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Purpose: Vemurafenib received approval for treatment of BRAF V600 variation metastatic melanoma in August 2011. This study analyzed Vemurafenib-related adverse events (AEs) to detect and characterize relevant safety signals using the real-word-data through the Food and Drug Administration Adverse Event Reporting System (FAERS). Methods: Disproportionality analyses, including the reporting odds ratio (ROR), the healthcare products regulatory agency (MHRA), the Bayesian confidence propagation neural network (BCPNN), and the multiitem gamma Poisson shrinker (MGPS) algorithms, were applied to quantify the signals of vemurafenib-related AEs. Results: Out of 8,042,244 reports gathered from the FAERS, 9554 reports of vemurafenib as the ‘primary suspected (PS)’ AEs were recognized. Vemurafenib-induced AEs occurrence targeted 23 system organ class (SOC). A total of 138 significant disproportionality PTs was simultaneously reserved according to the four algorithms. Unexpected significant AEs such as sarcoidosis and kidney fibrosis might also occur. The median onset time of vemurafenib-related AEs was 26 days (interquartile range [IQR] 8-97 days), and most of the cases occurred within the first one and two months after vemurafenib initiation. Conclusion: Our study detected potential new AEs signals and might provide powerful support for clinical monitoring and risk identification of vemurafenib.

Lipoproteins in the outer membrane of Gram-negative bacteria are involved in various vital physiological activities, including multidrug resistance. Synthesized in the cytoplasm and matured in the inner membrane, lipoproteins must be transported to the outer membrane through the Lol pathway mediated by the ATP-binding cassette transporter LolCDE in the inner membrane via an unknown mechanism. Here, we report cryo-EM structures of Escherichia coli LolCDE in apo, lipoprotein-bound, LolA-bound, ADP-bound and AMP-PNP-bound states at a resolution of 3.2–3.8 A, covering the complete lipoprotein transport cycle. Mutagenesis and in vivo viability assays verify features of the structures and reveal functional residues and structural characteristics of LolCDE. The results provide insights into the mechanisms of sorting and transport of outer-membrane lipoproteins and may guide the development of novel therapies against multidrug-resistant Gram-negative bacteria. Cryo-EM structures of the E. coli ABC transporter LolCDE in different functional states reveal mechanism of lipoprotein transport to the outer membrane of Gram-negative bacteria.

BACH1 (Brca1-Associated C-terminal Helicase) is an important DNA damage response factor, which is involved in DNA damage repair and maintenance of genomic stability. In this study, by using tandem protein affinity purification, we have identified BCLAF1 as a novel functional partner of BACH1. BCLAF1 constitutively interacts with BACH1 regardless of DNA damage. However, in response to DNA damage, along with BACH1, BCLAF1 is recruited to the DNA damage sites and the recruitment of BCLAF1 was regulated by BACH1 and BRCA1. Interestingly, BCLAF1 deficient cells are deficient for DSB-initiated HR, but RAD51 foci formation is intact following IR treatment. Taken together, these findings reveal that BCLAF1 is a functional binding partner of BACH1 playing a key role in DNA damage response.

Sepsis, a widely recognized disease, is characterized by multiple pathogen infections. Therefore, it is imperative to develop methods that can efficiently identify and neutralize pathogen species. Phage cocktail therapy utilizes the host specificity of phages to adapt to infect resistant bacteria. However, its low sterilization stability efficiency and lack of imaging units seriously restrict its application. Here, a novel strategy combining the aggregation-induced emission photosensitizer (AIE-PS) TBTCP-PMB with phages through a nucleophilic substitution reaction between benzyl bromide and sulfhydryl groups to remove pathogenic bacteria for sepsis treatment is proposed. This strategy retains the phage's host specificity while possessing AIE-PS characteristics with a fluorescence imaging function and reactive oxygen species (ROS) for detecting and sterilizing bacteria. This synergetic strategy combining phage cocktail therapy and photodynamic therapy (PDT) shows a strong "1 + 1 2" bactericidal efficacy and superior performance in sepsis mouse models with good biocompatibility. Furthermore, the strategy can quickly diagnose blood infections of clinical blood samples. This simple and accurate strategy provides a promising therapeutic platform for rapid pathogen detection and point-of-care diagnosis. Moreover, it presents a new method for expanding the library of antibacterial drugs to develop new strain identification and improve infectious disease treatment, thereby demonstrating strong translational potential.

