Structural Basis of the Mechanisms of Action and Immunity of Lactococcin A, a Class IId Bacteriocin.

Lactococcin A (LcnA), a class IId bacteriocin, induces membrane leakage and cell death by specifically binding to the membrane receptor-mannose phosphotransferase system (man-PTS), as is the case for pediocin-like (class IIa) bacteriocins. The cognate immunity protein of bacteriocins, which protects the producer cell from its own bacteriocin, recognizes and binds to the bacteriocin-man-PTS complex, consequently blocking membrane leakage. We previously deciphered the mode of action and immunity of class IIa bacteriocins. Here, we determined the structure of the ternary complex of LcnA, LciA (i.e., the immunity protein), and its receptor, i.e., the man-PTS of Lactococcus lactis (ll-man-PTS). An external loop on the membrane-located component IIC of ll-man-PTS was found to prevent specific binding of the N-terminal region of LcnA to the site recognized by pediocin-like bacteriocins. Thus, the N-terminal ß-sheet region of LcnA recognized an adjacent site on the extracellular side of ll-man-PTS, with the LcnA C-terminal hydrophobic helix penetrating into the membrane. The cytoplasmic cleft formed within the man-PTS Core and Vmotif domains induced by embedded LcnA from the periplasmic side is adopted by the appropriate angle between helices H3 and H4 of the N terminus of LciA. The flexible C terminus of LciA then blocks membrane leakage. To summarize, our findings reveal the molecular mechanisms of action and immunity of LcnA and LciA, laying a foundation for further design of class IId bacteriocins. IMPORTANCE Class IId (lactococcin-like) bacteriocins and class IIa (pediocin-like) bacteriocins share a few similarities: (i) both induce membrane leakage and cell death by specifically binding the mannose phosphotransferase system (man-PTS) on their target cells, and (ii) cognate immunity proteins recognize and bind to the bacteriocin-man-PTS complex to block membrane leakage. However, class IId bacteriocins lack the "pediocin box" motif, which is typical of class IIa bacteriocins, and basically target only lactococcal cells; in contrast, class IIa bacteriocins target diverse bacterial cells, but not lactococcal cells. We previously solved the structure of class IIa bacteriocin-receptor-immunity ternary complex from Lactobacillus sakei. Here, we determined the structure of the ternary complex of class IId bacteriocin LcnA, its cognate immunity protein LciA, and its receptor, the man-PTS of Lactococcus lactis. By comparing the interactions between man-PTS and class IIa and class IId bacteriocins, this study affords some clues to better understand the specificity of bacteriocins targeting the mannose phosphotransferase system.

A novel serum spherical lectin from lamprey reveals a more efficient mechanism of immune initiation and regulation in jawless vertebrates.

The innate immune system is the body's first line of defense against pathogens and involves antibody and complement system-mediated antigen removal. Immune-response-related complement molecules have been identified in lamprey, and the occurrence of innate immune response via the mannose-binding lectin-associated serine proteases of the lectin cascade has been reported. We have previously shown that lamprey (Lampetra japonica) serum can efficiently and specifically eliminate foreign pathogens. Therefore, we aimed to understand the immune mechanism of lamprey serum in this study. We identified and purified a novel spherical lectin (LSSL) from lamprey serum. LSSL had two structural calcium ions coordinated with conserved amino acids, as determined through cryogenic electron microscopy. LSSL showed high binding capacity with microbial and mammalian glycans and demonstrated agglutination activity against bacteria. Phylogenetic analysis revealed that LSSL was transferred from phage transposons to the lamprey genome via horizontal gene transfer. Furthermore, LSSL was associated with mannose-binding lectin-associated serine protease 1 and promoted the deposition of the C3 fragment on the surface of target cells upon binding. These results led us to conclude that LSSL initiates and regulates agglutination, resulting in exogenous pathogen and tumor cell eradication. Our observations will give a greater understanding of the origin and evolution of the complement system in higher vertebrates and lead to the identification of novel immune molecules and pathways for defense against pathogens and tumor cells.

