Destabilization of interaction between cytokinin signaling intermediates AHP1 and ARR4 modulates Arabidopsis development.

Eukaryotic two-component signaling involves the His-Asp-His-Asp multistep phosphorelay (MSP). In Arabidopsis thaliana, cytokinin-mediated MSP signaling intermediates include histidine kinases (HKs), histidine phosphotransfer proteins (Hpts) and response regulators (RRs). The structure-function relationship of interaction between Hpt (e.g. AHP1) and RR (e.g. ARR4) is poorly understood. Using a homology model and yeast two-hybrid analysis, we identified key amino acids of ARR4 at the AHP1-ΔARR4((16-175)) interaction interface. Mutating them in Arabidopsis (arr3,4,5,6,8,9 hextuple mutant background) and performing root length assays provided functional relevance, and coimmunoprecipitation (coIP) assay provided biochemical evidence for the interaction. The homology model mimics crystal structures of Hpt-RR complexes. Mutating selected interface residues of ARR4 either abolished or destabilized the interaction. D45A and Y96A mutations weakened interaction with AHP1, and exhibited weaker rescue of root elongation in the hextuple mutants. CoIP analysis using cytokinin-treated transgenic Arabidopsis seedlings provided biochemical evidence for weakened AHP1-ARR4 interaction. The relevance of the selected residues for the interaction was further validated in two independent pairs of Hpt-RR proteins from Arabidopsis and rice (Oryza sativa). Our data provide evidence of a link between Hpt-RR interaction affinity and regulation of downstream functions of RRs. This establishes a structure-function relationship for the final step of a eukaryotic MSP signal cascade.

Biophysical studies and NMR structure of YAP2 WW domain - LATS1 PPxY motif complexes reveal the basis of their interaction.

YES-associated protein (YAP) is a major effector protein of the Hippo tumor suppressor pathway, and is phosphorylated by the serine/threonine kinase LATS. Their binding is mediated by the interaction between WW domains of YAP and PPxY motifs of LATS. Their isoforms, YAP2 and LATS1 contain two WW domains and two PPxY motifs respectively. Here, we report the study of the interaction of these domains both in vitro and in human cell lines, to better understand the mechanism of their binding. We show that there is a reciprocal binding preference of YAP2-WW1 with LATS1-PPxY2, and YAP2-WW2 with LATS1-PPxY1. We solved the NMR structures of these complexes and identified several conserved residues that play a critical role in binding. We further created a YAP2 mutant by swapping the WW domains, and found that YAP2 phosphorylation at S127 by LATS1 is not affected by the spatial configuration of its WW domains. This is likely because the region between the PPxY motifs of LATS1 is unstructured, even upon binding with its partner. Based on our observations, we propose possible models for the interaction between YAP2 and LATS1.

Branched Peptide, B2088, Disrupts the Supramolecular Organization of Lipopolysaccharides and Sensitizes the Gram-negative Bacteria.

Dissecting the complexities of branched peptide-lipopolysaccharides (LPS) interactions provide rationale for the development of non-cytotoxic antibiotic adjuvants. Using various biophysical methods, we show that the branched peptide, B2088, binds to lipid A and disrupts the supramolecular organization of LPS. The disruption of outer membrane in an intact bacterium was demonstrated by fluorescence spectroscopy and checkerboard assays, the latter confirming strong to moderate synergism between B2088 and various classes of antibiotics. The potency of synergistic combinations of B2088 and antibiotics was further established by time-kill kinetics, mammalian cell culture infections model and in vivo model of bacterial keratitis. Importantly, B2088 did not show any cytotoxicity to corneal epithelial cells for at least 96 h continuous exposure or hemolytic activity even at 20 mg/ml. Peptide congeners containing norvaline, phenylalanine and tyrosine (instead of valine in B2088) displayed better synergism compared to other substitutions. We propose that high affinity and subsequent disruption of the supramolecular assembly of LPS by the branched peptides are vital for the development of non-cytotoxic antibiotic adjuvants that can enhance the accessibility of conventional antibiotics to the intracellular targets, decrease the antibiotic consumption and holds promise in averting antibiotic resistance.

Normal limits of ECG measurements related to atrial activity using a modified limb lead system.

OBJECTIVE: The present study was designed to derive the normal limits of a new ECG lead system aimed at enhancing the amplitude of atrial potentials through the use of bipolar chest leads. METHODS: Sixty healthy male subjects, mean age 38.85±8.76 years (range 25 to 58 years) were included in this study. In addition to a standard 12-lead ECG, a modified limb lead (MLL) ECG was recorded for 60 sec with the RA electrode placed in the 3rd right intercostal space slightly to the left of the mid-clavicular line, the LA electrode placed in the 5th right intercostal space slightly to the right of the mid-clavicular line and the LL electrode placed in the 5th right intercostal space on the mid- clavicular line. RESULTS: In the frontal plane, the modification of limb electrode positions produced significant changes compared to standard limb lead I and II. The mean P wave amplitude was 111±17µV in MLL I and 64±16µV in standard limb lead (SLL) I (p<0.001). Similarly it was 118±22µV in MLL II and 100±27µV in SLL II. No statistically significant changes were seen in V1-V6 due to modification of the Wilson central terminal electrode positions. CONCLUSION: The modification of limb electrode placement leads to changes in the amplitude of the P waves in the MLL leads I and II compared to SLL leads I and II in healthy subjects. These changes may be of importance in the detection of atrial electrical activity.