The YdiU Domain Modulates Bacterial Stress Signaling through Mn2+-Dependent UMPylation.

Sensing stressful conditions and adjusting the cellular metabolism to adapt to the environment are essential activities for bacteria to survive in variable situations. Here, we describe a stress-related protein, YdiU, and characterize YdiU as an enzyme that catalyzes the covalent attachment of uridine-5'-monophosphate to a protein tyrosine/histidine residue, an unusual modification defined as UMPylation. Mn2+ serves as an essential co-factor for YdiU-mediated UMPylation. UTP and Mn2+ binding converts YdiU to an aggregate-prone state facilitating the recruitment of chaperones. The UMPylation of chaperones prevents them from binding co-factors or clients, thereby impairing their function. Consistent with the recent finding that YdiU acts as an AMPylator, we further demonstrate that the self-AMPylation of YdiU padlocks its chaperone-UMPylation activity. A detailed mechanism is proposed based on the crystal structures of Apo-YdiU and YdiU-AMPNPP-Mn2+ and on molecular dynamics simulation models of YdiU-UTP-Mn2+ and YdiU-UTP-peptide. In vivo data demonstrate that YdiU effectively protects Salmonella from stress-induced ATP depletion through UMPylation.

Crystal structures of porcine STINGCBD-CDN complexes reveal the mechanism of ligand recognition and discrimination of STING proteins.

The cyclic dinucleotide (CDN)-stimulator of interferon genes (STING) pathway plays an important role in the detection of viral and bacterial pathogens in animals. Previous studies have shown that the metazoan second messenger cyclic [G(2',5')pA(3',5')p] (2',3'-cGAMP) generated by cyclic GMP-AMP synthase cGAS binds STING with high affinity compared with bacterial CDNs such as c-di-GMP, c-di-AMP, and 3',3'-cGAMP. Despite recent progress indicating that the CDN-binding domain (CBD) of dimeric STING binds asymmetric 2',3'-cGAMP preferentially over symmetric 3',3'-CDNs, it remains an open question whether STING molecules, such as human STING, adopt a symmetric dimeric conformation to efficiently engage its asymmetric ligand. Here, structural studies of the CBD from porcine STING (STINGCBD) in complex with CDNs at 1.76-2.6 Å resolution revealed that porcine STINGCBD, unlike its human and mouse counterparts, can adopt an asymmetric ligand-binding pocket to accommodate the CDNs. We observed that the extensive interactions and shape complementarity between asymmetric 2',3'-cGAMP and the ligand-binding pocket make it the most preferred ligand for porcine STING and that geometry constraints limit the binding between symmetric 3',3'-CDN and porcine STING. The ligand-discrimination mechanism of porcine STING observed here expands our understanding of how the CDN-STING pathway is activated and of its role in antiviral defense.

Mechanistic insight into 3-methylmercaptopropionate metabolism and kinetical regulation of demethylation pathway in marine dimethylsulfoniopropionate-catabolizing bacteria.

The vast majority of oceanic dimethylsulfoniopropionate (DMSP) is thought to be catabolized by bacteria via the DMSP demethylation pathway. This pathway contains four enzymes termed DmdA, DmdB, DmdC and DmdD/AcuH, which together catabolize DMSP to acetylaldehyde and methanethiol as carbon and sulfur sources respectively. While molecular mechanisms for DmdA and DmdD have been proposed, little is known of the catalytic mechanisms of DmdB and DmdC, which are central to this pathway. Here, we undertake physiological, structural and biochemical analyses to elucidate the catalytic mechanisms of DmdB and DmdC. DmdB, a 3-methylmercaptopropionate (MMPA)-coenzyme A (CoA) ligase, undergoes two sequential conformational changes to catalyze the ligation of MMPA and CoA. DmdC, a MMPA-CoA dehydrogenase, catalyzes the dehydrogenation of MMPA-CoA to generate MTA-CoA with Glu435 as the catalytic base. Sequence alignment suggests that the proposed catalytic mechanisms of DmdB and DmdC are likely widely adopted by bacteria using the DMSP demethylation pathway. Analysis of the substrate affinities of involved enzymes indicates that Roseobacters kinetically regulate the DMSP demethylation pathway to ensure DMSP functioning and catabolism in their cells. Altogether, this study sheds novel lights on the catalytic and regulative mechanisms of bacterial DMSP demethylation, leading to a better understanding of bacterial DMSP catabolism.

Author Correction: Molecular basis for the inhibition of the methyl-lysine binding function of 53BP1 by TIRR.

