WW Domains of the Yes-Kinase-Associated-Protein (YAP) Transcriptional Regulator Behave as Independent Units with Different Binding Preferences for PPxY Motif-Containing Ligands

YAP is a WW domain-containing effector of the Hippo tumor suppressor pathway, and the object of heightened interest as a potent oncogene and stemness factor. YAP has two major isoforms that differ in the number of WW domains they harbor. Elucidating the degree of co-operation between these WW domains is important for a full understanding of the molecular function of YAP. We present here a detailed biophysical study of the structural stability and binding properties of the two YAP WW domains aimed at investigating the relationship between both domains in terms of structural stability and partner recognition. We have carried out a calorimetric study of the structural stability of the two YAP WW domains, both isolated and in a tandem configuration, and their interaction with a set of functionally relevant ligands derived from PTCH1 and LATS kinases. We find that the two YAP WW domains behave as independent units with different binding preferences, suggesting that the presence of the second WW domain might contribute to modulate target recognition between the two YAP isoforms. Analysis of structural models and phage-display studies indicate that electrostatic interactions play a critical role in binding specificity. Together, these results are relevant to understand of YAP function and open the door to the design of highly specific ligands of interest to delineate the functional role of each WW domain in YAP signaling.     

Binding of the Lactococcal Drug Dependent Transcriptional Regulator LmrR to Its Ligands and Responsive Promoter Regions

The heterodimeric ABC transporter LmrCD from Lactococcus lactis is able to extrude several different toxic compounds from the cell, fulfilling a role in the intrinsic and induced drug resistance. The expression of the lmrCD genes is regulated by the multi-drug binding repressor LmrR, which also binds to its own promoter to autoregulate its own expression. Previously, we reported the crystal structure of LmrR in the presence and absence of the drugs Hoechst 33342 and daunomycin. Analysis of the mechanism how drugs control the repressor activity of LmrR is impeded by the fact that these drugs also bind to DNA. Here we identified, using X-ray crystallography and fluorescence, that riboflavin binds into the drug binding cavity of LmrR, adopting a similar binding mode as Hoechst 33342 and daunomycin. Microscale thermophoresis was employed to quantify the binding affinity of LmrR to its responsive promoter regions and to evaluate the cognate site of LmrR in the lmrCD promoter region. Riboflavin reduces the binding affinity of LmrR for the promoter regions. Our results support a model wherein drug binding to LmrR relieves the LmrR dependent repression of the lmrCD genes.     

Automated multiple ligand screening by frontal affinity chromatography-mass spectrometry (FAC-MS)

High-throughput screening (HTS) efforts to discover "hits" typically rely on the large-scale parallel screening of individual compounds with attempts to screen mixtures of compounds typically and, unfortunately, giving rise to false positives and false negatives due to the nature of the HTS readout (% inhibition/activation above a defined threshold) that makes deconvolution virtually intractable. Bioaffinity screening methods have emerged as an alternative or orthogonal method to classic HTS. One of these methods, frontal affinity chromatography coupled to mass spectrometry detection (FAC-MS), although still a relatively new technique, is turning out to be a viable screening tool. However, to push FAC-MS more to the forefront as a moderate primary HTS system (or a secondary screening assay), automation needs to be addressed. An automated FAC-MS system is described using 2 columns containing immobilized hERbeta, whereby while 1 column is being regenerated, the other is being used. The authors are extrapolating that in a continuous 24-h operation, the number of ligands screened could potentially approach 10,000. In addition, preliminary structure-activity relationship binding information (typically not seen in early primary HTS) can be obtained by observing the rank order of the library members in the various mixtures.     

Role of Methionine in Biosynthesis of Prodigiosin by Serratia marcescens

Methionine alone did not allow biosynthesis of prodigiosin (2-methyl-3-amyl-6-methoxyprodigiosene) in nonproliferating cells (NPC) of Serratia marcescens strain Nima. However, when methionine was added to NPC synthesizing prodigiosin in the presence of other amino acids, the lag period for synthesis of prodigiosin was shortened, an increased amount of the pigment was formed, and the optimal concentrations of the other amino acids were reduced. Less prodigiosin was synthesized when addition of methionine was delayed beyond 4 h. The specific activity of prodigiosin synthesized by addition of (14)CH(3)-methionine was 40 to 50 times greater than that synthesized from methionine-2-(14)C or (14)COOH-methionine. NPC of mutant OF of S. marcescens synthesized norprodigiosin (2-methyl-3-amyl-6-hydroxyprodigiosene), and the specific activity of this pigment synthesized in the presence of (14)CH(3)-methionine was only 5 to 13 times greater than that synthesized from methionine-2-(14)C or (14)COOH-methionine. A particulate, cell-free extract of mutant WF of S. marcescens methylated norprodigiosin to form prodigiosin. When the extract was added to NPC of mutant OF synthesizing norprodigiosin in the presence of (14)CH(3)-methionine, the prodigiosin formed had 80% greater specific activity than the norprodigiosin synthesized in the absence of the extract. The C6 hydroxyl group of norprodigiosin was methylated in the presence of the extract and methionine. Biosynthesis of prodigiosin by NPC of strain Nima also was augmented by addition of S-adenosylmethionine. Various analogues of methionine such as norleucine, norvaline, ethionine, and alpha-methylmethionine did not affect biosynthesis of prodigiosin by NPC either in the presence or absence of methionine.     

Mechanism for Attenuation of DNA Binding by MarR Family Transcriptional Regulators by Small Molecule Ligands

Members of the multiple antibiotic resistance regulator (MarR) family control gene expression in a variety of metabolic processes in bacteria and archaea. Hypothetical uricase regulator (HucR), which belongs to the ligand-responsive branch of the MarR family, regulates uricase expression in Deinococcus radiodurans by binding a shared promoter region between uricase and HucR genes. We show here that HucR responds only to urate and, to a lesser extent, to xanthine by attenuated DNA binding, compared to other intermediates of purine degradation. Using molecular-dynamics-guided mutational analysis, we identified the ligand-binding site in HucR. Electrophoretic mobility shift assays and intrinsic Trp fluorescence have identified W20 from the N-terminal helix and R80 from helix 3, which serves as a scaffold for the DNA recognition helix, as being essential for ligand binding. Using structural data combined with in silico and in vitro analyses, we propose a mechanism for the attenuation of DNA binding in which a conformational change initiated by charge repulsion due to a bound ligand propagates to DNA recognition helices. This mechanism may apply generally to MarR homologs that bind anionic phenolic ligands.