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1.
Legionella pneumophila, the intracellular pathogen that can cause severe pneumonia known as Legionnaire's disease, translocates close to 300 effectors inside the host cell using Dot/Icm type IVB secretion system. The structure and function for the majority of these effector proteins remains unknown. Here, we present the crystal structure of the L. pneumophila effector Lem10. The structure reveals a multidomain organization with the largest C‐terminal domain showing strong structural similarity to the HD protein superfamily representatives. However, Lem10 lacks the catalytic His‐Asp residue pair and does not show any in vitro phosphohydrolase enzymatic activity, typical for HD proteins. While the biological function of Lem10 remains elusive, our analysis shows that similar distinct features are shared by a significant number of HD domains found in Legionella proteins, including the SidE family of effectors known to play an important role during infection. Taken together our data point to the presence of a specific group of non‐catalytic Legionella HD domains, dubbed LHDs, which are involved in pathogenesis. Proteins 2015; 83:2319–2325. © 2015 Wiley Periodicals, Inc.  相似文献   

2.
The DrrA protein of Legionella pneumophila is involved in mistargeting of endoplasmic reticulum‐derived vesicles to Legionella‐containing vacuoles through recruitment of the small GTPase Rab1. To this effect, DrrA binds specifically to phosphatidylinositol 4‐phosphate (PtdIns(4)P) lipids on the cytosolic surface of the phagosomal membrane shortly after infection. In this study, we present the atomic structure of the PtdIns(4)P‐binding domain of a protein (DrrA) from a human pathogen. A detailed kinetic investigation of its interaction with PtdIns(4)P reveals that DrrA binds to this phospholipid with, as yet unprecedented, high affinity, suggesting that DrrA can sense a very low abundance of the lipid.  相似文献   

3.
Biogenesis of a specialized organelle that supports intracellular replication of Legionella pneumophila involves the fusion of secretory vesicles exiting the endoplasmic reticulum (ER) with phagosomes containing this bacterial pathogen. Here, we investigated host plasma membrane SNARE proteins to determine whether they play a role in trafficking of vacuoles containing L. pneumophila. Depletion of plasma membrane syntaxins by RNA interference resulted in delayed acquisition of the resident ER protein calnexin and enhanced retention of Rab1 on phagosomes containing virulent L. pneumophila, suggesting that these SNARE proteins are involved in vacuole biogenesis. Plasma membrane‐localized SNARE proteins syntaxin 2, syntaxin 3, syntaxin 4 and SNAP23 localized to vacuoles containing L. pneumophila. The ER‐localized SNARE protein Sec22b was found to interact with plasma membrane SNAREs on vacuoles containing virulent L. pneumophila, but not on vacuoles containing avirulent mutants of L. pneumophila. The addition of α‐SNAP and N‐ethylmaleimide‐sensitive factor (NSF) to the plasma membrane SNARE complexes formed by virulent L. pneumophila resulted in the dissociation of Sec22b, indicating functional pairing between these SNAREs. Thus, L. pneumophila stimulates the non‐canonical pairing of plasma membrane t‐SNAREs with the v‐SNARE Sec22b to promote fusion of the phagosome with ER‐derived vesicles. The mechanism by which L. pneumophila promotes pairing of plasma membrane syntaxins and Sec22b could provide unique insight into how the secretory vesicles could provide an additional membrane reserve subverted during phagosome maturation.  相似文献   

4.
Lpg0406, a hypothetical protein from Legionella pneumophila, belongs to carboxymuconolactone decarboxylase (CMD) family. We determined the crystal structure of lpg0406 both in its apo and reduced form. The structures reveal that lpg0406 forms a hexamer and have disulfide exchange properties. The protein has an all‐helical fold with a conserved thioredoxin‐like active site CXXC motif and a proton relay system similar to that of alkylhydroperoxidase from Mycobacterium tuberculosis (MtAhpD), suggesting that lpg0406 might function as an enzyme with peroxidase activity and involved in antioxidant defense. A comparison of the size and the surface topology of the putative substrate‐binding region between lpg0406 and MtAhpD indicates that the two enzymes accommodate the different substrate preferences. The structural findings will enhance understanding of the CMD family protein structure and its various functions.  相似文献   

5.
The pathogenic bacteria Legionella pneumophila is known to cause Legionnaires' Disease, a severe pneumonia that can be fatal to immunocompromised individuals and the elderly. Shohdy et al. identified the L. pneumophila vacuole sorting inhibitory protein VipF as a putative N‐acetyltransferase based on sequence homology. We have characterized the basic structural and functional properties of VipF to confirm this original functional assignment. Sequence conservation analysis indicates two putative CoA‐binding regions within VipF. Homology modeling and small angle X‐ray scattering suggest a monomeric, dual‐domain structure joined by a flexible linker. Each domain contains the characteristic beta‐splay motif found in many acetyltransferases, suggesting that VipF may contain two active sites. Docking experiments suggest reasonable acetyl‐CoA binding locations within each beta‐splay motif. Broad substrate screening indicated that VipF is capable of acetylating chloramphenicol and both domains are catalytically active. Given that chloramphenicol is not known to be N‐acetylated, this is a surprising finding suggesting that VipF is capable of O‐acetyltransferase activity. Proteins 2016; 84:1422–1430. © 2016 Wiley Periodicals, Inc.  相似文献   