The original version of this Article contained an error in the author affiliations. Xiaochun Yu was incorrectly associated with College of Life Sciences, Hebei University, Baoding 071000 Hebei, China.This has now been corrected in both the PDF and HTML versions of the Article.

Structure-function analyses reveal the mechanism of the ARH3-dependent hydrolysis of ADP-ribosylation.

ADP-ribosylation of proteins plays key roles in multiple biological processes, including DNA damage repair. Recent evidence suggests that serine is an important acceptor for ADP-ribosylation, and that serine ADP-ribosylation is hydrolyzed by ADP-ribosylhydrolase 3 (ARH3 or ADPRHL2). However, the structural details in ARH3-mediated hydrolysis remain elusive. Here, we determined the structure of ARH3 in a complex with ADP-ribose (ADPR). Our analyses revealed a group of acidic residues in ARH3 that keep two Mg2+ ions at the catalytic center for hydrolysis of Ser-linked ADP-ribosyl group. In particular, dynamic conformational changes involving Glu41 were observed in the catalytic center. Our observations suggest that Mg2+ ions together with Glu41 and water351 are likely to mediate the cleavage of the glycosidic bond in the serine-ADPR substrate. Moreover, we found that ADPR is buried in a groove and forms multiple hydrogen bonds with the main chain and side chains of ARH3 residues. On the basis of these structural findings, we used site-directed mutagenesis to examine the functional roles of key residues in the catalytic pocket of ARH3 in mediating the hydrolysis of ADP-ribosyl from serine and DNA damage repair. Moreover, we noted that ADPR recognition is essential for the recruitment of ARH3 to DNA lesions. Taken together, our study provides structural and functional insights into the molecular mechanism by which ARH3 hydrolyzes the ADP-ribosyl group from serine and contributes to DNA damage repair.

Molecular basis for the inhibition of the methyl-lysine binding function of 53BP1 by TIRR.

53BP1 performs essential functions in DNA double-strand break (DSB) repair and it was recently reported that Tudor interacting repair regulator (TIRR) negatively regulates 53BP1 during DSB repair. Here, we present the crystal structure of the 53BP1 tandem Tudor domain (TTD) in complex with TIRR. Our results show that three loops from TIRR interact with 53BP1 TTD and mask the methylated lysine-binding pocket in TTD. Thus, TIRR competes with histone H4K20 methylation for 53BP1 binding. We map key interaction residues in 53BP1 TTD and TIRR, whose mutation abolishes complex formation. Moreover, TIRR suppresses the relocation of 53BP1 to DNA lesions and 53BP1-dependent DNA damage repair. Finally, despite the high-sequence homology between TIRR and NUDT16, NUDT16 does not directly interact with 53BP1 due to the absence of key residues required for binding. Taken together, our study provides insights into the molecular mechanism underlying TIRR-mediated suppression of 53BP1-dependent DNA damage repair.

The archaeal ATPase PINA interacts with the helicase Hjm via its carboxyl terminal KH domain remodeling and processing replication fork and Holliday junction.

PINA is a novel ATPase and DNA helicase highly conserved in Archaea, the third domain of life. The PINA from Sulfolobus islandicus (SisPINA) forms a hexameric ring in crystal and solution. The protein is able to promote Holliday junction (HJ) migration and physically and functionally interacts with Hjc, the HJ specific endonuclease. Here, we show that SisPINA has direct physical interaction with Hjm (Hel308a), a helicase presumably targeting replication forks. In vitro biochemical analysis revealed that Hjm, Hjc, and SisPINA are able to coordinate HJ migration and cleavage in a concerted way. Deletion of the carboxyl 13 amino acid residues impaired the interaction between SisPINA and Hjm. Crystal structure analysis showed that the carboxyl 70 amino acid residues fold into a type II KH domain which, in other proteins, functions in binding RNA or ssDNA. The KH domain not only mediates the interactions of PINA with Hjm and Hjc but also regulates the hexameric assembly of PINA. Our results collectively suggest that SisPINA, Hjm and Hjc work together to function in replication fork regression, HJ formation and HJ cleavage.

Crystal structures of manganese-dependent transcriptional repressor MntR (Rv2788) from Mycobacterium tuberculosis in apo and manganese bound forms.

