M. Brett Waddell

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. 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.

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. 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.

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. 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, p27s 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.

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.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.

Architecture of the Bacteriophage T4 Activator MotA/Promoter DNA Interaction during Sigma Appropriation

Background: No structure exists of the bacteriophage T4 activator MotA with DNA. Results: Using FeBABE, physical models, and ICM Molsoft, we determined how MotA interacts with DNA within the transcription complex. Conclusion: The unusual “double-wing” motif in MotACTD sits within the DNA major groove. Significance: FeBABE analyses together with structures can be used to determine protein-DNA architecture within multiprotein complexes. 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 σ70 subunit of host RNA polymerase, while a T4 activator, MotA, engages the C terminus of σ70 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 MotANTD, MotACTD, 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 MotACTD 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.

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. 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 β-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 β-hairpin based MDM2 inhibitors.

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 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.

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

Abstract 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 β-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.