An Enzymatic Oxidation Cascade Converts δ-Thiolactone Anthracene to Anthraquinone in the Biosynthesis of Anthraquinone-Fused Enediynes.

Anthraquinone-fused enediynes are anticancer natural products featuring a DNA-intercalating anthraquinone moiety. Despite recent insights into anthraquinone-fused enediyne (AQE) biosynthesis, the enzymatic steps involved in anthraquinone biogenesis remain to be elucidated. Through a combination of in vitro and in vivo studies, we demonstrated that a two-enzyme system, composed of a flavin adenine dinucleotide (FAD)-dependent monooxygenase (DynE13) and a cofactor-free enzyme (DynA1), catalyzes the final steps of anthraquinone formation by converting δ-thiolactone anthracene to hydroxyanthraquinone. We showed that the three oxygen atoms in the hydroxyanthraquinone originate from molecular oxygen (O2), with the sulfur atom eliminated as H2S. We further identified the key catalytic residues of DynE13 and A1 by structural and site-directed mutagenesis studies. Our data support a catalytic mechanism wherein DynE13 installs two oxygen atoms with concurrent desulfurization and decarboxylation, whereas DynA1 acts as a cofactor-free monooxygenase, installing the final oxygen atom in the hydroxyanthraquinone. These findings establish the indispensable roles of DynE13 and DynA1 in AQE biosynthesis and unveil novel enzymatic strategies for anthraquinone formation.

Inhibition of falcilysin from Plasmodium falciparum by interference with its closed-to-open dynamic transition.

In the absence of an efficacious vaccine, chemotherapy remains crucial to prevent and treat malaria. Given its key role in haemoglobin degradation, falcilysin constitutes an attractive target. Here, we reveal the mechanism of enzymatic inhibition of falcilysin by MK-4815, an investigational new drug with potent antimalarial activity. Using X-ray crystallography, we determine two binary complexes of falcilysin in a closed state, bound with peptide substrates from the haemoglobin α and ß chains respectively. An antiparallel ß-sheet is formed between the substrate and enzyme, accounting for sequence-independent recognition at positions P2 and P1. In contrast, numerous contacts favor tyrosine and phenylalanine at the P1' position of the substrate. Cryo-EM studies reveal a majority of unbound falcilysin molecules adopting an open conformation. Addition of MK-4815 shifts about two-thirds of falcilysin molecules to a closed state. These structures give atomic level pictures of the proteolytic cycle, in which falcilysin interconverts between a closed state conducive to proteolysis, and an open conformation amenable to substrate diffusion and products release. MK-4815 and quinolines bind to an allosteric pocket next to a hinge region of falcilysin and hinders this dynamic transition. These data should inform the design of potent inhibitors of falcilysin to combat malaria.

Cryo-EM structure of the entire FtsH-HflKC AAA protease complex.

The membrane-bound AAA protease FtsH is the key player controlling protein quality in bacteria. Two single-pass membrane proteins, HflK and HflC, interact with FtsH to modulate its proteolytic activity. Here, we present structure of the entire FtsH-HflKC complex, comprising 12 copies of both HflK and HflC, all of which interact reciprocally to form a cage, as well as four FtsH hexamers with periplasmic domains and transmembrane helices enclosed inside the cage and cytoplasmic domains situated at the base of the cage. FtsH K61/D62/S63 in the ß2-ß3 loop in the periplasmic domain directly interact with HflK, contributing to complex formation. Pull-down and in vivo enzymatic activity assays validate the importance of the interacting interface for FtsH-HflKC complex formation. Structural comparison with the substrate-bound human m-AAA protease AFG3L2 offers implications for the HflKC cage in modulating substrate access to FtsH. Together, our findings provide a better understanding of FtsH-type AAA protease holoenzyme assembly and regulation.

Structural analyses of the AAA+ ATPase domain of the transcriptional regulator GtrR in the BDSF quorum-sensing system in Burkholderia cenocepacia.