The pathogenic Mycobacterium tuberculosis encodes two members of the DtxR family metalloregulators, IdeR and MntR. IdeR represses gene expression in response to ferrous iron, while MntR (Rv2788) functions as a manganese-dependent transcriptional repressor, which represses the expression of manganese transporter genes to maintain manganese homeostasis. Although the structural study towards IdeR is in-depth, there is no MntR structure available. Herein, we report both apo and manganese bound forms of MntR structures from M. tuberculosis. MntR has evolved into two metal ion binding sites like other DtxR proteins and for the first time, we captured the two sites fully occupied by its natural ions with one Mn2+ ion at the first site and two Mn2+ ions at the second binding site (binuclear manganese cluster). The conformation change of MntR resulting from manganese binding could prime the MntR for DNA binding, which is a conserved activation mechanism among DtxR family.

NrnC, an RNase D-Like Protein From Agrobacterium, Is a Novel Octameric Nuclease That Specifically Degrades dsDNA but Leaves dsRNA Intact.

NrnC from Agrobacterium tumefaciens (At_NrnC, UniProt accession number A9CG28) is a nuclease containing a single DEDDy domain. Here, we determined the structures of both the apo and metal-ion-bound forms of At_NrnC. Although the overall structure of the At_NrnC protomer is similar to that of the RNase D exonuclease domain, nuclease assays unexpectedly revealed that At_NrnC possesses remarkably different substrate specificity. In contrast to RNase D, which degrades both single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA), At_NrnC hydrolyses ssRNA, single-stranded DNA (ssDNA), and double-stranded DNA (dsDNA) with high efficiency but does not degrade dsRNA. Crystal packing analysis and biochemical data indicated that At_NrnC forms an octameric hollow cylindrical structure that allows ssRNA, ssDNA, and dsDNA, but not dsRNA, to enter the central tunnel where the multiple active sites perform hydrolysis. This novel structural feature confers a high processivity and is responsible for the preference of At_NrnC for longer dsDNA substrates.

Salmonella STM1697 coordinates flagella biogenesis and virulence by restricting flagellar master protein FlhD4C2 from recruiting RNA polymerase.

Salmonella reduces flagella biogenesis to avoid detection within host cells by a largely unknown mechanism. We identified an EAL-like protein STM1697 as required and sufficient for this process. STM1697 surges to a high level after Salmonella enters host cells and restrains the expression of flagellar genes by regulating the function of flagellar switch protein FlhD4C2, the transcription activator of all other flagellar genes. Unlike other anti-FlhD4C2 factors, STM1697 does not prevent FlhD4C2 from binding to target DNA. A 2.0 Å resolution STM1697-FlhD structure reveals that STM1697 binds the same region of FlhD as STM1344, but with weaker affinity. Further experiments show that STM1697 regulates flagella biogenesis by restricting FlhD4C2 from recruiting RNA polymerase and the regulatory effect of STM1697 on flagellar biogenesis and virulence are all achieved by interaction with FlhD. Finally, we describe a novel mechanism mediated by STM1697 in which Salmonella can inhibit the production of flagella antigen and escape from the host immune system.

Structural basis for the regulation of chemotaxis by MapZ in the presence of c-di-GMP.

The bacterial second messenger cyclic diguanylate monophosphate (c-di-GMP) mediates multiple aspects of bacterial physiology through binding to various effectors. In some cases, these effectors are single-domain proteins which only contain a PilZ domain. It remains largely unknown how single-domain PilZ proteins function and regulate their downstream targets. Recently, a single-domain PilZ protein, MapZ (PA4608), was identified to inhibit the activity of the methyltransferase CheR1. Here, crystal structures of the C-terminal domain of CheR1 containing SAH and of CheR1 in complex with c-di-GMP-bound MapZ are reported. It was observed that the binding site of MapZ in CheR1 partially overlaps with the SAH/SAM-binding pocket. Consequently, binding of MapZ blocks SAH/SAM binding. This provides direct structural evidence on the mechanism of inhibition of CheR1 by MapZ in the presence of c-di-GMP.

Crystal structure of the crenarchaeal ExoIII AP endonuclease SisExoIII reveals a conserved disulfide bond endowing the protein with thermostability.

