SummaryClustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and the associated proteins (Cas) comprise a system of adaptive immunity against viruses and plasmids in prokaryotes. Cas1 is a CRISPR-associated protein that is common to all CRISPR-containing prokaryotes but its function remains obscure. Here we show that the purified Cas1 protein of Escherichia coli (YgbT) exhibits nuclease activity against single-stranded and branched DNAs including Holliday junctions, replication forks and 5Ј-flaps. The crystal structure of YgbT and sitedirected mutagenesis have revealed the potential active site. Genome-wide screens show that YgbT physically and genetically interacts with key components of DNA repair systems, including recB, recC and ruvB. Consistent with these findings, the ygbT deletion strain showed increased sensitivity to DNA damage and impaired chromosomal segregation. Similar phenotypes were observed in strains with deletion of CRISPR clusters, suggesting that the function of YgbT in repair involves interaction with the CRISPRs. These results show that YgbT belongs to a novel, structurally distinct family of nucleases acting on branched DNAs and suggest that, in addition to antiviral immunity, at least some components of the CRISPR-Cas system have a function in DNA repair.

IpaH proteins are E3 ubiquitin ligases delivered by the type III secretion apparatus into host cells upon infection of humans by the Gram-negative pathogen Shigella flexneri. These proteins comprise a variable leucine-rich repeat-containing N-terminal domain and a conserved C-terminal domain harboring an invariant cysteine residue that is crucial for activity. IpaH homologs are encoded by diverse animal and plant pathogens. Here we demonstrate that the IpaH C-terminal domain carries the catalytic activity for ubiquitin transfer and that the N-terminal domain carries the substrate specificity. The structure of the IpaH C-terminal domain, determined to 2.65-Å resolution, represents an all-helical fold bearing no resemblance to previously defined E3 ubiquitin ligases. The conserved and essential cysteine residue lies on a flexible, surface-exposed loop surrounded by conserved acidic residues, two of which are crucial for IpaH activity. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptType III secretion (T3S) systems are used by numerous Gram-negative pathogenic bacteria to deliver effector proteins into the cells of their human, animal or plant hosts. T3S systems comprise the T3S apparatus (T3SA) that form syringe-like structures that span the bacterial envelope and extend like a needle from the bacterial surface, translocators that transit through the T3SA and form a pore within the target cell membrane, effectors that transit through the T3SA and the pore into the cytosol of host cells, and specific chaperones and transcription regulators for secretion and transcription of these effectors 1,2 . Some effectors target the actin cytoskeleton to promote entry or inhibit phagocytosis of the bacterium, whereas other effectors interfere with the host's innate immune responses 1 .The ubiquitin pathway is a common target of bacterial effectors. This pathway involves one ubiquitin-activating enzyme (E1), a limited number of ubiquitin-conjugating enzymes (E2s) and many ubiquitin-ligating enzymes (E3s). The C-terminal glycine residue of 76-residue ubiquitin is first charged via a thioester linkage onto a cysteine residue of E1 and then transferred to a cysteine residue of an E2. E3s recruit an E2 or a subset of E2s for ubiquitin transfer to specific substrates. Two classes of E3s are differentiated on the basis of their mechanism of action and on sequence or structural similarities. RING (really interesting new gene) and U-box (a modified RING motif) domain-containing E3s act as adaptor-like molecules by bringing a ubiquitinated E2 and the substrate into sufficiently close proximity to promote the ubiquitination of the substrate. In contrast, HECT (homologous to E6-associated protein C terminus) domain-containing E3s possess an essential cysteine residue that acts as an acceptor for the ubiquitin carried by the E2 before its transfer to the substrate. The N-and C-terminal domains of HECT E3s are usually involved in substrate binding and catalytic activity, respectively 3 .Several T3S effector...

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.

