Trimethylation of lysine residue K4 of histone H3 (H3K4me3) strongly correlates with active promoters for RNA polymerase II-transcribed genes. Several reader proteins, including the basal transcription factor TFIID, for this nucleosomal mark have been identified. Its TAF3 subunit specifically binds the H3K4me3 mark via its conserved plant homeodomain (PHD) finger. Here, we report the solution structure of the TAF3-PHD finger and its complex with an H3K4me3 peptide. Using a combination of NMR, mutagenesis, and affinity measurements, we reveal the structural basis of binding affinity, methylation-state specificity, and crosstalk with asymmetric dimethylation of R2. A unique local structure rearrangement in the K4me3-binding pocket of TAF3 due to a conserved sequence insertion underscores the requirement for cation-pi interactions by two aromatic residues. Interference by asymmetric dimethylation of arginine 2 suggests that a H3R2/K4 "methyl-methyl" switch in the histone code dynamically regulates TFIID-promoter association.

The genome of the filamentous fungus Aspergillus niger is rich in genes encoding pectinases, a broad class of enzymes that have been extensively studied due to their use in industrial applications. The sequencing of the A. niger genome provided more knowledge concerning the individual pectinolytic genes, but little is known about the regulatory genes involved in pectin degradation. Understanding regulation of the pectinolytic genes provides a tool to optimize the production of pectinases in this industrially important fungus. This study describes the identification and characterization of one of the activators of pectinase-encoding genes, RhaR. Inactivation of the gene encoding this regulator resulted in down-regulation of genes involved in the release of L-rhamnose from the pectin substructure rhamnogalacturonan I, as well as catabolism of this monosaccharide. The rhaR disruptant was unable to grow on L-rhamnose, but only a small reduction in growth on pectin was observed. This is likely caused by the presence of a second, so far unknown regulator that responds to the presence of D-galacturonic acid.

Leukocyte-associated immunoglobulinlike receptor-1 (LAIR-1), one of the most widely spread immune receptors, attenuates immune cell activation when bound to specific sites in collagen. The collagenbinding domain of LAIR-1 is homologous to that of glycoprotein VI (GPVI), a collagen receptor crucial for platelet activation. Because LAIR-1 and GPVI also display overlapping collagen-binding specificities, a common structural basis for collagen recognition would appear likely. Therefore, it is crucial to gain insight into the molecular interaction of both receptors with their ligand to prevent unwanted cross-reactions during therapeutic intervention. We determined the crystal structure of LAIR-1 and mapped its collagen-binding site by nuclear magnetic resonance (NMR) titrations and mutagenesis. Our data identify R59, E61, and W109 as key residues for collagen interaction. These residues are strictly conserved in LAIR-1 and GPVI alike; however, they are located outside the previously proposed GPVI collagenbinding site. Our data provide evidence for an unanticipated mechanism of collagen recognition common to LAIR-1 and GPVI. This fundamental insight will contribute to the exploration of specific means of intervention in collagen-induced signaling in immunity and hemostasis. (Blood.

DesK is a bacterial thermosensor protein involved in maintaining membrane fluidity in response to changes in environmental temperature. Most likely, the protein is activated by changes in membrane thickness, but the molecular mechanism of sensing and signaling is still poorly understood. Here we aimed to elucidate the mode of action of DesK by studying the so-called "minimal sensor DesK" (MS-DesK), in which sensing and signaling are captured in a single transmembrane segment. This simplified version of the sensor allows investigation of membrane thickness-dependent protein-lipid interactions simply by using synthetic peptides, corresponding to the membrane-spanning parts of functional and nonfunctional mutants of MS-DesK incorporated in lipid bilayers with varying thicknesses. The lipid-dependent behavior of the peptides was investigated by circular dichroism, tryptophan fluorescence, and molecular modeling. These experiments were complemented with in vivo functional studies on MS-DesK mutants. Based on the results, we constructed a model that suggests a new mechanism for sensing in which the protein is present as a dimer and responds to an increase in bilayer thickness by membrane incorporation of a C-terminal hydrophilic motif. This results in exposure of three serines on the same side of the transmembrane helices of MS-DesK, triggering a switching of the dimerization interface to allow the formation of a serine zipper. The final result is activation of the kinase state of MS-DesK.thermosensing | two-component system | lipid-protein interaction | helix-helix interaction | transmembrane helix dimerization

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Antimicrobial peptides are naturally occurring short amphipathic proteins, innate to the immune system and shown to induce selective lytic activity towards microbial pathogens. Protegrin-1 is an 18-residue, cationic, b-sheet antimicrobial peptide stabilized by two disulfide bonds. Concentration-dependent structural transformations of supported lipid bilayer patches as a result of peptide-membrane interactions have been visualized through the use of atomic force microscopy. A three-stage concentration-dependent transformation has been characterized, which begins with edge instability, followed by pore formation and worm-like micelle formation. This suggests that protegrin-1 acts to lower the line-energy at the edge of the bilayer. Membrane and lipid characteristics, including fluidity, charge and acyl chain length, can alter the activity of antimicrobial peptides. To identify the importance of both acyl-chain length and fluidity on the activity of protegrin-1, these two variables were decoupled. When the bilayers are examined at the same relative fluidity levels, they demonstrate the three-stage transformation observed on a fluid control bilayer, in contrast to the structural transformations that were observed in the gel phase bilayers. This suggests that fluidity exhibits a large influence on the transformations that occur as a result of protegrin-1. To examine the importance of acyl-chain length, the activity of antimicrobial peptides was studied using unsaturated bilayers. Our results indicate that the longer chain bilayers are less susceptible to disruption. This could be due to the hydrophobic mismatch between protegrin-1 and the thicker hydrophobic portion of longer chain lipid bilayers . These results highlight the importance of subtle membrane characteristics in the activity of antimicrobial peptides towards bacterial cells. Lipid bilayers with cholesterol are more accurate eukaryotic cell mimics and will allow examination of the selective preference of antimicrobial peptide activity.

M2 is a pH-dependent matrix protein from influenza virus widely known for its role in viral uncoating and the target of the amantadine flu drug that prevents proton transport. An additional role played by M2 relies on collective effects where clusters of M2-homotetramer proteins have been hypothesized to induce local membrane curvature effects (Rossman et al., Cell 142, pp. 902-913, 2010). We use molecular dynamics simulations with the MARTINI coarse grained parametrization to study a system comprised by two model membranes linked by an hour-glass shaped structure. We study the role played by M2 homotetramers in the scission step, leading to lipid-bilayer separation. The geometry of the model and the membrane-lipid interaction details are further used to adjust parameters on a Helfrich-like functional in order to propose a minimal model for viral scission at the mesoscopic scale.

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