Global transcriptional regulator downstream RpfR (GtrR) is a key downstream regulator for quorum-sensing signaling molecule cis-2-dodecenoic acid (BDSF). As a bacterial enhancer-binding protein (bEBP), GtrR is composed of an N-terminal receiver domain, a central ATPases associated with diverse cellular activities (AAA+) ATPase σ54 -interaction domain, and a C-terminal helix-turn-helix DNA-binding domain. In this work, we solved its AAA+ ATPase domain in both apo and GTP-bound forms. The structure revealed how GtrR specifically recognizes GTP. In addition, we also revealed that GtrR has moderate GTPase activity in vitro in the absence of its activation signal. Finally, we found the residues K170, D236, R311, and R357 in GtrR that are crucial to its biological function, any single mutation leading to completely abolishing GtrR activity.

Translational GTPase BipA Is Involved in the Maturation of a Large Subunit of Bacterial Ribosome at Suboptimal Temperature.

BPI-inducible protein A (BipA), a highly conserved paralog of the well-known translational GTPases LepA and EF-G, has been implicated in bacterial motility, cold shock, stress response, biofilm formation, and virulence. BipA binds to the aminoacyl-(A) site of the bacterial ribosome and establishes contacts with the functionally important regions of both subunits, implying a specific role relevant to the ribosome, such as functioning in ribosome biogenesis and/or conditional protein translation. When cultured at suboptimal temperatures, the Escherichia coli bipA genomic deletion strain (ΔbipA) exhibits defects in growth, swimming motility, and ribosome assembly, which can be complemented by a plasmid-borne bipA supplementation or suppressed by the genomic rluC deletion. Based on the growth curve, soft agar swimming assay, and sucrose gradient sedimentation analysis, mutation of the catalytic residue His78 rendered plasmid-borne bipA unable to complement its deletion phenotypes. Interestingly, truncation of the C-terminal loop of BipA exacerbates the aforementioned phenotypes, demonstrating the involvement of BipA in ribosome assembly or its function. Furthermore, tandem mass tag-mass spectrometry analysis of the ΔbipA strain proteome revealed upregulations of a number of proteins (e.g., DeaD, RNase R, CspA, RpoS, and ObgE) implicated in ribosome biogenesis and RNA metabolism, and these proteins were restored to wild-type levels by plasmid-borne bipA supplementation or the genomic rluC deletion, implying BipA involvement in RNA metabolism and ribosome biogenesis. We have also determined that BipA interacts with ribosome 50S precursor (pre-50S), suggesting its role in 50S maturation and ribosome biogenesis. Taken together, BipA demonstrates the characteristics of a bona fide 50S assembly factor in ribosome biogenesis.

Ribosome Protection Proteins-"New" Players in the Global Arms Race with Antibiotic-Resistant Pathogens.

Bacteria have evolved an array of mechanisms enabling them to resist the inhibitory effect of antibiotics, a significant proportion of which target the ribosome. Indeed, resistance mechanisms have been identified for nearly every antibiotic that is currently used in clinical practice. With the ever-increasing list of multi-drug-resistant pathogens and very few novel antibiotics in the pharmaceutical pipeline, treatable infections are likely to become life-threatening once again. Most of the prevalent resistance mechanisms are well understood and their clinical significance is recognized. In contrast, ribosome protection protein-mediated resistance has flown under the radar for a long time and has been considered a minor factor in the clinical setting. Not until the recent discovery of the ATP-binding cassette family F protein-mediated resistance in an extensive list of human pathogens has the significance of ribosome protection proteins been truly appreciated. Understanding the underlying resistance mechanism has the potential to guide the development of novel therapeutic approaches to evade or overcome the resistance. In this review, we discuss the latest developments regarding ribosome protection proteins focusing on the current antimicrobial arsenal and pharmaceutical pipeline as well as potential implications for the future of fighting bacterial infections in the time of "superbugs."

Structural Basis and Function of the N Terminus of SARS-CoV-2 Nonstructural Protein 1.

