Ever since Ranganathan and coworkers subjected the covariation of amino acid residues in the postsynaptic density-95/Discs large/Zonula occludens 1 (PDZ) domain family to a statistical correlation analysis, PDZ domains have represented a paradigmatic family to explore single domain protein allostery. Nevertheless, several theoretical and experimental studies in the past two decades have contributed contradicting results with regard to structural localization of the allosteric networks, or even questioned their actual existence in PDZ domains. In this review, we first describe theoretical and experimental approaches that were used to probe the energetic network(s) in PDZ domains. We then compare the proposed networks for two well-studied PDZ domains namely the third PDZ domain from PSD-95 and the second PDZ domain from PTP-BL. Our analysis highlights the contradiction between the different methods and calls for additional work to better understand these allosteric phenomena.

Nuclear activated β-catenin plays a causative role in colorectal cancers (CRC) but remains an elusive therapeutic target. Using human CRC cells harboring different Wnt/β-catenin pathway mutations in APC/KRAS or β-catenin/KRAS genes, and both genetic and pharmacological knockdown approaches, we show that oncogenic β-catenin signaling negatively regulates the expression of NHERF1 (Na+/H+ exchanger 3 regulating factor 1), a PDZ-adaptor protein that is usually lost or downregulated in early dysplastic adenomas to exacerbate nuclear β-catenin activity. Chromatin immunoprecipitation (ChIP) assays demonstrated that β-catenin represses NHERF1 via TCF4 directly, while the association between TCF1 and the Nherf1 promoter increased upon β-catenin knockdown. To note, the occurrence of a cytostatic survival response in settings of single β-catenin-depleted CRC cells was abrogated by combining NHERF1 inhibition via small hairpin RNA (shRNA) or RS5517, a novel PDZ1-domain ligand of NHERF1 that prevented its ectopic nuclear entry. Mechanistically, dual NHERF1/β-catenin targeting promoted an autophagy-to-apoptosis switch consistent with the activation of Caspase-3, the cleavage of PARP and reduced levels of phospho-ERK1/2, Beclin-1, and Rab7 autophagic proteins compared with β-catenin knockdown alone. Collectively, our data unveil novel β-catenin/TCF-dependent mechanisms of CRC carcinogenesis, also offering preclinical proof of concept for combining β-catenin and NHERF1 pharmacological inhibitors as a mechanism-based strategy to augment apoptotic death of CRC cells refractory to current Wnt/β-catenin-targeted therapeutics.

d-Fructose 6-phosphate aldolase (FSA) catalyzes the asymmetric cross-aldol addition of phenylacetaldehyde and hydroxyacetone. We conducted structure-guided saturation mutagenesis of noncatalytic active-site residues to produce new FSA variants, with the goal of widening the substrate scope of the wild-type enzyme toward a range of para- and meta-substituted arylated aldehydes. After a single generation of mutagenesis and selection, enzymes with diverse substrate selectivity scopes were identified. The kinetic parameters and stereoselectivities for a subset of enzyme/substrate combinations were determined for the reactions in both the aldol addition and cleavage reaction directions. The achieved collection of new aldolase enzymes provides new tools for controlled asymmetric synthesis of substituted aldols.