Lipopolysaccharides (LPS) of Gram-negative bacteria are critical for the defence against cytotoxic substances and must be transported from the inner membrane (IM) to the outer membrane (OM) through a bridge formed by seven membrane proteins (LptBFGCADE). The IM component LptB2FG powers the process through a yet unclarified mechanism. Here we report three high-resolution cryo-EM structures of LptB2FG alone and complexed with LptC (LptB2FGC), trapped in either the LPS- or AMP-PNP-bound state. The structures reveal conformational changes between these states and substrate binding with or without LptC. We identify two functional transmembrane arginine-containing loops interacting with the bound AMP-PNP and elucidate allosteric communications between the domains. AMP-PNP binding induces an inward rotation and shift of the transmembrane helices of LptFG and LptC to tighten the cavity, with the closure of two lateral gates, to eventually expel LPS into the bridge. Functional assays reveal the functionality of the LptF and LptG periplasmic domains. Our findings shed light on the LPS transport mechanism.

The highly asymmetric outer membrane of Gram-negative bacteria functions in the defense against cytotoxic substances, such as antibiotics. The Mla pathway maintains outer membrane lipid asymmetry by transporting phospholipids between the inner and outer membranes. It comprises six Mla proteins, MlaFEDBCA, including the ABC transporter MlaFEDB, which functions via an unknown mechanism. Here we determine cryo-EM structures of Escherichia coli MlaFEDB in an apo state and bound to phospholipid, ADP or AMP-PNP to a resolution of 3.3–4.1 A and establish a proteoliposome-based transport system that includes MlaFEDB, MlaC and MlaA–OmpF to monitor the transport direction of phospholipids. In vitro transport assays and in vivo membrane permeability assays combined with mutagenesis identify functional residues that not only recognize and transport phospholipids but also regulate the activity and structural stability of the MlaFEDB complex. Our results provide mechanistic insights into the Mla pathway, which could aid antimicrobial drug development. Cryo-EM structures, along with transport assays with proteoliposomes and bacterial growth analyses, show that the Escherichia coli Mla complex mediates ATP-dependent retrograde transport of phospholipids.

Small molecular PD‐1 inhibitors are lacking in current immuno‐oncology clinic. PD‐1/PD‐L1 antibody inhibitors currently approved for clinical usage block interaction between PD‐L1 and PD‐1 to enhance cytotoxicity of CD8+ cytotoxic T lymphocyte (CTL). Whether other steps along the PD‐1 signaling pathway can be targeted remains to be determined. Here, we report that methylene blue (MB), an FDA‐approved chemical for treating methemoglobinemia, potently inhibits PD‐1 signaling. MB enhances the cytotoxicity, activation, cell proliferation, and cytokine‐secreting activity of CTL inhibited by PD‐1. Mechanistically, MB blocks interaction between Y248‐phosphorylated immunoreceptor tyrosine‐based switch motif (ITSM) of human PD‐1 and SHP2. MB enables activated CTL to shrink PD‐L1 expressing tumor allografts and autochthonous lung cancers in a transgenic mouse model. MB also effectively counteracts the PD‐1 signaling on human T cells isolated from peripheral blood of healthy donors. Thus, we identify an FDA‐approved chemical capable of potently inhibiting the function of PD‐1. Equally important, our work sheds light on a novel strategy to develop inhibitors targeting PD‐1 signaling axis., PD‐1 inhibitors that are currently used in the clinic exhibit toxicity and limited patient response rate. This study identifies methylene blue (MB), an FDA‐approved chemical for treating methemoglobinemia, as a new potent PD‐1 inhibitor.

The asymmetric phospholipid outer membrane (OM) of Gram-negative bacteria serves as the first line of defense against cytotoxic substances such as antibiotics. The Mla pathway is known to maintain the lipid asymmetry of the OM by transporting phospholipids between the inner and outer membranes. Six Mla proteins MlaFEDBCA are involved, with the ABC transporter MlaFEDB acts through a mechanism yet to be elucidated. Here we determine cryo-EM structures of MlaFEDB in apo, phospholipid-, ADP- or AMP-PNP-bound state to 3.3-3.75 Å resolution and establish a proteoliposome-based transport system containing MlaFEDB, MlaC and MlaA/OmpF to reveal the transport direction of phospholipids. Mutagenetic in vitro transport assays and in vivo sensitivity assays reveal functional residues which recognize and transport phospholipids as well as regulate the activity and structural stability of the MlaFEDB complex. Our work provides molecular basis for understanding the mechanism of the Mla pathway which could be targeted for antimicrobial drug development.

