An order-to-disorder structural switch activates the FoxM1 transcription factor

Intrinsically disordered transcription factor transactivation domains (TADs) function through structural plasticity, adopting ordered conformations when bound to transcriptional co-regulators. Many transcription factors contain a negative regulatory domain (NRD) that suppresses recruitment of transcriptional machinery through autoregulation of the TAD. We report the solution structure of an autoinhibited NRD-TAD complex within FoxM1, a critical activator of mitotic gene expression. We observe that while both the FoxM1 NRD and TAD are primarily intrinsically disordered domains, they associate and adopt a structured conformation. We identify how Plk1 and Cdk kinases cooperate to phosphorylate FoxM1, which releases the TAD into a disordered conformation that then associates with the TAZ2 or KIX domains of the transcriptional co-activator CBP. Our results support a mechanism of FoxM1 regulation in which the TAD undergoes switching between disordered and different ordered structures

Structural Basis of BRCC36 Function in DNA Repair and Immune Regulation

In mammals, ∼100 deubiquitinases act on ∼20,000 intracellular ubiquitination sites. Deubiquitinases are commonly regarded as constitutively active, with limited regulatory and targeting capacity. The BRCA1-A and BRISC complexes serve in DNA double-strand break repair and immune signaling and contain the lysine-63 linkage-specific BRCC36 subunit that is functionalized by scaffold subunits ABRAXAS and ABRO1, respectively. The molecular basis underlying BRCA1-A and BRISC function is currently unknown. Here we show that in the BRCA1-A complex structure, ABRAXAS integrates the DNA repair protein RAP80 and provides a high-affinity binding site that sequesters the tumor suppressor BRCA1 away from the break site. In the BRISC structure, ABRO1 binds SHMT2α, a metabolic enzyme enabling cancer growth in hypoxic environments, which we find prevents BRCC36 from binding and cleaving ubiquitin chains. Our work explains modularity in the BRCC36 DUB family, with different adaptor subunits conferring diversified targeting and regulatory functions.ISSN:1097-2765ISSN:1097-416

Structural basis of divergent cyclin-dependent kinase activation by Spy1/RINGO proteins.

Cyclin-dependent kinases (Cdks) are principal drivers of cell division and are an important therapeutic target to inhibit aberrant proliferation. Cdk enzymatic activity is tightly controlled through cyclin interactions, posttranslational modifications, and binding of inhibitors such as the p27 tumor suppressor protein. Spy1/RINGO (Spy1) proteins bind and activate Cdk but are resistant to canonical regulatory mechanisms that establish cell-cycle checkpoints. Cancer cells exploit Spy1 to stimulate proliferation through inappropriate activation of Cdks, yet the mechanism is unknown. We have determined crystal structures of the Cdk2-Spy1 and p27-Cdk2-Spy1 complexes that reveal how Spy1 activates Cdk. We find that Spy1 confers structural changes to Cdk2 that obviate the requirement of Cdk activation loop phosphorylation. Spy1 lacks the cyclin-binding site that mediates p27 and substrate affinity, explaining why Cdk-Spy1 is poorly inhibited by p27 and lacks specificity for substrates with cyclin-docking sites. We identify mutations in Spy1 that ablate its ability to activate Cdk2 and to proliferate cells. Our structural description of Spy1 provides important mechanistic insights that may be utilized for targeting upregulated Spy1 in cancer

Protein Interactions in Genome Maintenance as Novel Antibacterial Targets

<div><p>Antibacterial compounds typically act by directly inhibiting essential bacterial enzyme activities. Although this general mechanism of action has fueled traditional antibiotic discovery efforts for decades, new antibiotic development has not kept pace with the emergence of drug resistant bacterial strains. These limitations have severely restricted the therapeutic tools available for treating bacterial infections. Here we test an alternative antibacterial lead-compound identification strategy in which essential protein-protein interactions are targeted rather than enzymatic activities. Bacterial single-stranded DNA-binding proteins (SSBs) form conserved protein interaction “hubs” that are essential for recruiting many DNA replication, recombination, and repair proteins to SSB/DNA nucleoprotein substrates. Three small molecules that block SSB/protein interactions are shown to have antibacterial activity against diverse bacterial species. Consistent with a model in which the compounds target multiple SSB/protein interactions, treatment of <i>Bacillus subtilis</i> cultures with the compounds leads to rapid inhibition of DNA replication and recombination, and ultimately to cell death. The compounds also have unanticipated effects on protein synthesis that could be due to a previously unknown role for SSB/protein interactions in translation or to off-target effects. Our results highlight the potential of targeting protein-protein interactions, particularly those that mediate genome maintenance, as a powerful approach for identifying new antibacterial compounds.</p> </div

MPTA, BCBP, and CFAM are bactericidal.