The crystal structure of Atg18 reveals a new binding site for Atg2 in Saccharomyces cerevisiae.

Macroautophagy (hereafter referred to as autophagy) is a highly conserved catabolic eukaryotic pathway that is critical for stress responses and homeostasis. Atg18, one of the core proteins involved in autophagy, belongs to the PROPPIN family and is composed of seven WD40 repeats. Together with Atg2, Atg18 participates in the elongation of phagophores and the recycling of Atg9 in yeast. Despite extensive studies on the PROPPIN family, the structure of Atg18 from Saccharomyces cerevisiae has not been determined. Here, we report the structure of ScAtg18 at a resolution of 2.8 Å. Based on bioinformatics and structural analysis, we found that the 7AB loop of ScAtg18 is extended in Atg18, in comparison to other members of the PROPPIN family. Genetic analysis revealed that the 7AB loop of ScAtg18 is required for autophagy. Biochemical and biophysical experiments indicated that the 7AB loop of ScAtg18 is critical for interaction with ScAtg2 and the recruitment of ScAtg2 to the autophagy-initiating site. Collectively, our results show that the 7AB loop of ScAtg18 is a new binding site for Atg2 and is of functional importance to autophagy.

Gold nanocage assemblies for selective second harmonic generation imaging of cancer cell.

Second harmonic generation (SHG) imaging using near infrared laser light is the key to improving penetration depths, leading to biological understanding. Unfortunately, currently SHG imaging techniques have limited capability due to the poor signal-to-noise ratio, resulting from the low SHG efficiency of available dyes. Targeted tumor imaging over nontargeted tissues is also a challenge that needs to be overcome. Driven by this need, in this study, the development of two-photon SHG imaging of live cancer cell lines selectively by enhancement of the nonlinear optical response of gold nanocage assemblies is reported. Experimental results show that two-photon scattering intensity can be increased by few orders of magnitude by just developing nanoparticle self-assembly. Theoretical modeling indicates that the field enhancement values for the nanocage assemblies can explain, in part, the enhanced nonlinear optical properties. Our experimental data also show that A9 RNA aptamer conjugated gold nanocage assemblies can be used for targeted SHG imaging of the LNCaP prostate cancer cell line. Experimental results with the HaCaT normal skin cell lines show that bioconjugated nanocage-based assemblies demonstrate SHG imaging that is highly selective and will be able to distinguish targeted cancer cell lines from other nontargeted cell types. After optimization, this reported SHG imaging assay could have considerable application for biology.

Plasmonic resonances in self-assembled reduced symmetry gold nanorod structures.

Self-assembled plasmonic Dolmen structures consisting of small gold nanorods (length = 50 nm and diameter = 20 nm) with a few nanometer gaps are observed to show coherent effects of super-radiance and characteristics of Fano resonance due to the significantly reduced symmetry of the structure. Relative to previous larger structures from top-down electron-beam lithography, the single crystallinity and atomically smooth surfaces of these self-assembled plasmonic structures result in 50% narrower resonances, and the small gaps with associated strong coupling enable observation of multiple dark and bright modes. By tilting the cap monomer with respect to the base dimer an order of magnitude increase in E-field enhancement at the Fano dip is obtained. In addition, a spectrally broad mode is observed indicating the strong impact of the geometry of the structure on the nature of coupled modes. The highly localized electric near-fields in the gaps will enable strong light matter interactions and the narrow resonances will be useful for improved figure of merits in inexpensive chemical and biosensing.

Plasmon-induced transparency in the visible region via self-assembled gold nanorod heterodimers.

