Liquid-crystalline ordering of antimicrobial peptide-DNA complexes controls TLR9 activation.

Double-stranded DNA (dsDNA) can trigger the production of type I interferon (IFN) in plasmacytoid dendritic cells (pDCs) by binding to endosomal Toll-like receptor-9 (TLR9; refs 1-5). It is also known that the formation of DNA-antimicrobial peptide complexes can lead to autoimmune diseases via amplification of pDC activation. Here, by combining X-ray scattering, computer simulations, microscopy and measurements of pDC IFN production, we demonstrate that a broad range of antimicrobial peptides and other cationic molecules cause similar effects, and elucidate the criteria for amplification. TLR9 activation depends on both the inter-DNA spacing and the multiplicity of parallel DNA ligands in the self-assembled liquid-crystalline complex. Complexes with a grill-like arrangement of DNA at the optimum spacing can interlock with multiple TLR9 like a zipper, leading to multivalent electrostatic interactions that drastically amplify binding and thereby the immune response. Our results suggest that TLR9 activation and thus TLR9-mediated immune responses can be modulated deterministically.This work is supported by NSF grants DMR1411329 and DMR1106106, EU grants ARG-ERC-COLSTRUCTION 227758 and ITN-COMPLOIDS 234810, by the Herchel Smith Fund, and by the Slovenian Research Agency through Grant P1-0055, and the Swiss National Science Foundation (FN 310030-144072). X-ray research was conducted at Stanford Synchrotron Radiation Lightsource, SLAC National Laboratory, supported by the US DOE Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515, the Advanced Light Source, supported by the US DOE Office of Basic Energy Sciences under Contract No. DE-AC02-05CH11231, and at the UCLA CNSI.This is the author accepted manuscript. The final version is available from NPG at http://www.nature.com/nmat/journal/v14/n7/full/nmat4298.html

c-di-GMP modulates type IV MSHA pilus retraction and surface attachment in Vibrio cholerae.

Biofilm formation by Vibrio cholerae facilitates environmental persistence, and hyperinfectivity within the host. Biofilm formation is regulated by 3',5'-cyclic diguanylate (c-di-GMP) and requires production of the type IV mannose-sensitive hemagglutinin (MSHA) pilus. Here, we show that the MSHA pilus is a dynamic extendable and retractable system, and its activity is directly controlled by c-di-GMP. The interaction between c-di-GMP and the ATPase MshE promotes pilus extension, whereas low levels of c-di-GMP correlate with enhanced retraction. Loss of retraction facilitated by the ATPase PilT increases near-surface roaming motility, and impairs initial surface attachment. However, prolonged retraction upon surface attachment results in reduced MSHA-mediated surface anchoring and increased levels of detachment. Our results indicate that c-di-GMP directly controls MshE activity, thus regulating MSHA pilus extension and retraction dynamics, and modulating V. cholerae surface attachment and colonization

c-di-GMP modulates type IV MSHA pilus retraction and surface attachment in Vibrio cholerae

Biofilm formation by Vibrio cholerae facilitates environmental persistence, and hyperinfectivity within the host. Biofilm formation is regulated by 3’,5’-cyclic diguanylate (c-di-GMP) and requires production of the type IV mannose-sensitive hemagglutinin (MSHA) pilus. Here, we show that the MSHA pilus is a dynamic extendable and retractable system, and its activity is directly controlled by c-di-GMP. The interaction between c-di-GMP and the ATPase MshE promotes pilus extension, whereas low levels of c-di-GMP correlate with enhanced retrac- tion. Loss of retraction facilitated by the ATPase PilT increases near-surface roaming motility, and impairs initial surface attachment. However, prolonged retraction upon surface attach- ment results in reduced MSHA-mediated surface anchoring and increased levels of detachment. Our results indicate that c-di-GMP directly controls MshE activity, thus reg- ulating MSHA pilus extension and retraction dynamics, and modulating V. cholerae surface attachment and colonization

Studies of the gelsolin(150--169)-phosphatidylinositol 4,5-bisphosphate interaction and the filamentous actin-magnesium ion interaction