Polyamines are essential in all branches of life. Spermidine synthase (putrescine aminopropyltransferase, PAPT) catalyzes the biosynthesis of spermidine, a ubiquitous polyamine. The crystal structure of the PAPT from Thermotoga maritima (TmPAPT) has been solved to 1.5 Å resolution in the presence and absence of AdoDATO (S-adenosyl-1,8-diamino-3-thiooctane), a compound containing both substrate and product moieties. This, the first structure of an aminopropyltransferase, reveals deep cavities for binding substrate and cofactor, and a loop that envelops the active site. The AdoDATO binding site is lined with residues conserved in PAPT enzymes from bacteria to humans, suggesting a universal catalytic mechanism. Other conserved residues act sterically to provide a structural basis for polyamine specificity. The enzyme is tetrameric; each monomer consists of a C-terminal domain with a Rossmann-like fold and an Nterminal β-stranded domain. The tetramer is assembled using a novel barrel-type oligomerization motif.The nearly ubiquitous polyamines (putrescine, spermidine and spermine) are polycationic mediators of cell proliferation and differentiation 1 whose functions likely provide both stability and neutralization for nucleic acids. The abundance of polyamines is tightly regulated through biosynthesis, degradation, uptake and efflux 2 . The biosynthesis of polyamines is carried out by three highly conserved polyamine biosynthetic enzymes ( Fig. 1): ornithine decarboxylase, putrescine amino-propyltransferase (PAPT) and spermidine aminopropyltransferase (SAPT). The strong correlation of polyamine synthesis with cell growth renders these aminopropyl transferases (APTs) attractive targets for the development of antiproliferative therapeutics. The APTs are known to be inhibited by their common nucleoside product, as well as other nucleoside analogs. Most inhibitors, such as the nucleoside-polyamine adduct 4 , is also a potent inhibitor of PAPT.The catalytic mechanism of polyamine biosynthesis is unclear, partly because there is no detailed structural information for this class of enzymes. To provide structural insights into the mechanism of spermidine synthesis, the crystal structure of PAPT from Thermotoga maritima (TmPAPT) was determined at high resolution in ligand-free and AdoDATO-bound states. These structures provide insight into the function of the enzyme and its potential mechanism of action. The identification of conserved active site residues should enable accurate models of the mammalian enzymes. The PAPT foldThe structure of TmPAPT was solved using multiwavelength anomalous diffraction (MAD) from selenomethionine (SeMet)-containing crystals. The TmPAPT monomer consists of two domains: an N-terminal domain, composed of six β-strands, and a Rossmann-like C-terminal domain (Fig. 2). The larger C-terminal catalytic core domain (residues 75-296) consists of a seven-stranded β-sheet with a strand order of 9↑, 8↑, 7↑, 10↑, 11↑, 13↓, 12↑ flanked by nine α-helices. This domain resembles a topology observed in...

Type III effectors are virulence factors of Gram-negative bacterial pathogens delivered directly into host cells by the type III secretion nanomachine where they manipulate host cell processes such as the innate immunity and gene expression. Here, we show that the novel type III effector XopL from the model plant pathogen Xanthomonas campestris pv. vesicatoria exhibits E3 ubiquitin ligase activity in vitro and in planta, induces plant cell death and subverts plant immunity. E3 ligase activity is associated with the C-terminal region of XopL, which specifically interacts with plant E2 ubiquitin conjugating enzymes and mediates formation of predominantly K11-linked polyubiquitin chains. The crystal structure of the XopL C-terminal domain revealed a single domain with a novel fold, termed XL-box, not present in any previously characterized E3 ligase. Mutation of amino acids in the central cavity of the XL-box disrupts E3 ligase activity and prevents XopL-induced plant cell death. The lack of cysteine residues in the XL-box suggests the absence of thioester-linked ubiquitin-E3 ligase intermediates and a non-catalytic mechanism for XopL-mediated ubiquitination. The crystal structure of the N-terminal region of XopL confirmed the presence of a leucine-rich repeat (LRR) domain, which may serve as a protein-protein interaction module for ubiquitination target recognition. While the E3 ligase activity is required to provoke plant cell death, suppression of PAMP responses solely depends on the N-terminal LRR domain. Taken together, the unique structural fold of the E3 ubiquitin ligase domain within the Xanthomonas XopL is unprecedented and highlights the variation in bacterial pathogen effectors mimicking this eukaryote-specific activity.