The phenomenon of plasmon-induced transparency holds immense potential for high sensitivity sensors and optical information processing due to the extreme dispersion and slowing of light within a narrow spectral window. Unfortunately plasmonic metamaterials demonstrating this effect has been restricted to infrared and greater wavelengths due to requisite precision in structure fabrication. Here we report a novel metamaterial synthesized by bottom-up self-assembly of gold nanorods. The small dimensions (≤ 50/20 nm, length/diameter), atomically smooth surfaces, and nanometer resolution enable the first demonstration of plasmon-induced transparency at visible wavelengths. The slow-down factors within the reduced symmetry heterodimer cluster are comparable to longer wavelength counterparts. The inherent spectral tunability and facile large-scale integration afforded by self-assembled metamaterials will open a new paradigm for physically realizable on-chip photonic device designs.

Architecture of the bacteriophage lambda tail.

Bacteriophage lambda has a double-stranded DNA genome and a long, flexible, non-contractile tail encoded by a contiguous block of 11 genes downstream of the head genes. The tail allows host recognition and delivery of viral DNA from the head shell to the cytoplasm of the infected cell. Here, we present a high-resolution structure of the tail complex of bacteriophage lambda determined by cryoelectron microscopy. Most component proteins of the lambda tail were determined at the atomic scale. The structure sheds light on the molecular organization of the extensively studied tail of bacteriophage lambda.

Pronounced effects of anisotropy on plasmonic properties of nanorings fabricated by electron beam lithography.

Gold nanoring dimers were fabricated via EBL with dimensions of 127.6 ± 2.5 and 57.8 ± 2.3 nm for the outer and inner diameters, respectively, with interparticle separations ranging from 17.8 ± 3.4 to 239.2 ± 3.7 nm. The coupling between the inner and outer surfaces of a single nanoring renders it very sensitive to any anisotropy. We found that anisotropy in the particle geometry and anisotropy introduced by the substrate combine to create very unique spectral features in this system.

Nicking mechanism underlying the DNA phosphorothioate-sensing antiphage defense by SspE.

DNA phosphorothioate (PT) modification, with a nonbridging phosphate oxygen substituted by sulfur, represents a widespread epigenetic marker in prokaryotes and provides protection against genetic parasites. In the PT-based defense system Ssp, SspABCD confers a single-stranded PT modification of host DNA in the 5'-CPSCA-3' motif and SspE impedes phage propagation. SspE relies on PT modification in host DNA to exert antiphage activity. Here, structural and biochemical analyses reveal that SspE is preferentially recruited to PT sites mediated by the joint action of its N-terminal domain (NTD) hydrophobic cavity and C-terminal domain (CTD) DNA binding region. PT recognition enlarges the GTP-binding pocket, thereby increasing GTP hydrolysis activity, which subsequently triggers a conformational switch of SspE from a closed to an open state. The closed-to-open transition promotes the dissociation of SspE from self PT-DNA and turns on the DNA nicking nuclease activity of CTD, enabling SspE to accomplish self-nonself discrimination and limit phage predation, even when only a small fraction of modifiable consensus sequences is PT-protected in a bacterial genome.

Structural and functional insight into the effect of AFF4 dimerization on activation of HIV-1 proviral transcription.

Super elongation complex (SEC) is a positive regulator of RNA polymerase II, which is required for HIV-1 proviral transcription. AFF1/4 is the scaffold protein that recruits other components of SEC and forms dimer depending on its THD domain (TPRL with Handle Region Dimerization Domain). Here we report the crystal structure of the human AFF4-THD at the resolution of 2.4 Å. The α4, α5, and α6 of one AFF4-THD mediate the formation of a dimer and pack tightly against the equivalent part of the second molecule in the dimer of AFF-THD. Mutagenesis analysis revealed that single mutations of either Phe1014 or Tyr1096 of AFF4 to alanine impair the formation of the AFF4 dimer. In addition, transactivation assay also indicated that Phe1014 and Tyr1096 of AFF4 are critical to the transactivation activity of AFF4. Interestingly, the corresponding residues Phe1063 and Tyr1145 in AFF1 have an effect on the transactivation of HIV-1 provirus. However, such mutations of AFF1/4 have no effect on the interaction of AFF1/4 with other subunits of the SEC. Together, our data demonstrated that the dimerization of AFF1/4 is essential to transactivation of HIV-1 provirus.