A 20-amino acid segment, G(150-169), of the actin-binding protein gelsolin is the binding site for both filamentous actin (F-actin) and the phospholipid phosphatidyl-inositol 4,5-bisphosphate (PIP\sb2). The goal of this work is to study the interaction between G(150-169) and PIP\sb2. Circular dichroism (CD) experiments show that G(150-169) undergoes a coil-helix transition in the presence of PIP\sb2 and in TFE-H\sb2O co-solvent. The TFE-induced helical structure of the peptide is studied as a model for the PIP\sb2-induced structure using two-dimensional NMR spectroscopy. The region from Pro-154 to Lys-166 is found to be helical, and the N-terminus forms a turn. SDS micelle solution is then used as a model lipid environment. The structure of G(150-169) in SDS micelle solution studied by NMR is somewhat different from that in TFE co-solvent, and the peptide is involved in hydrophobic interactions with SDS micelles. Finally, the direct interaction between G(150-169) and PIP\sb2 is studied using mixed SDS/PIP\sb2 micelle solution. The peptide structure is found to be similar to that observed in SDS micelle solution. The effect of the binding of G(150-169) on the esterphosphate groups of PIP\sb2 is measured by the \sp{31}P longitudinal relaxation time. The monoesterphosphates on the inositol ring are immobilized by the binding of the peptide, whereas the diesterphosphate is not at all affected. These results explain the specificity of the binding of PIP\sb2 to G(150-169). The interaction between Mg\sp{2+} and actin filaments is studied by \sp{25}Mg NMR. The dynamics of the Mg\sp{2+} ions in actin filament solution demonstrate that F-actin possesses polyelectrolyte properties. The displacement of Mg\sp{2+} by Co(NH\sb3)\sb6\sp{3+} suggests that the binding of Mg\sp{2+} to F-actin is non-specific

Fundamental processes of protein folding: Measuring the energetic balance between helix formation and hydrophobic interactions

Theories of protein folding often consider contributions from three fundamental elements: loops, hydrophobic interactions, and secondary structures. The pathway of protein folding, the rate of folding, and the final folded structure should be predictable if the energetic contributions to folding of these fundamental factors were properly understood. αtα is a helix-turn-helix peptide that was developed by de novo design to provide a model system for the study of these important elements of protein folding. Hydrogen exchange experiments were performed on selectively 15N-labeled αtα and used to calculate the stability of hydrogen bonds within the peptide. The resulting pattern of hydrogen bond stability was analyzed using a version of Lifson-Roig model that was extended to include a statistical parameter for tertiary interactions. This parameter, x, represents the additional statistical weight conferred upon a helical state by a tertiary contact. The hydrogen exchange data is most closely fit by the XHC model with an x parameter of 9.25. Thus the statistical weight of a hydrophobic tertiary contact is ∼5.8× the statistical weight for helix formation by alanine. The value for the x parameter derived from this study should provide a basis for the understanding of the relationship between hydrophobic cluster formation and secondary structure formation during the early stages of protein folding

Functional Specialization in Vibrio cholerae Diguanylate Cyclases: Distinct Modes of Motility Suppression and c-di-GMP Production.

Vibrio cholerae biofilm formation and associated motility suppression are correlated with increased concentrations of cyclic diguanylate monophosphate (c-di-GMP), which are in turn driven by increased levels and/or activity of diguanylate cyclases (DGCs). To further our understanding of how c-di-GMP modulators in V. cholerae individually and collectively influence motility with cellular resolution, we determined how DGCs CdgD and CdgH impact intracellular c-di-GMP levels, motility, and biofilm formation. Our results indicated that CdgH strongly influences swim speed distributions; cells in which cdgH was deleted had higher average swim speeds than wild-type cells. Furthermore, our results suggest that CdgD, rather than CdgH, is the dominant DGC responsible for postattachment c-di-GMP production in biofilms. Lipopolysaccharide (LPS) biosynthesis genes were found to be extragenic bypass suppressors of the motility phenotypes of strains ΔcdgD and ΔcdgH We compared the motility regulation mechanism of the DGCs with that of Gmd, an LPS O-antigen biosynthesis protein, and discovered that comodulation of c-di-GMP levels by these motility effectors can be positively or negatively cooperative rather than simply additive. Taken together, these results suggest that different environmental and metabolic inputs orchestrate DGC responses of V. cholerae via c-di-GMP production and motility modulation.IMPORTANCE Cyclic diguanylate monophosphate (c-di-GMP) is a broadly conserved bacterial signaling molecule that affects motility, biofilm formation, and virulence. Although it has been known that high intracellular concentrations of c-di-GMP correlate with motility suppression and biofilm formation, how the 53 predicted c-di-GMP modulators in Vibrio cholerae collectively influence motility is not understood in detail. Here we used a combination of plate assays and single-cell tracking methods to correlate motility and biofilm formation outcomes with specific enzymes involved in c-di-GMP synthesis in Vibrio cholerae, the causative agent of the disease cholera