AP endonuclease recognizes and cleaves apurinic/apyrimidinic (AP) sites and plays a critical role in base excision repair. Many ExoIII and EndoIV family AP endonucleases have been characterized both biochemically and structurally in Eukaryote and Bacteria. However, relatively fewer have been studied in Euryarchaeota and there is no such report on an AP endonuclease from Crenarchaeota. Here we report, for the first time, the crystal structure of a crenarchaeal ExoIII AP endonuclease, SisExoIII, from Sulfolobus islandicus REY15A. SisExoIII comprises a two-layer core formed by 10 ß-sheets and a shell formed by 9 surrounding α-helices. A disulfide bond connecting ß8 and ß9 is formed by Cys142 and Cys215. This intra-molecular linkage is conserved among crenarchaeal ExoIII homologs and site-directed mutagenesis revealed that it endows the protein with thermostability, however, disruption of the disulfide bond only has a slight effect on the AP endonuclease activity. We also observed that several key residues within the catalytic center including conserved Glu35 and Asn9 show different conformation compared with known ExoIII proteins and form various intra-molecular salt bridges. The protein possesses three putative DNA binding loops with higher flexibility and hydrophobicity than those of ExoIIIs from other organisms. These features may result in low AP endonuclease activity and defect of exonuclease activity of SisExoIII. The study has deepened our understanding in the structural basis of crenarchaeal ExoIII catalysis and clarified a role of the disulfide bond in maintaining protein thermostability.

cybLuc: An Effective Aminoluciferin Derivative for Deep Bioluminescence Imaging.

To enhance the efficiency of firefly luciferase/luciferin bioluminescence imaging, a series of N-cycloalkylaminoluciferins (cyaLucs) were developed by introducing lipophilic N-cycloalkylated substitutions. The experimental results demonstrate that these cyaLucs are effective substrates for native firefly luciferase (Fluc) and can produce elevated bioluminescent signals in vitro, in cellulo, and in vivo. It should be noted that, in animal studies, N-cyclobutylaminoluciferin (cybLuc) at 10 µM (0.1 mL), which is 0.01% of the standard dose of d-luciferin (dLuc) used in mouse imaging, can radiate 20-fold more bioluminescent light than d-luciferin (dLuc) or aminoluciferin (aLuc) at the same concentration. Longer in vivo emission imaging using cybLuc suggests that it can be used for long-time observation. Regarding the mechanism of cybLuc, our cocrystal structure data from firefly luciferase with oxidized cybLuc suggested that oxidized cybLuc fits into the same pocket as oxyluciferin. Most interestingly, our results demonstrate that the sensitivity of cybLuc in brain tumor imaging contributes to its extended application in deep tissues.

A Novel Enterovirus 71 (EV71) Virulence Determinant: The 69th Residue of 3C Protease Modulates Pathogenicity.

Human enterovirus type 71 (EV71), the major causative agent of hand-foot-and-mouth disease, has been known to cause fatal neurological complications. Unfortunately, the reason for neurological complications that have been seen in fatal cases of the disease and the relationship between EV71 virulence and viral genetic sequences remains largely undefined. The 3C protease (3Cpro) of EV71 plays an irreplaceable role in segmenting the precursor polyprotein during viral replication, and intervening with host life activity during viral infection. In this study, for the first time, the 69th residue of 3C protease has been identified as a novel virulence determinant of EV71. The recombinant virus with single point variation, in the 69th of 3Cpro, exhibited obvious decline in replication, and virulence. We further determined the crystal structure of 3C N69D at 1.39 Ǻ resolution and found that conformation of 3C N69D demonstrated significant changes compared with a normal 3C protein, in the substrate-binding site and catalytic active site. Strikingly, one of the switch loops, essential in fixing substrates, adopts an open conformation in the 3C N69D-rupintrivir complex. Consistent with this apparent structural disruption, the catalytic activity of 3C N69D decreased sharply for host derived and viral derived substrates, detected for both in vitro and in vivo. Interestingly, in addition to EV71, Asp69 was also found in 3C proteases of other virus strains, such as CAV16, and was conserved in nearly all C type human rhinovirus. Overall, we identified a natural virulence determinant of 3C protease and revealed the mechanism of attenuated virulence is mediated by N69D substitution. Our data provides new insight into the enzymatic mechanism of a subdued 3C protease and suggests a theoretical basis for virulence determinantion of picornaviridae.

Crystal structure of PvdO from Pseudomonas aeruginosa.

Pyoverdine I (PVDI) is a water-soluble fluorescein siderophore with strong iron chelating ability from the gram-negative pathogen Pseudomonas aeruginosa PAO1. Compared to common siderophores, PVDI is a relatively large compound whose synthesis requires a group of enzymes with different catalytic activities. In addition to four nonribosomal peptide synthetases (NRPS) which are responsible for the production of the peptide backbone of PVDI, several additional enzymes are associated with the modification of the side chains. PvdO is one of these enzymes and participates in PVDI precursor maturation in the periplasm. We determined the crystal structure of PvdO at 1.24 Å resolution. The PvdO structure shares a common fold with some FGly-generating enzymes (FGE) and is stabilized by Ca2+. However, the catalytic residues in FGE are not observed in PvdO, indicating PvdO adopts a unique catalytic mechanism.