Inward-facing conformation of l-ascorbate transporter suggests an elevator mechanism.

Various bacteria can ferment vitamin C (l-ascorbate) under anaerobic conditions via the phosphoenolpyruvate-dependent phosphotransferase system (PTS). The PTSasc system is composed of two soluble energy-coupling proteins (EI and HPr) and an enzyme II complex (EIIA, EIIB, and EIIC) for the anaerobic uptake of ascorbate and its phosphorylation to l-ascorbate 6-phosphate in vivo. Crystal structures of the ascorbate-bound EIIC component from Escherichia coli are available in outward-open and occluded conformations, suggesting a possible elevator mechanism of membrane transport. Despite these advances, it remains unclear how EIIC actually transports the substrate across the membrane and interacts with EIIB, which transfers its phosphate group to the EIIC-embedding ascorbate. Here, we present the crystal structure of the EIICasc component from Pasteurella multocida in the inward-facing conformation. By comparing three conformational states, we confirmed the original proposed model: the ascorbate translocation can be achieved by a rigid-body movement of the substrate-binding core domain relative to the V motif domain, which brings along the transmembrane helices TM2 and TM7 of the V motif domain to undergo a winding at the pivotal positions. Together with an in vivo transport assay, we completed the picture of the transport cycle of the ascorbate superfamily of membrane-spanning EIIC components of the PTS system.

Computational model of hepatitis B virus DNA polymerase: molecular dynamics and docking to understand resistant mutations.

Hepatitis B virus (HBV) DNA polymerase (HDP) is a pharmacological target of intense interest. Of the seven agents approved in USA for the treatment of HBV infections, five are HDP inhibitors. However, resistance development against HDP inhibitors, such as lamivudine and adefovir, has severely hurt their efficacy to treat HBV. As a step toward understanding the mechanism of resistance development and for gaining detailed insights about the active site of the enzyme, we have built a homology model of HDP which is an advance over previously reported ones. Validation using various techniques, including PROSTAT, PROCHECK, and Verify-3D profile, proved the model to be stereochemically significant. The stability of the model was studied using a 5 ns molecular dynamics simulation. The model was found to be sufficiently stable after the initial 2.5 ns with overall root mean squared deviation (RMSD) of 4.13 A. The homology model matched the results of experimental mutation studies of HDP reported in the literature, including those of antiviral-resistant mutations. Our model suggests the significant role of conserved residues, such as rtLys32, in binding of the inhibitors, contrary to previous studies. The model provides an explanation for the inactivity of some anti-HIV molecules which are inactive against HDP. Conformational changes which occurred in certain binding pocket amino acids helped to explain the better binding of some of the inhibitors in comparison to the substrates.

Solvation in supercritical water.

Solvation in supercritical water under equilibrium and nonequilibrium conditions is studied via molecular dynamics simulations. The influence of solute charge distributions and solvent density on the solvation structures and dynamics is examined with a diatomic probe solute molecule. It is found that the solvation structure varies dramatically with the solute dipole moment, especially in low-density water, in accord with many previous studies on ion solvation. This electrostrictive effect has important consequences for solvation dynamics. In the case of a nonequilibrium solvent relaxation, if there are sufficiently many water molecules close to the solute at the outset of the relaxation, the solvent response measured as a dynamic Stokes shift is almost completely governed by inertial rotations of these water molecules. By contrast, in the opposite case of a low local solvent density near the solute, not only rotations but also translations of water molecules play an important role in solvent relaxation dynamics. The applicability of a linear response is found to be significantly restricted at low water densities.