Autophagy is an essential component of innate immunity, enabling the detection and elimination of intracellular pathogens. Legionella pneumophila, an intracellular pathogen that can cause a severe pneumonia in humans, is able to modulate autophagy through the action of effector proteins that are translocated into the host cell by the pathogen's Dot/Icm type IV secretion system. Many of these effectors share structural and sequence similarity with eukaryotic proteins. Indeed, phylogenetic analyses have indicated their acquisition by horizontal gene transfer from a eukaryotic host. Here we report that L. pneumophila translocates the effector protein sphingosine-1 phosphate lyase (LpSpl) to target the host sphingosine biosynthesis and to curtail autophagy. Our structural characterization of LpSpl and its comparison with human SPL reveals high structural conservation, thus supporting prior phylogenetic analysis. We show that LpSpl possesses S1P lyase activity that was abrogated by mutation of the catalytic site residues. L. pneumophila triggers the reduction of several sphingolipids critical for macrophage function in an LpSpl-dependent and -independent manner. LpSpl activity alone was sufficient to prevent an increase in sphingosine levels in infected host cells and to inhibit autophagy during macrophage infection. LpSpl was required for efficient infection of A/J mice, highlighting an important virulence role for this effector. Thus, we have uncovered a previously unidentified mechanism used by intracellular pathogens to inhibit autophagy, namely the disruption of host sphingolipid biosynthesis.Legionella pneumophila | sphingosine-1-phosphate lyase | autophagy | sphingolipids | virulence T he Gram-negative intracellular bacterium Legionella pneumophila is an opportunistic human pathogen responsible for Legionnaires' disease. The bacteria are naturally found in freshwater systems where they replicate within protozoan hosts (1). It is thought that the adaptation to replication within amoebas has equipped L. pneumophila with the factors required to replicate successfully within human macrophages following opportunistic infection (2). Through genome sequencing, we have discovered that L. pneumophila encodes a high number and variety of proteins similar in sequence to eukaryotic proteins that are never or rarely found in other prokaryotic genomes (3). Subsequent phylogenetic analyses have suggested that many of these proteins were acquired by horizontal gene transfer (3, 4). One of these proteins exhibits a high degree of similarity to eukaryotic sphingosine-1 phosphate lyase (SPL). The L. pneumophila SPL homolog (LpSpl encoded by gene lpp2128, lpg2176, or legS2) is conserved in all L. pneumophila strains sequenced to date, but absent from Legionella longbeachae (SI Appendix, Table S1). Phylogenetic analysis of SPL sequences showed that the L. pneumophila spl gene was most likely acquired early during evolution by horizontal gene transfer from a protozoan organism (4, 5). With the increase in genome sequences available...

The structure of SurE provided information about the protein's fold, oligomeric state, and active site. The protein possessed magnesium-dependent acid phosphatase activity, but the physiologically relevant substrate(s) remains to be identified. The importance of three of the assigned active site residues in catalysis was confirmed by site-directed mutagenesis.

Phenazines produced by Pseudomonas and Streptomyces spp. are heterocyclic nitrogen-containing metabolites with antibiotic, antitumor, and antiparasitic activity. The antibiotic properties of pyocyanin, produced by Pseudomonas aeruginosa, were recognized in the 1890s, although this blue phenazine is now known to be a virulence factor in human disease. Despite their biological significance, the biosynthesis of phenazines is not fully understood. Here we present structural and functional studies of PhzF, an enzyme essential for phenazine synthesis in Pseudomonas spp. PhzF shares topology with diaminopimelate epimerase DapF but lacks the same catalytic residues. The structure of PhzF in complex with its substrate, trans-2,3-dihydro-3-hydroxyanthranilic acid, suggests that it is an isomerase using the conserved glutamate E45 to abstract a proton from C3 of the substrate. The proton is returned to C1 of the substrate after rearrangement of the double-bond system, yielding an enol that converts to the corresponding ketone. PhzF is a dimer that may be bifunctional, providing a shielded cavity for ketone dimerization via double Schiff-base formation to produce the phenazine scaffold. Our proposed mechanism is supported by mass and NMR spectroscopy. The results are discussed in the context of related structures and protein sequences of unknown biochemical function.active site residues ͉ catalytic activity ͉ x-ray structure