Lipopolysaccharide (LPS) is an important component of the outer membrane (OM) of Gram-negative bacteria, playing essential roles in protecting bacteria from harsh environments, in drug resistance and in pathogenesis. LPS is synthesized in the cytoplasm and translocated to the periplasmic side of the inner membrane (IM), where it matures. Seven lipopolysaccharide transport proteins, LptA-G, form a trans‑envelope complex that is responsible for LPS extraction from the IM and transporting it across the periplasm to the OM. The LptD/E of the complex transports LPS across the OM and inserts it into the outer leaflet of the OM. In this review we focus upon structural and mechanistic studies of LPS transport proteins, with a particular focus upon the LPS ABC transporter LptB2FG. This ATP binding cassette transporter complex consists of twelve transmembrane segments and has a unique mechanism whereby it extracts LPS from the periplasmic face of the IM through a pair of lateral gates and then powers trans‑periplasmic transport to the OM through a slide formed by either of the periplasmic domains of LptF or LptG, LptC, LptA and the N-terminal domain of LptD. The structural and functional studies of the seven lipopolysaccharide transport proteins provide a platform to explore the unusual mechanisms of LPS extraction, transport and insertion from the inner membrane to the outer membrane. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.

Inflammatory bowel diseases (IBD), composed mainly of Crohn’s disease (CD) and ulcerative colitis (UC), are strongly implicated in the development of intestinal inflammation lesions. Its exact etiology and pathogenesis are still undetermined. Recently accumulating evidence supports that group 3 innate lymphoid cells (ILC3) are responsible for gastrointestinal mucosal homeostasis through moderate generation of IL-22, IL-17, and GM-CSF in the physiological state. ILC3 contribute to the progression and aggravation of IBD while both IL-22 and IL-17, along with IFN-γ, are overexpressed by the dysregulation of NCR− ILC3 or NCR+ ILC3 function and the bias of NCR+ ILC3 towards ILC1 as well as regulatory ILC dysfunction in the pathological state. Herein, we feature the group 3 innate lymphoid cells’ development, biological function, maintenance of gut homeostasis, mediation of IBD occurrence, and potential application to IBD therapy.

Significance Gut bacteria play a key role in health and disease, but the molecular mechanisms underpinning their interaction with the host remain elusive. The serine-rich repeat proteins (SRRPs) are a family of adhesins identified in many Gram-positive pathogenic bacteria. We previously showed that beneficial bacterial species found in the gut also express SRRPs and that SRRP was required for the ability of Lactobacillus reuteri strain to colonize mice. Here, our structural and biochemical data reveal that L. reuteri SRRP adopts a β-solenoid fold not observed in other structurally characterized SRRPs and functions as an adhesin via a pH-dependent mechanism, providing structural insights into the role of these adhesins in biofilm formation of gut symbionts., Lactobacillus reuteri, a Gram-positive bacterial species inhabiting the gastrointestinal tract of vertebrates, displays remarkable host adaptation. Previous mutational analyses of rodent strain L. reuteri 100-23C identified a gene encoding a predicted surface-exposed serine-rich repeat protein (SRRP100-23) that was vital for L. reuteri biofilm formation in mice. SRRPs have emerged as an important group of surface proteins on many pathogens, but no structural information is available in commensal bacteria. Here we report the 2.00-Å and 1.92-Å crystal structures of the binding regions (BRs) of SRRP100-23 and SRRP53608 from L. reuteri ATCC 53608, revealing a unique β-solenoid fold in this important adhesin family. SRRP53608-BR bound to host epithelial cells and DNA at neutral pH and recognized polygalacturonic acid (PGA), rhamnogalacturonan I, or chondroitin sulfate A at acidic pH. Mutagenesis confirmed the role of the BR putative binding site in the interaction of SRRP53608-BR with PGA. Long molecular dynamics simulations showed that SRRP53608-BR undergoes a pH-dependent conformational change. Together, these findings provide mechanistic insights into the role of SRRPs in host–microbe interactions and open avenues of research into the use of biofilm-forming probiotics against clinically important pathogens.