Nonstructural protein 1 (Nsp1) of severe acute respiratory syndrome coronaviruses (SARS-CoVs) is an important pathogenic factor that inhibits host protein translation by means of its C terminus. However, its N-terminal function remains elusive. Here, we determined the crystal structure of the N terminus (amino acids [aa] 11 to 125) of SARS-CoV-2 Nsp1 at a 1.25-Å resolution. Further functional assays showed that the N terminus of SARS-CoVs Nsp1 alone loses the ability to colocalize with ribosomes and inhibit protein translation. The C terminus of Nsp1 can colocalize with ribosomes, but its protein translation inhibition ability is significantly weakened. Interestingly, fusing the C terminus of Nsp1 with enhanced green fluorescent protein (EGFP) or other proteins in place of its N terminus restored the protein translation inhibitory ability to a level equivalent to that of full-length Nsp1. Thus, our results suggest that the N terminus of Nsp1 is able to stabilize the binding of the Nsp1 C terminus to ribosomes and act as a nonspecific barrier to block the mRNA channel, thus abrogating host mRNA translation.

Structure of Arabidopsis CESA3 catalytic domain with its substrate UDP-glucose provides insight into the mechanism of cellulose synthesis.

Cellulose is synthesized by cellulose synthases (CESAs) from the glycosyltransferase GT-2 family. In plants, the CESAs form a six-lobed rosette-shaped CESA complex (CSC). Here we report crystal structures of the catalytic domain of Arabidopsis thaliana CESA3 (AtCESA3CatD) in both apo and uridine diphosphate (UDP)-glucose (UDP-Glc)-bound forms. AtCESA3CatD has an overall GT-A fold core domain sandwiched between a plant-conserved region (P-CR) and a class-specific region (C-SR). By superimposing the structure of AtCESA3CatD onto the bacterial cellulose synthase BcsA, we found that the coordination of the UDP-Glc differs, indicating different substrate coordination during cellulose synthesis in plants and bacteria. Moreover, structural analyses revealed that AtCESA3CatD can form a homodimer mainly via interactions between specific beta strands. We confirmed the importance of specific amino acids on these strands for homodimerization through yeast and in planta assays using point-mutated full-length AtCESA3. Our work provides molecular insights into how the substrate UDP-Glc is coordinated in the CESAs and how the CESAs might dimerize to eventually assemble into CSCs in plants.

Structural basis of a novel repressor, SghR, controlling Agrobacterium infection by cross-talking to plants.

Agrobacterium tumefaciens infects various plants and causes crown gall diseases involving temporal expression of virulence factors. SghA is a newly identified virulence factor enzymatically releasing salicylic acid from its glucoside conjugate and controlling plant tumor development. Here, we report the structural basis of SghR, a LacI-type transcription factor highly conserved in Rhizobiaceae family, regulating the expression of SghA and involved in tumorigenesis. We identified and characterized the binding site of SghR on the promoter region of sghA and then determined the crystal structures of apo-SghR, SghR complexed with its operator DNA, and ligand sucrose, respectively. These results provide detailed insights into how SghR recognizes its cognate DNA and shed a mechanistic light on how sucrose attenuates the affinity of SghR with DNA to modulate the expression of SghA. Given the important role of SghR in mediating the signaling cross-talk during Agrobacterium infection, our results pave the way for structure-based inducer analog design, which has potential applications for agricultural industry.

Structural basis of human full-length kindlin-3 homotrimer in an auto-inhibited state.