Crystal structure and biochemical features of dye-decolorizing peroxidase YfeX from Escherichia coli O157 Asp143 and Arg232 play divergent roles toward different substrates.

YfeX from Escherichia coli O157 is a bacterial dye-decolorizing peroxidase that represents both dye-decoloring activity and typical peroxidase activity. We reported the crystal structure of YfeX bound to heme at 2.09 Å resolution. The YfeX monomer resembles a ferredoxin-like fold and contains two domains. The three conserved residues surrounding the heme group are His215, Asp143 and Arg232. His215 functions as the proximal axial ligand of the heme iron atom. Biochemical data show that the catalytic significance of the conserved Asp143 and Arg232 depends on the substrate types and that YfeX may adopt various catalytic mechanisms toward divergent substrates. In addition, it is observed that an access tunnel spans from the protein molecular surface to the heme distal region, it serves as the passageway for the entrance and binding of the H2O2.

Crystal Structure of Murein-Tripeptide Amidase MpaA from Escherichia coli O157 at 2.6 Å Resolution.

Peptidoglycan (PG) is an essential component of the cell wall, and undergoes reconstruction by various PG hydrolases during cell growth, development and division. The murein- tripeptide (Mtp) amidase MpaA belongs to PG hydrolase family and is responsible for cleaving the γ-D-Glumeso- Dap amide bond in the Mtp released during PG turnover. The current paper reports the crystal structure of MpaA from Escherichia coli (E. coli) O157 at 2.6 Å resolution. The asymmetric unit consists of two protein molecules and each monomer represents the common α/ß fold of metallocarboxypeptidases (MCP). The Tyr133-Asp143 loop appears to mediate the entrance and binding of the substrate into the active groove. A structural comparison of MpaA with its homologue from Vibrio harveyi showed that MpaA has narrower active pocket entrance with a smaller surface opening, which is determined by the Val204-Thr211 loop. The reported structure provides a starting point for the molecular mechanism of MpaA in a significant human pathogen.

Crystal structure and molecular mechanism of an aspartate/glutamate racemase from Escherichia coli O157.

EcL-DER, the aspartate/glutamate racemase from the pathogen Escherichia coli O157, exhibits racemase activity for l-aspartate and l-glutamate. This study reports the crystal structures of apo-EcL-DER, the EcL-DER-l-aspartate and the EcL-DER-d-aspartate complexes. The EcL-DER structure contains two domains, forming pseudo-mirror symmetry in the active site. A unique catalytic pair consisting of Thr(83) and Cys(197) exists in the active site. The characteristic conformations of l-Asp and d-Asp in the active site provide a straight structural evidence for the racemization mechanism of EcL-DER. In addition, the diversity of catalytic pairs implies that PLP-independent amino acid racemases adopt various catalytic mechanisms and are classified into different subgroups.

Structural insights into the T6SS effector protein Tse3 and the Tse3-Tsi3 complex from Pseudomonas aeruginosa reveal a calcium-dependent membrane-binding mechanism.

The opportunistic pathogen Pseudomonas aeruginosa uses the type VI secretion system (T6SS) to deliver the muramidase Tse3 into the periplasm of rival bacteria to degrade their peptidoglycan (PG). Concomitantly, P. aeruginosa uses the periplasm-localized immunity protein Tsi3 to prevent potential self-intoxication caused by Tse3, and thus gains an edge over rival bacteria in fierce niche competition. Here, we report the crystal structures of Tse3 and the Tse3-Tsi3 complex. Tse3 contains an annexin repeat-like fold at the N-terminus and a G-type lysozyme fold at the C-terminus. One loop in the N-terminal domain (Loop 12) and one helix (α9) from the C-terminal domain together anchor Tse3 and the Tse3-Tsi3 complex to membrane in a calcium-dependent manner in vitro, and this membrane-binding ability is essential for Tse3's activity. In the C-terminal domain, a Y-shaped groove present on the surface likely serves as the PG binding site. Two calcium-binding motifs are also observed in the groove and these are necessary for Tse3 activity. In the Tse3-Tsi3 structure, three loops of Tsi3 insert into the substrate-binding groove of Tse3, and three calcium ions present at the interface of the complex are indispensable for the formation of the Tse3-Tsi3 complex.