6.
The methylation of U1498 located in the 16S ribosomal RNA of Escherichia coli is an important modification affecting ribosomal activity. RsmE methyltransferases methylate specifically this position in a mechanism that requires an S‐adenosyl‐L‐methionine (AdoMet) molecule as cofactor. Here we report the structure of Apo and AdoMet‐bound Lpg2936 from Legionella pneumophila at 1.5 and 2.3 Å, respectively. The protein comprises an N‐terminal PUA domain and a C‐terminal SPOUT domain. The latter is responsible for protein dimerization and cofactor binding. Comparison with similar structures suggests that Lpg2936 is an RsmE‐like enzyme that can target the equivalent of U1498 in the L. pneumophila ribosomal RNA, thereby potentially enhancing ribosomal activity during infection‐mediated effector production. The multiple copies of the enzyme found in both structures reveal a flexible conformation of the bound AdoMet ligand. Isothermal titration calorimetry measurements suggest an asymmetric two site binding mode. Our results therefore also provide unprecedented insights into AdoMet/RsmE interaction, furthering our understanding of the RsmE catalytic mechanism.  相似文献   

7.
Abebe T  Skadsen RW  Kaeppler HF 《Planta》2005,221(2):170-183
The lemma and palea (lemma/palea), which form the husk of barley (Hordeum vulgare L.) seeds, constitutively express high levels of defense-related genes, relative to leaves [Abebe et al. (2004) Crop Sci 44:942–950]. One of these genes, Lem2, is expressed mainly in the lemma/palea and coleoptile and is strongly upregulated by salicylic acid (SA) and its functional analog 2,6-dichloroisonicotinic acid . Induction by SA was rapid, occurring within 4 h of treatment. However, Lem2 is not responsive to methyl jasmonate (MeJA) or wounding and is downregulated by drought, dehydration, and abscisic acid. These results suggest that Lem2 is involved in systemic acquired resistance. Sequence analysis showed that LEM2 is a jacalin-related lectin (JRL)-like protein with two domains. Consistent with northern and western blot data, transient expression analyses using Lem2::gfp constructs showed strong expression in lemmas and a trace expression in leaves. Successive 5 deletions of the 1,414 bp upstream region gradually weakened promoter strength, as measured by real-time PCR. Promoter deletion studies also revealed that the –75/+70 region (containing the TATA box, 5 UTR, and a SA-response element) determines tissue specificity and that the distal promoter region simply enhances expression. Southern analysis indicated that Morex barley has at least three copies of the Lem2 gene arranged in tandem on chromosome 5(1H) Bin 02, near the short arm telomere. Lem2 is not present in the barley cultivars Steptoe, Harrington, Golden Promise, and Q21861.Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U. S. Department of Agriculture  相似文献   

8.
Legionella pneumophila is an intracellularly surviving pathogen that releases about 270 different proteins into the host cell during infection. A set of secreted proteins takes control of the vesicular trafficking regulator Rab1. Legionella LepB inactivates Rab1 by acting as a GTPase‐activating protein (GAP). We present the crystal structure of the Rab1b:LepB complex together with a thorough biochemical analysis and show that the GAP domain of LepB consists of an unusual fold. LepB acts by an atypical RabGAP mechanism that is reminiscent of classical GAPs and therefore sets the protein apart from mammalian TBC‐like GAPs. Surprisingly, LepB can function as a GAP for Rab3, Rab8, Rab13 and Rab35, too, suggesting that it has a broader cellular role than previously thought.  相似文献   

9.
Human infections by the intracellular bacterial pathogen Legionella pneumophila result in a severe form of pneumonia, the Legionnaire's disease. L. pneumophila utilizes a Type IVb secretion (T4bS) system termed “dot/icm” to secrete protein effectors to the host cytoplasm. The dot/icm system is powered at least in part by a functionally critical AAA+ ATPase, a protein called DotB, thought to belong to the VirB11 family of proteins. Here we present the crystal structure of DotB at 3.19 Å resolution, in its hexameric form. We observe that DotB is in fact a structural intermediate between VirB11 and PilT family proteins, with a PAS‐like N‐terminal domain coupled to a RecA‐like C‐terminal domain. It also shares critical structural elements only found in PilT. The structure also reveals two conformers, termed α and β, with an αβαβαβ configuration. The existence of α and β conformers in this class of proteins was confirmed by solving the structure of DotB from another bacterial pathogen, Yersinia, where, intriguingly, we observed an ααβααβ configuration. The two conformers co‐exist regardless of the nucleotide‐bound states of the proteins. Our investigation therefore reveals that these ATPases can adopt a wider range of conformational states than was known before, shedding new light on the extraordinary spectrum of conformations these ATPases can access to carry out their function. Overall, the structure of DotB provides a template for further rational drug design to develop more specific antibiotics to tackle Legionnaire's disease. PDB Code(s): Will ; be ; provided  相似文献   