Lactobacillus reuteri, a Gram-positive bacterial species inhabiting the gastrointestinal tract of vertebrates, displays remarkable host adaptation. Previous mutational analyses of rodent strain L. reuteri 100-23C identified a gene encoding a predicted surface-exposed serine-rich repeat protein (SRRP100-23) that was vital for L. reuteri biofilm formation in mice. SRRPs have emerged as an important group of surface proteins on many pathogens, but no structural information is available in commensal bacteria. Here we report the 2.00-A and 1.92-A crystal structures of the binding regions (BRs) of SRRP100-23 and SRRP53608 from L. reuteri ATCC 53608, revealing a unique β-solenoid fold in this important adhesin family. SRRP53608-BR bound to host epithelial cells and DNA at neutral pH and recognized polygalacturonic acid (PGA), rhamnogalacturonan I, or chondroitin sulfate A at acidic pH. Mutagenesis confirmed the role of the BR putative binding site in the interaction of SRRP53608-BR with PGA. Long molecular dynamics simulations showed that SRRP53608-BR undergoes a pH-dependent conformational change. Together, these findings provide mechanistic insights into the role of SRRPs in host–microbe interactions and open avenues of research into the use of biofilm-forming probiotics against clinically important pathogens.

The crystal structure of the major outer membrane protein U (OmpU) from Vibrio cholerae has been determined, which exhibits distinct structural features from other structurally characterized porins and provides the structural basis for the bacterial invasion and phage recognition., Vibrio cholerae causes a severe disease that kills thousands of people annually. The outer membrane protein OmpU is the most abundant outer membrane protein in V. cholerae, and has been identified as an important virulence factor that is involved in host-cell interaction and recognition, as well as being critical for the survival of the pathogenic V. cholerae in the host body and in harsh environments. The mechanism of these processes is not well understood owing to a lack of the structure of V. cholerae OmpU. Here, the crystal structure of the V. cholerae OmpU trimer is reported to a resolution of 2.2 Å. The protomer forms a 16-β-stranded barrel with a noncanonical N-terminal coil located in the lumen of the barrel that consists of residues Gly32–Ser42 and is observed to participate in forming the second gate in the pore. By mapping the published functional data onto the OmpU structure, the OmpU structure reinforces the notion that the long extracellular loop L4 with a β-hairpin-like motif may be critical for host-cell binding and invasion, while L3, L4 and L8 are crucially implicated in phage recognition by V. cholerae.

Arenaviruses cause severe hemorrhagic fever diseases in humans, and there are limited preventative and therapeutic measures against these diseases. Previous structural and functional analyses of arenavirus nucleoproteins (NPs) revealed a conserved DEDDH exoribonuclease (RNase) domain that is important for type I interferon (IFN) suppression, but the biological roles of the NP RNase in viral replication and host immune suppression have not been well characterized. Infection of guinea pigs with Pichinde virus (PICV), a prototype arenavirus, can serve as a surrogate small animal model for arenavirus hemorrhagic fevers. In this report, we show that mutation of each of the five RNase catalytic residues of PICV NP diminishes the IFN suppression activity and slightly reduces the viral RNA replication activity. Recombinant PICVs with RNase catalytic mutations can induce high levels of IFNs and barely grow in IFN-competent A549 cells, in sharp contrast to the wild-type (WT) virus, while in IFN-deficient Vero cells, both WT and mutant viruses can replicate at relatively high levels. Upon infection of guinea pigs, the RNase mutant viruses stimulate strong IFN responses, fail to replicate productively, and can become WT revertants. Serial passages of the RNase mutants in vitro can also generate WT revertants. Thus, the NP RNase function is essential for the innate immune suppression that allows the establishment of a productive early viral infection, and it may be partly involved in the process of viral RNA replication. IMPORTANCE Arenaviruses, such as Lassa, Lujo, and Machupo viruses, can cause severe and deadly hemorrhagic fever diseases in humans, and there are limited preventative and treatment options against these diseases. Development of broad-spectrum antiviral drugs depends on a better mechanistic understanding of the conserved arenavirus proteins in viral infection. The nucleoprotein (NPs) of all arenaviruses carry a unique exoribonuclease (RNase) domain that has been shown to be critical for the suppression of type I interferons. However, the functional roles of the NP RNase in arenavirus replication and host immune suppression have not been characterized systematically. Using a prototype arenavirus, Pichinde virus (PICV), we characterized the viral growth and innate immune suppression of recombinant RNase-defective mutants in both cell culture and guinea pig models. Our study suggests that the NP RNase plays an essential role in the suppression of host innate immunity, and possibly in viral RNA replication, and that it can serve as a novel target for developing antiviral drugs against arenavirus pathogens.

