The WW domain is a new protein module with two highly conserved tryptophans that binds proline-rich peptide motifs in vitro. It is present in a number of signalling and regulatory proteins, often in several copies. Here we investigate the solution structure of the WW domain of human YAP65 (for Yes kinase-associated protein) in complex with proline-rich peptides containing the core motif PPxY. The structure of the domain with the bound peptide GTPPPPYTVG is a slightly curved, three-stranded, antiparallel beta-sheet. Two prolines pack against the first tryptophan, forming a hydrophobic buckle on the convex side of the sheet. The concave side has three exposed hydrophobic residues (tyrosine, tryptophan and leucine) which form the binding site for the ligand. A non-conserved isoleucine in the amino-terminal flanking region covers a hydrophobic patch and stabilizes the WW domain of human YAP65 in vitro. The structure of the WW domain differs from that of the SH3 domain and reveals a new design for a protein module that uses stacked aromatic surface residues to arrange a binding site for proline-rich peptides.

Syntrophins are modular proteins belonging to the dystrophin associated glycoprotein complex and are thought to be involved in the regulation of the muscular system. Screening of peptide libraries revealed selectivity of the synotrophin PDZ domain toward the motif R/K/Q-E-S/T-X-V-COO- found to be highly conserved in the alpha-subunit C-terminus of vertebrate voltage gated sodium channels (VGSCs). The solution structure of the domain in complex with the peptide G-V-K-E-S-L-V shows specific interactions between the conserved residues in the peptide and syntrophin-characteristic residues in the domain. We propose that syntrophins localize VGSCs to the dystrophin network through its PDZ domain.

SYNOPSISHighly oriented fibers of Li-, Na-, K-, and CsDNA were prepared with a previously developed wet spinning method. The procedure gave a large number of equivalent fiber bundle samples (reference length, Lo, typically = 12-15 cm) for systematic measurements of the fiber length L in ethanol-water solutions, using a simple mechanochemical set up. The decrease in relative length L / Lo with increasing ethanol concentration at room temperature gave evidence for the B-A transition centered at 76% ( v / v ) ethanol for NaDNA fibers and at 80 and 84% ethanol for Kand CsDNA fibers. A smaller decrease in L / L o of LiDNA fibers was attributed to the B-C transition centered at 80% ethanol. In a second type of experiment with DNA fibers in ethanol-water solutions, the heat-induced helix-coil transition, or melting, revealed itself in a marked contraction of the DNA fibers. The melting temperature T,, decreased linearly with increasing ethanol concentration for fibers in the B-DNA ethanol concentration region. In the B-A transition region, Na-and KUNA fibers showed a local maximum in T,. On further increase of the ethanol concentration, the A-DNA region followed with an even steeper linear decrease in T,. The dependence on the identity of the counterion is discussed with reference to the model for groove binding of cations in B-DNA developed by Skuratovskii and co-workers and to the results from Raman studies of the interhelical bonds in A-DNA performed by Lindsay and co-workers. An attempt to apply the theory of Chogovadze and Frank-Kamenetskii on DNA melting in the B-A transition region to the curves failed. However, for Na-and KDNA the T , dependence in and around the A-B transition region could be expressed as a weighted mean value of T , of Aand B-DNA. On further increase of the ethanol concentration, above 84% ethanol for LiDNA and above about 90% ethanol for Na-, K-, and CsDNA, a drastic change occurred. T , increased and a few percentages higher ethanol concentrations were found to stabilize the DNA fibers so that they did not melt at all, not even at the upper temperature limit of the experiments ( -80°C). This is interpreted as being due to the strong aggregation induced by these high ethanol concentrations and to the formation of P-DNA. Many features of the results are compatible with the counterion-water affinity model. In another series of measurements, T,,, of DNA fibers in 75% ethanol was measured at various salt concentrations. No salt effect was observed (with the exception of LiDNA at low salt concentrations). This result is supported hy calculations within the Poisson-Roltzmann cylindrical cell model.

The glycolytic PFKFB3 enzyme is widely overexpressed in cancer cells and an emerging anti-cancer target. Here, we identify PFKFB3 as a critical factor in homologous recombination (HR) repair of DNA double-strand breaks. PFKFB3 rapidly relocates into ionizing radiation (IR)-induced nuclear foci in an MRN-ATM-γH2AX-MDC1-dependent manner and co-localizes with DNA damage and HR repair proteins. PFKFB3 relocalization is critical for recruitment of HR proteins, HR activity, and cell survival upon IR. We develop KAN0438757, a small molecule inhibitor that potently targets PFKFB3. Pharmacological PFKFB3 inhibition impairs recruitment of ribonucleotide reductase M2 and deoxynucleotide incorporation upon DNA repair, and reduces dNTP levels. Importantly, KAN0438757 induces radiosensitization in transformed cells while leaving non-transformed cells unaffected. In summary, we identify a key role for PFKFB3 enzymatic activity in HR repair and present KAN0438757, a selective PFKFB3 inhibitor that could potentially be used as a strategy for the treatment of cancer.

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