10.
11.
Human interleukin‐22 (IL‐22) is a member of the IL‐10 cytokine family that has recently been shown to have major therapeutic potential. IL‐22 is an unusual cytokine as it does not act directly on immune cells. Instead, IL‐22 controls the differentiation, proliferation and antimicrobial protein expression of epithelial cells, thereby maintaining epithelial barrier function. In this study, we transiently expressed human IL‐22 in Nicotiana benthamiana plants and investigated the role of N‐glycosylation on protein folding and biological activity. Expression levels of IL‐22 were up to 5.4 μg/mg TSP, and N‐glycan analysis revealed the presence of the atypical Lewis A structure. Surprisingly, upon engineering of human‐like N‐glycans on IL‐22 by co‐expressing mouse FUT8 in ΔXT/FT plants a strong reduction in Lewis A was observed. Also, core α1,6‐fucoylation did not improve the biological activity of IL‐22. The combination of site‐directed mutagenesis of Asn54 and in vivo deglycosylation with PNGase F also revealed that N‐glycosylation at this position is not required for proper protein folding. However, we do show that the presence of a N‐glycan on Asn54 contributes to the atypical N‐glycan composition of plant‐produced IL‐22 and influences the N‐glycan composition of N‐glycans on other positions. Altogether, our data demonstrate that plants offer an excellent tool to investigate the role of N‐glycosylation on folding and activity of recombinant glycoproteins, such as IL‐22.  相似文献   

12.
Pseudomonas aeruginosa is an opportunistic pathogen commonly found in humans and other organisms and is an important cause of infection especially in patients with compromised immune defense mechanisms. The PA3611 gene of P. aeruginosa PAO1 encodes a secreted protein of unknown function, which has been recently classified into a small Pseudomonas‐specific protein family called DUF4146. As part of our effort to extend structural coverage of novel protein space and provide a structure‐based functional insight into new protein families, we report the crystal structure of PA3611, the first structural representative of the DUF4146 protein family. Proteins 2014; 82:1086–1092. © 2013 Wiley Periodicals, Inc.  相似文献   

13.
The FIC motif and the eukaryotic‐like ankyrin repeats are found in many bacterial type IV effectors, yet little is known about how these domains enable bacteria to modulate host cell functions. Bacterial FIC domains typically bind ATP and transfer adenosine monophosphate moiety onto target proteins. The ankyrin repeat‐containing protein AnkX encoded by the intracellular pathogen Legionella pneumophila is unique in that its FIC domain binds to CDP‐choline and transfers a phosphocholine residue onto proteins in the Rab1 GTPase family. By determining the structures of unbound AnkX and AnkX with bound CDP‐choline, CMP/phosphocholine and CMP, we demonstrate that the orientation of substrate binding in relation to the catalytic FIC motif enables this protein to function as a phosphocholinating enzyme rather than a nucleotidyl transferase. Additionally, the structure reveals that the ankyrin repeats mediate scaffolding interactions that resemble those found in protein–protein interactions, but are unprecedented in intramolecular interactions. Together with phosphocholination experiments, our structures unify a general phosphoryl transferase mechanism common to all FIC enzymes that should be conserved from bacteria to human.  相似文献   

14.
FK506‐binding protein 22 (FKBP22) from the psychrotophic bacterium Shewanella sp. SIB1 (SIB1 FKBP22) is a homodimeric protein with peptidyl prolyl cis‐trans isomerase (PPIase) activity. Each monomer consists of the N‐terminal domain responsible for dimerization and C‐terminal catalytic domain. To reveal interactions at the dimer interface of SIB1 FKBP22, the crystal structure of the N‐domain of SIB1 FKBP22 (SN‐FKBP22, residues 1‐68) was determined at 1.9 Å resolution. SN‐FKBP22 forms a dimer, in which each monomer consists of three helices (α1, α2, and α3N). In the dimer, two monomers have head‐to‐head interactions, in which residues 8–64 of one monomer form tight interface with the corresponding residues of the other. The interface is featured by the presence of a Val‐Leu knot, in which Val37 and Leu41 of one monomer interact with Val41 and Leu37 of the other, respectively. To examine whether SIB1 FKBP22 is dissociated into the monomers by disruption of this knot, the mutant protein V37R/L41R‐FKBP22, in which Val37 and Leu41 of SIB1 FKBP22 are simultaneously replaced by Arg, was constructed and biochemically characterized. This mutant protein was indistinguishable from the SIB1 FKBP22 derivative lacking the N‐domain in oligomeric state, far‐UV CD spectrum, thermal denaturation curve, PPIase activity, and binding ability to a folding intermediate of protein, suggesting that the N‐domain of V37R/L41R‐FKBP22 is disordered. We propose that a Val‐Leu knot at the dimer interface of SIB1 FKBP22 is important for dimerization and dimerization is required for folding of the N‐domain.  相似文献   