We describe methods to express the nucleoprotein (NP) of Lassa fever virus (LASV) in E. coli, to purify and crystallize it using the sitting-drop vapor diffusion method. The crystals were screened using Rigaku micro-007 X-ray generator and a dataset was collected at a resolution of 2.36 Å. The crystals belong to space group P3, with the unit cell parameters a = b = 176.35 Å, c = 56.40 Å, α = β = 90°, and γ = 120°. Using the X-ray diffraction method, we constructed a three-dimensional structure of the LASV NP that should aid in the development of novel therapeutic strategies against this virus, for which vaccine and effective treatment modalities are currently unavailable.

Outer membrane β-barrel proteins play important roles in importing nutrients, exporting wastes and conducting signals in Gram-negative bacteria, mitochondria and chloroplasts. The outer membrane proteins are inserted and assembled into the outer membrane by OMP85 family proteins. In Escherichia coli , the b-barrel assembly machinery (BAM) contains four lipoproteins BamB, BamC, BamD and BamE, and one outer membrane protein BamA, forming a "top hat"-like structure. Structural and functional studies of the E. coli BAM machinery have revealed that the rotation of periplasmic ring may trigger the barrel b1C-b6C scissor-like movement that promote the unfolded outer membrane protein insertion without using ATP. Here we report the BamA C-terminal barrel structure of Salmonella enterica Typhimurium str. LT2 and functional assays, which reveal that the BamA's C-terminal residue Trp, the b16C strand of the barrel and the periplasmic turns are critical for the functionality of BamA. These findings indicate that the unique b16C and the periplasmic turns of BamA are important for the out membrane insertion and assembly. The periplasmic turns might mediate the rotation of the periplasmic ring to the scissor-like movement of BamA b1C-b6C, triggering the outer membrane protein insertion. These results are important for understanding the outer membrane protein insertion in Gram-negative bacteria, as well as in mitochondria and chloroplasts.

Lipopolysaccharide (LPS) is the main component of the outer membrane of Gram-negative bacteria, which plays an essential role in protecting the bacteria from harsh conditions and antibiotics. LPS molecules are transported from the inner membrane to the outer membrane by seven LPS transport proteins. LptB is vital in hydrolyzing ATP to provide energy for LPS transport, however this mechanism is not very clear. Here we report wild-type LptB crystal structure in complex with ATP and Mg(2+), which reveals that its structure is conserved with other nucleotide-binding proteins (NBD). Structural, functional and electron microscopic studies demonstrated that the ATP binding residues, including K42 and T43, are crucial for LptB's ATPase activity, LPS transport and the vitality of Escherichia coli cells with the exceptions of H195A and Q85A; the H195A mutation does not lower its ATPase activity but impairs LPS transport, and Q85A does not alter ATPase activity but causes cell death. Our data also suggest that two protomers of LptB have to work together for ATP hydrolysis and LPS transport. These results have significant impacts in understanding the LPS transport mechanism and developing new antibiotics.

Lipopolysaccharide (LPS) is essential for most Gram-negative bacteria and has crucial roles in protection of the bacteria from harsh environments and toxic compounds, including antibiotics. Seven LPS transport proteins (that is, LptA–LptG) form a trans-envelope protein complex responsible for the transport of LPS from the inner membrane to the outer membrane, the mechanism for which is poorly understood. Here we report the first crystal structure of the unique integral membrane LPS translocon LptD–LptE complex. LptD forms a novel 26-stranded β-barrel, which is to our knowledge the largest β-barrel reported so far. LptE adopts a roll-like structure located inside the barrel of LptD to form an unprecedented two-protein ‘barrel and plug’ architecture. The structure, molecular dynamics simulations and functional assays suggest that the hydrophilic O-antigen and the core oligosaccharide of the LPS may pass through the barrel and the lipid A of the LPS may be inserted into the outer leaflet of the outer membrane through a lateral opening between strands β1 and β26 of LptD. These findings not only help us to understand important aspects of bacterial outer membrane biogenesis, but also have significant potential for the development of novel drugs against multi-drug resistant pathogenic bacteria.\ud \ud