Kindlin-1, -2, and -3 directly bind integrin ß cytoplasmic tails to regulate integrin activation and signaling. Despite their functional significance and links to several diseases, structural information on full-length kindlin proteins remains unknown. Here, we report the crystal structure of human full-length kindlin-3, which reveals a novel homotrimer state. Unlike kindlin-3 monomer, which is the major population in insect and mammalian cell expression systems, kindlin-3 trimer does not bind integrin ß cytoplasmic tail as the integrin-binding pocket in the F3 subdomain of 1 protomer is occluded by the pleckstrin homology (PH) domain of another protomer, suggesting that kindlin-3 is auto-inhibited upon trimer formation. This is also supported by functional assays in which kindlin-3 knockout K562 erythroleukemia cells reconstituted with the mutant kindlin-3 containing trimer-disrupting mutations exhibited an increase in integrin-mediated adhesion and spreading on fibronectin compared with those reconstituted with wild-type kindlin-3. Taken together, our findings reveal a novel mechanism of kindlin auto-inhibition that involves its homotrimer formation.

Agrobacteria reprogram virulence gene expression by controlled release of host-conjugated signals.

It is highly intriguing how bacterial pathogens can quickly shut down energy-costly infection machinery once successful infection is established. This study depicts that mutation of repressor SghR increases the expression of hydrolase SghA in Agrobacterium tumefaciens, which releases plant defense signal salicylic acid (SA) from its storage form SA ß-glucoside (SAG). Addition of SA substantially reduces gene expression of bacterial virulence. Bacterial vir genes and sghA are differentially transcribed at early and later infection stages, respectively. Plant metabolite sucrose is a signal ligand that inactivates SghR and consequently induces sghA expression. Disruption of sghA leads to increased vir expression in planta and enhances tumor formation whereas mutation of sghR decreases vir expression and tumor formation. These results depict a remarkable mechanism by which A. tumefaciens taps on the reserved pool of plant signal SA to reprogram its virulence upon establishment of infection.

Structural analyses unravel the molecular mechanism of cyclic di-GMP regulation of bacterial chemotaxis via a PilZ adaptor protein.

The bacterial second messenger cyclic di-GMP (c-di-GMP) has emerged as a prominent mediator of bacterial physiology, motility, and pathogenicity. c-di-GMP often regulates the function of its protein targets through a unique mechanism that involves a discrete PilZ adaptor protein. However, the molecular mechanism for PilZ protein-mediated protein regulation is unclear. Here, we present the structure of the PilZ adaptor protein MapZ cocrystallized in complex with c-di-GMP and its protein target CheR1, a chemotaxis-regulating methyltransferase in Pseudomonas aeruginosa This cocrystal structure, together with the structure of free CheR1, revealed that the binding of c-di-GMP induces dramatic structural changes in MapZ that are crucial for CheR1 binding. Importantly, we found that restructuring and repositioning of two C-terminal helices enable MapZ to disrupt the CheR1 active site by dislodging a structural domain. The crystallographic observations are reinforced by protein-protein binding and single cell-based flagellar motor switching analyses. Our studies further suggest that the regulation of chemotaxis by c-di-GMP through MapZ orthologs/homologs is widespread in proteobacteria and that the use of allosterically regulated C-terminal motifs could be a common mechanism for PilZ adaptor proteins. Together, the findings provide detailed structural insights into how c-di-GMP controls the activity of an enzyme target indirectly through a PilZ adaptor protein.

Crystal structures of CRISPR-associated Csx3 reveal a manganese-dependent deadenylation exoribonuclease.

In prokaryotes, the CRISPR/Cas system is known to target and degrade invading phages and foreign genetic elements upon subsequent infection. However, the structure and function of many Cas proteins remain largely unknown, due to the high diversity of Cas proteins. Here we report 3 crystal structures of Archaeoglobus fulgidus Csx3 (AfCsx3) in free form, in complex with manganese ions and in complex with a single-stranded RNA (ssRNA) fragment, respectively. AfCsx3 harbors a ferredoxin-like fold and forms dimer both in the crystal and in solution. Our structure-based biochemical analysis demonstrates that the RNA binding sites and cleavage sites are located at 2 separate surfaces within the AfCsx3 dimer, suggesting a model to bind, tether and cleave the incoming RNA substrate. In addition, AfCsx3 displays robust 3'-deadenylase activity in the presence of manganese ions, which strongly suggests that AfCsx3 functions as a deadenylation exonuclease. Taken together, our results indicate that AfCsx3 is a Cas protein involved in RNA deadenylation and provide a framework for understanding the role of AfCsx3 in the Type III-B CRISPR/Cas system.

