Domain-Selective BET Ligands Yield Next-Generation Synthetic Genome Readers/Regulators with Nonidentical Cellular Functions.

SynTEF1, a prototype synthetic genome reader/regulator (SynGR), was designed to target GAA triplet repeats and restore the expression of frataxin () 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.

Structural basis of BAK activation in mitochondrial apoptosis initiation.

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.

Bromodomain-Selective BET Inhibitors Are Potent Antitumor Agents against MYC-Driven Pediatric Cancer.

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.

Low-Barrier and Canonical Hydrogen Bonds Modulate Activity and Specificity of a Catalytic Triad.

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.

Dynamic anticipation by Cdk2/Cyclin A-bound p27 mediates signal integration in cell cycle regulation.

p27 is an intrinsically disordered protein (IDP) that inhibits cyclin-dependent kinase (Cdk)/cyclin complexes (e.g., Cdk2/cyclin A), causing cell cycle arrest.

The phage T4 MotA transcription factor contains a novel DNA binding motif that specifically recognizes modified DNA.

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.

Flexibility is important for inhibition of the MDM2/p53 protein-protein interaction by cyclic β-hairpins.

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.

MicroRNAs Form Triplexes with Double Stranded DNA at Sequence-Specific Binding Sites; a Eukaryotic Mechanism via which microRNAs Could Directly Alter Gene Expression.

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.

Design, Synthesis and Evaluation of 2,5-Diketopiperazines as Inhibitors of the MDM2-p53 Interaction.

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 identification, analysis and structure-based development of novel inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase.

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.

Architecture of the bacteriophage T4 activator MotA/promoter DNA interaction during sigma appropriation.

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.

A short linear motif in BNIP3L (NIX) mediates mitochondrial clearance in reticulocytes.

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.

Structure of a glomulin-RBX1-CUL1 complex: inhibition of a RING E3 ligase through masking of its E2-binding surface.

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.

Electrostatically accelerated coupled binding and folding of intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) are now recognized to be prevalent in biology, and many potential functional benefits have been discussed. However, the frequent requirement of peptide folding in specific interactions of IDPs could impose a kinetic bottleneck, which could be overcome only by efficient folding upon encounter. Intriguingly, existing kinetic data suggest that specific binding of IDPs is generally no slower than that of globular proteins.

On the mechanism of action of SJ-172550 in inhibiting the interaction of MDM4 and p53.

SJ-172550 (1) was previously discovered in a biochemical high throughput screen for inhibitors of the interaction of MDMX and p53 and characterized as a reversible inhibitor (J. Biol. Chem.

Catalysis and sulfa drug resistance in dihydropteroate synthase.

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.

Mechanism of cell cycle entry mediated by the intrinsically disordered protein p27(Kip1).

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.

Structures of SPOP-substrate complexes: insights into molecular architectures of BTB-Cul3 ubiquitin ligases.

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.

Heterodimerization of the sialidase NEU1 with the chaperone protective protein/cathepsin A prevents its premature oligomerization.

Lysosomal neuraminidase-1 (NEU1) forms a multienzyme complex with beta-galactosidase and protective protein/cathepsin A (PPCA). Because of its association with PPCA, which acts as a molecular chaperone, NEU1 is transported to the lysosomal compartment, catalytically activated, and stabilized. However, the mode(s) of association between these two proteins both en route to the lysosome and in the multienzyme complex has remained elusive.

Structural dissection of a gating mechanism preventing misactivation of ubiquitin by NEDD8's E1.

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.

Structure of a SUMO-binding-motif mimic bound to Smt3p-Ubc9p: conservation of a non-covalent ubiquitin-like protein-E2 complex as a platform for selective interactions within a SUMO pathway.

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.

Thermodynamic benchmark study using Biacore technology.

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.

Cdk-inhibitory activity and stability of p27Kip1 are directly regulated by oncogenic tyrosine kinases.

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.