Lipopolysaccharide (LPS) is an essential component of the outer membranes (OM) of most Gram-negative bacteria, which plays a crucial role in protection of the bacteria from toxic compounds and harsh conditions. The LPS is biosynthesized at the cytoplasmic side of inner membrane (IM), and then transported across the aqueous periplasmic compartment and assembled correctly at the outer membrane. This process is accomplished by seven LPS transport proteins (LptA-G), but the transport mechanism remains poorly understood. Here, we present findings by pull down assays in which the periplasmic component LptA interacts with both the IM complex LptBFGC and the OM complex LptDE in vitro, but not with complex LptBFG. Using purified Lpt proteins, we have successfully reconstituted the seven transport proteins as a complex in vitro. In addition, the LptC may play an essential role in regulating the conformation of LptBFG to secure the lipopolysaccharide from the inner membrane. Our results contribute to the understanding of lipopolysaccharide transport mechanism and will provide a platform to study the detailed mechanism of the LPS transport in vitro.

Background: Arenaviral nucleoproteins play a critical role in innate immune suppression. Results: Structures of Lassa nucleoprotein in complex with triphosphate dsRNA and Tacaribe virus nucleoprotein have been determined. Conclusion: Both Lassa and Tacaribe nucleoproteins can strongly inhibit IFN-β production by degrading immune-stimulatory dsRNA. Significance: A unique immune suppression mode of arenaviral nucleoproteins has been revealed., A hallmark of severe Lassa fever is the generalized immune suppression, the mechanism of which is poorly understood. Lassa virus (LASV) nucleoprotein (NP) is the only known 3′-5′ exoribonuclease that can suppress type I interferon (IFN) production possibly by degrading immune-stimulatory RNAs. How this unique enzymatic activity of LASV NP recognizes and processes RNA substrates is unknown. We provide an atomic view of a catalytically active exoribonuclease domain of LASV NP (LASV NP-C) in the process of degrading a 5′ triphosphate double-stranded (ds) RNA substrate, a typical pathogen-associated molecular pattern molecule, to induce type I IFN production. Additionally, we provide for the first time a high-resolution crystal structure of an active exoribonuclease domain of Tacaribe arenavirus (TCRV) NP. Coupled with the in vitro enzymatic and cell-based interferon suppression assays, these structural analyses strongly support a unified model of an exoribonuclease-dependent IFN suppression mechanism shared by all known arenaviruses. New knowledge learned from these studies should aid the development of therapeutics against pathogenic arenaviruses that can infect hundreds of thousands of individuals and kill thousands annually.

This work was supported by the Medical Research Council (grant no. G1100110/1 to C.D.) and the Wellcome Trust (career development fellowship WT083501MA to C.D. and program grant 079810 and project grant 091783 to R.M.E.). R.M.E. is a Wellcome Trust senior investigator. Schmallenberg virus (SBV), a newly emerged orthobunyavirus (family Bunyaviridae), has spread rapidly across Europe and has caused congenital abnormalities in the offspring of cattle, sheep, and goats. Like other orthobunyaviruses, SBV contains a tripartite negative-sense RNA genome that encodes four structural and two nonstructural proteins. The nucleoprotein (N) encapsidates the three viral genomic RNA segments and plays a crucial role in viral RNA transcription and replication. Here we report the crystal structure of the bacterially expressed SBV nucleoprotein to a 3.06-angstrom resolution. The protomer is composed of two domains (N-terminal and C-terminal domains) with flexible N-terminal and C-terminal arms. The N protein has a novel fold and forms a central positively charged cleft for genomic RNA binding. The nucleoprotein purified under native conditions forms a tetramer, while the nucleoprotein obtained following denaturation and refolding forms a hexamer. Our structural and functional analyses demonstrate that both N-terminal and C-terminal arms are involved in N-N interaction and oligomerization and play an essential role in viral RNA synthesis, suggesting a novel mechanism for viral RNA encapsidation and transcription. Publisher PDF

The outer membrane (OM) of Gram-negative bacteria is a unique asymmetric lipid bilayer in which the outer leaflet is composed of lipopolysaccharide (LPS) and the inner leaflet is formed by glycerophospholipid (GPL). The OM plays a fundamental role in protecting Gram-negative bacteria from harsh environments and toxic compounds. The transport and assembly pathways for phospholipids of bacterial OM are unknown. Cardiolipin (CL) plays an important role in OM biogenesis and pathogenesis, and the inner membrane (IM) protein PbgA, containing five transmembrane domains and a globular domain in periplasm has been recently identified as a CL transporter from the IM to the OM with an unknown mechanism. Here we present the first two crystal structures of soluble periplasmic globular domain of PbgA from S. typhimurium and E. coli, which revealed that the globular domains of PbgA resemble the structures of the arylsulfatase protein family and contains a novel core hydrophobic pocket that may be responsible for binding and transporting CLs. Our structural and functional studies shed an important light on the mechanism of CL transport in Gram-negative bacteria from the IM to the OM, which offers great potential for the development of novel antibiotics against multi-drug resistant bacterial infections.