<p>Time kill curves of <i>B. subtilis</i> PY79 treated with (<b>A</b>) controls (Dimethyl sulfoxide (DMSO), mitomycin C (MMC), nalidixic acid (NA)), (<b>B</b>) MPTA, (<b>C</b>) BCBP, or (<b>D</b>) CFAM at 0.5×, 1×, 2×, or 4× MIC levels. Data points are the mean from three independent experiments with error bars representing one standard deviation of the mean.</p

Structural basis for LIN54 recognition of CHR elements in cell cycle-regulated promoters.

The MuvB complex recruits transcription factors to activate or repress genes with cell cycle-dependent expression patterns. MuvB contains the DNA-binding protein LIN54, which directs the complex to promoter cell cycle genes homology region (CHR) elements. Here we characterize the DNA-binding properties of LIN54 and describe the structural basis for recognition of a CHR sequence. We biochemically define the CHR consensus as TTYRAA and determine that two tandem cysteine rich regions are required for high-affinity DNA association. A crystal structure of the LIN54 DNA-binding domain in complex with a CHR sequence reveals that sequence specificity is conferred by two tyrosine residues, which insert into the minor groove of the DNA duplex. We demonstrate that this unique tyrosine-mediated DNA binding is necessary for MuvB recruitment to target promoters. Our results suggest a model in which MuvB binds near transcription start sites and plays a role in positioning downstream nucleosomes

RecA-GFP foci are reduced following treatment of cells with small molecules that inhibit interaction with SSB.

a<p>A small percentage of cells treated with each of the compounds exhibited RecA-GFP localization which was punctate and/or membrane associated, which is not consistent with discrete, nucleoid associated foci. These localizations were scored, but excluded from the calculation shown above. The number of cells with mislocalized RecA-GFP foci are as follows for each compound: MPTA, 28; BCBP, 30; CFAM, 9.</p

MPTA, BCBP, and CFAM suppress growth after compound removal.

<p>Post antibiotic effect of <i>B. subtilis</i> PY79 treated with (<b>A</b>) MPTA, (<b>B</b>) BCBP, or (<b>B</b>) CFAM, after 1 hour of treatment with 1×, 2×, or 4× MIC levels. CFUs were measured at time zero (just prior to treatment) and at the times indicated after treatment. Data points are the mean from three independent experiments with error bars representing one standard deviation of the mean.</p

The SSB PPI inhibitors block DNA replication and protein translation in <i>B. subtilis</i> prior to cell death.

<p>(<b>A</b>) Incorporation of [³H] thymidine is measured in a pulse experiment in the absence or presence of MPTA (18 µM), BCBP (16 µM), CFAM (48 µM), or Nalidixic acid (NA, 25 µg/mL). Compounds were added to the culture at the 20 min time point. Duplicate samples were measured for each time point and each experiment was repeated in triplicate. All samples were normalized to the time zero reading with the data point as the mean and error bars are one standard deviation from the mean. The line connects the data points for simplicity and does not represent the amount of incorporation. (<b>B</b>) Incorporation of [³H] Leucine is measured in a pulse experiment in the absence or presence of MPTA (18 µM), BCBP (16 µM), CFAM (48 µM), or Nalidixic acid (NA, 25 µg/mL). Compounds were added to the culture at the 20 min time point. Duplicate samples were measured for each time point and each experiment was repeated in triplicate. All samples were normalized to the time zero reading with the data point as the mean and error bars are one standard deviation from the mean. The line connects the data points for simplicity and does not represent the amount of incorporation. (<b>C</b>) CFU/ml of <i>B. subtilis</i> under the same conditions as in <i>A</i> and <i>B</i>. Data points are the mean from all experiments with error bars representing one standard deviation of the mean.</p