in this benchmark study to characterize the thermodynamics of small-molecule inhibitor-enzyme interactions using Biacore instruments. Participants were provided with reagents (the enzyme carbonic anhydrase II, which was immobilized onto the sensor surface, and four sulfonamide-based inhibitors) and were instructed to collect response data from 6 to 36 degrees C. van't Hoff enthalpies and entropies were calculated from the temperature dependence of the binding constants. The equilibrium dissociation and thermodynamic constants determined from the Biacore analysis matched the values determined using isothermal titration calorimetry. These results demonstrate that immobilization of the enzyme onto the sensor surface did not alter the thermodynamics of these interactions. This benchmark study also provides insights into the opportunities and challenges in carrying out thermodynamic studies using optical biosensors.