All Gram-negative bacteria, mitochondria and chloroplasts have outer membrane proteins (OMPs) that perform many fundamental biological processes. The OMPs in Gram-negative bacteria are inserted and folded into the outer membrane by the β-barrel assembly machinery (BAM). The mechanism involved is poorly understood, owing to the absence of a structure of the entire BAM complex. Here we report two crystal structures of the Escherichia coli BAM complex in two distinct states: an inward-open state and a lateral-open state. Our structures reveal that the five polypeptide transport-associated domains of BamA form a ring architecture with four associated lipoproteins, BamB–BamE, in the periplasm. Our structural, functional studies and molecular dynamics simulations indicate that these subunits rotate with respect to the integral membrane β-barrel of BamA to induce movement of the β-strands of the barrel and promote insertion of the nascent OMP.

Lassa virus, the causative agent of Lassa fever, causes thousands of deaths annually and is a biological threat agent, for which there is no vaccine and limited therapy. The nucleoprotein (NP) of Lassa virus has essential roles in viral RNA synthesis and immune suppression, the molecular mechanisms of which are poorly understood. Here we report the crystal structure of Lassa virus NP at 1.80 A resolution, which reveals amino (N)- and carboxy (C)-terminal domains with structures unlike any of the reported viral NPs. The N domain folds into a novel structure with a deep cavity for binding the m7GpppN cap structure that is required for viral RNA transcription, whereas the C domain contains 3'-5' exoribonuclease activity involved in suppressing interferon induction. To our knowledge this is the first X-ray crystal structure solved for an arenaviral NP, which reveals its unexpected functions and indicates unique mechanisms in cap binding and immune evasion. These findings provide great potential for vaccine and drug development.

(Chemical Equation Presented) It takes two: Both a lysine and a glutamate residue in the active site of tryptophan halogenase are essential for its chlorination activity. A mechanism for the regioselective enzymatic chlorination of tryptophan involving both amino acids is suggested (see scheme). © 2008 Wiley-VCH Verlag GmbH & Co. KGaA.

In Mycobacterium tuberculosis, two related Z-prenyl diphosphate synthases, E,Z-farnesyl diphosphate synthase (Rv1086) and decaprenyl diphosphate synthase (Rv2361c), work in series to synthesize decaprenyl phosphate (C(50)) from isopentenyl diphosphate and E-geranyl diphosphate. Decaprenyl phosphate plays a central role in the biosynthesis of essential mycobacterial cell wall components, such as the mycolyl-arabinogalactan-peptidoglycan complex and lipoarabinomannan; thus, its synthesis has attracted considerable interest as a potential therapeutic target. Rv1086 is a unique prenyl diphosphate synthase in that it adds only one isoprene unit to geranyl diphosphate, generating the 15-carbon product (E,Z-farnesyl diphosphate). Rv2361c then adds a further seven isoprene units to E,Z-farnesyl diphosphate in a processive manner to generate the 50-carbon prenyl diphosphate, which is then dephosphorylated to generate a carrier for activated sugars. The molecular basis for chain-length discrimination by Rv1086 during synthesis is unknown. We also report the structure of apo Rv1086 with citronellyl diphosphate bound and with the product mimic E,E-farnesyl diphosphate bound. We report the structures of Rv2361c in the apo form, with isopentenyl diphosphate bound and with a substrate analogue, citronellyl diphosphate. The structures confirm the enzymes are very closely related. Detailed comparison reveals structural differences that account for chain-length control in Rv1086. We have tested this hypothesis and have identified a double mutant of Rv1086 that makes a range of longer lipid chains.