The role of Litopenaeus vannamei p38 in white spot syndrome virus infection.

p38 as a member of MAPK family, has been conversed from yeast to mammals. It has been implicated in numerous biological processes, including the responses to stress and inflammation. In this study, three closely related p38 MAPK homologs, lvp38a, lvp38b and lvp38c, which differ only in an internal 25-amino acid sequence, have been cloned from Litopenaeus vannamei. Three isoforms shared p38 conserved TGY motif and catalytic center, as well as had maximum identities to human p38α and Drosophila p38b. Tissue distribution revealed that lvp38a and lvp38b were ubiquitously expressed in most tissues, while lvp38c showed at relatively low levels and in a tissue-specific manner. Western blotting analysis showed that lvp38 was activated by phosphorylation during WSSV infection. Furthermore, silencing lvp38 mediated by specific dsRNA in shrimps promoted white spot syndrome virus (WSSV) replication and viral gene transcription at the early stage. These results demonstrated that lvp38 was involved in WSSV infection and might participate in host defense at the early stage.

Molecular cloning and characterization of a cDNA encoding extracellular signal-regulated kinase from Litopenaeus vannamei.

Extracellular signal-regulated kinase (ERK) is a serine/threonine-specific kinase, which is activated by downstream signaling molecules of cellular activation, cytokine and chemokine stimulation and various other stimuli. Here we cloned an ERK gene from Litopenaeus vannamei and designated it as lverk. The lverk cDNA contained an open reading frame of 1098 bp encoding 365 amino acids. LVERK had a conserved TEY motif and serine/threonine protein kinase (S_TKc) domain, and close phylogenetic relationship to Penaeus monodon and Marsupenaeus japonicus ERK. Immunofluorescence staining analysis showed that following serum stimulation LVERK was located in cytoplasm and nucleus, but phospho-LVERK was prominently in nucleus, suggesting conserved ERK signaling module occurred in shrimp cells. Then during the white spot syndrome virus (WSSV) infection, LVERK and phospho-LVERK increased at the early stage of infection. Once silencing of lverk in vivo, the replication of WSSV was obviously inhibited. Moreover, treatment of mitogen-activated protein kinase kinase inhibitor in vitro could result in reduction of WSSV proliferation and delay of viral early gene transcription. Our results indicated a role of LVERK involved in WSSV infection. Understanding how WSSV influences ERK signaling pathway to dismantle an effective immune response may lead to insight into pathogenic progression and possible disease control.

A novel JNK from Litopenaeus vannamei involved in white spot syndrome virus infection.

The c-Jun N-terminal kinase (JNK), a member of MAP kinases, is a serine/threonine-specific protein kinase which responds to extracellular stimuli and regulate various cellular activities. It is well documented in innate immune responses and reported to be involved in various viral infections of mammals. In present study, we cloned JNK homolog in a crustacean, Litopenaeus vannamei (designated as LvJNK) and studied its role in white spot syndrome virus (WSSV) infection. Sequence analysis displayed that LvJNK shared high similarity with other members of the JNK subfamily, including the conserved TPY motif and serine/threonine protein kinase (S_TKc) domain. Western blot analysis showed that the activation of LvJNK took place in WSSV infection. LvJnk silencing mediated by specific dsRNA in shrimps could significantly inhibit the proliferation of the virus. Moreover, inhibition of shrimp JNK signaling pathway by specific inhibitor resulted in the reduction of WSSV replication and the delay of WSSV gene transcription. These results indicate for the first time that shrimp JNK is activated in response to WSSV infection and WSSV could benefit from JNK activation. It may facilitate our understanding of the molecular mechanism of virus infection and provided a potential target for preventing the WSSV infection.