Capsular polysaccharides (CPSs) are essential virulence determinants of many pathogenic bacteria. Escherichia coli group 1 CPSs provide paradigms for widespread surface polysaccharide assembly systems in Gram-negative bacteria. In these systems, complex carbohydrate polymers must be exported across the periplasm and outer membrane to the cell surface. Group 1 CPS export requires oligomers of the outer membrane protein, Wza, for translocation across the outer membrane. Assembly also depends on Wzc, an inner membrane tyrosine autokinase known to regulate export and synthesis of group 1 CPS. Here, we provide a structural view of a complex comprising Wzc and Wza that spans the periplasm, connecting the inner and outer membranes. Examination of transmembrane sections of the complex suggests that the periplasm is compressed at the site of complex formation. An important feature of CPS production is the coupling of steps involved in biosynthesis and export. We propose that the Wza–Wzc complex provides the structural and regulatory core of a larger macromolecular machine. We suggest a mechanism by which CPS may move from the periplasm through the outer membrane.

The striking feature of carbohydrates is their constitutional, conformational and configurational diversity. Biology has harnessed this diversity and manipulates carbohydrate residues in a variety of ways, one of which is epimerization. RmlC catalyzes the epimerization of the C3' and C5' positions of dTDP-6-deoxy-D-xylo-4-hexulose, forming dTDP-6-deoxy-L-lyxo-4-hexulose. RmlC is the third enzyme of the rhamnose pathway, and represents a validated anti-bacterial drug target. Although several structures of the enzyme have been reported, the mechanism and the nature of the intermediates have remained obscure. Despite its relatively small size (22 kDa), RmlC catalyzes four stereospecific proton transfers and the substrate undergoes a major conformational change during the course of the transformation. Here we report the structure of RmlC from several organisms in complex with product and product mimics. We have probed site-directed mutants by assay and by deuterium exchange. The combination of structural and biochemical data has allowed us to assign key residues and identify the conformation of the carbohydrate during turnover. Clear knowledge of the chemical structure of RmlC reaction intermediates may offer new opportunities for rational drug design.

The SUMO (small ubiquitin-like modifier)-specific protease SENP1 (sentrin-specific protease 1) can process the three forms of SUMO to their mature forms and deconjugate SUMO from modified substrates. It has been demonstrated previously that SENP1 processed SUMO-1 more efficiently than SUMO-2, but displayed little difference in its ability to deconjugate the different SUMO paralogues from modified substrates. To determine the basis for this substrate specificity, we have determined the crystal structure of SENP1 in isolation and in a transition-state complex with SUMO-2. The interface between SUMO-2 and SENP1 has a relatively poor complementarity, and most of the recognition is determined by interaction between the conserved C-terminus of SUMO-2 and the cleft in the protease. Although SENP1 is rather similar in structure to the related protease SENP2, these proteases have different SUMO-processing activities. Electrostatic analysis of SENP1 in the region where the C-terminal peptide, removed during maturation, would project indicates that it is the electrostatic complementarity between this region of SENP1 and the C-terminal peptides of the various SUMO paralogues that mediates selectivity.

Regioselective halogenation of electron rich substrates is catalysed by flavin-dependent halogenases. Thienodolin produced by Streptomyces albogriseolus contains a chlorine atom in the 6-position of the indole ring system and is believed to be derived from tryptophan. Using the gene of the tryptophan 7-halogenase (PrnA) from the pyrrolnitrin biosynthetic gene cluster the gene for a tryptophan 6-halogenase was cloned, sequenced and heterologously overexpressed in Pseudomonas strains. In vitro activity of the purified enzyme could only be shown in a two-component enzyme system consisting of the halogenase, a flavin reductase, NADH, FAD and halide ions. The enzyme catalyses the regioselective chlorination and bromination of l- and d-tryptophan. In its native form the enzyme is probably a homodimer with a relative molecular mass of the subunits of 63 600 (including the His-tag). Transformation of the pyrrolnitrin producer Pseudomonas chlororaphis ACN with a plasmid containing the tryptophan 6-halogenase gene ...

Chlorinated natural products include vancomycin and cryptophycin A. Their biosynthesis involves regioselective chlorination by flavin-dependent halogenases. We report the structural characterization of tryptophan 7-halogenase (PrnA), which regioselectively chlorinates tryptophan. Tryptophan and flavin adenine dinucleotide (FAD) are separated by a 10 angstrom–long tunnel and bound by distinct enzyme modules. The FAD module is conserved in halogenases and is related to flavin-dependent monooxygenases. On the basis of biochemical studies, crystal structures, and by analogy with monooxygenases, we predict that FADH 2 reacts with O 2 to make peroxyflavin, which is decomposed by Cl – . The resulting HOCl is guided through the tunnel to tryptophan, where it is activated to participate in electrophilic aromatic substitution.