15.
The effect of five antibiotics: carbenicillin, chloramphenicol, cefotaxime, kanamycin and hygromycin on the organogenesis from callus cultures of Coryphantha elphantidens (Lem.) Lem. have been studied. Carbenicillin and cefotaxime stimulated shoot regeneration from callus. All antibiotics under study suppressed rooting of in vitro formed shoots. After five sequential subcultures on kanamycin supplemented medium, antibiotic resistant callus was obtained. To study the impact of kanamycin on resistant callus, total protein content was also studied. Selected callus showed a remarkable increase in callus mass. Antibiotic resistant plants have been selected by screening callus pieces on kanamycin supplemented media. Total protein content increased with subsequent subcultures in kanamycin resistant callus. The kanamycin selected shoots withstood the stability test after 2 months on antibiotic free medium. Plants were raised from the callus, which formed roots in 20 mg dm–3 kanamycin, which was under study.  相似文献   

16.
Symbioses among co‐evolving taxa are often marked by genome reductions such as a loss of protein‐coding genes in at least one of the partners as a means of reducing redundancy or intergenomic conflict. To explore this phenomenon in an iconic yet under‐studied group of obligate symbiotic organisms, mitochondrial genomes of 22 newly sequenced and annotated species of lichenized fungi were compared to 167 mitochondrial genomes of nonlichenized fungi. Our results demonstrate the first broad‐scale loss of atp9 from mitochondria of lichenized fungi. Despite key functions in mitochondrial energy production, we show that atp9 has been independently lost in three different lineages spanning 10 of the 22 studied species. A search for predicted, functional copies of atp9 among genomes of other symbionts involved in each lichen revealed the full‐length, presumably functional copies of atp9 in either the photosynthetic algal partner or in other symbiotic fungi in all 10 instances. Together, these data yield evidence of an obligate symbiotic relationship in which core genomic processes have been streamlined, likely due to co‐evolution.  相似文献   

17.
It is generally accepted that naturally existing functional domains can serve as building blocks for complex protein structures, and that novel functions can arise from assembly of different combinations of these functional domains. To inform our understanding of protein evolution and explore the modular nature of protein structure, two model enzymes were chosen for study, purT‐encoded glycinamide ribonucleotide formyltransferase (PurT) and purK‐encoded N5‐carboxylaminoimidazole ribonucleotide synthetase (PurK). Both enzymes are found in the de novo purine biosynthetic pathway of Escherichia coli. In spite of their low sequence identity, PurT and PurK share significant similarity in terms of tertiary structure, active site organization, and reaction mechanism. Their characteristic three domain structures categorize both PurT and PurK as members of the ATP‐grasp protein superfamily. In this study, we investigate the exchangeability of individual protein domains between these two enzymes and the in vivo and in vitro functional properties of the resulting hybrids. Six domain‐swapped hybrids were unable to catalyze full wild‐type reactions, but each hybrid protein could catalyze partial reactions. Notably, an additional loop replacement in one of the domain‐swapped hybrid proteins was able to restore near wild‐type PurK activity. Therefore, in this model system, domain‐swapped proteins retained the ability to catalyze partial reactions, but further modifications were required to efficiently couple the reaction intermediates and achieve catalysis of the full reaction. Implications for understanding the role of domain swapping in protein evolution are discussed.  相似文献   

18.
The FK506‐binding protein (FKBP) family consists of proteins with a variety of protein–protein interaction domains and versatile cellular functions. It is assumed that all members are peptidyl‐prolyl cis–trans isomerases with the enzymatic function attributed to the FKBP domain. Six members of this family localize to the mammalian endoplasmic reticulum (ER). Four of them, FKBP22 (encoded by the FKBP14 gene), FKBP23 (FKBP7), FKBP60 (FKBP9), and FKBP65 (FKBP10), are unique among all FKBPs as they contain the EF‐hand motifs. Little is known about the biological roles of these proteins, but emerging genetics studies are attracting great interest to the ER resident FKBPs, as mutations in genes encoding FKBP10 and FKBP14 were shown to cause a variety of matrix disorders. Although the structural organization of the FKBP‐type domain as well as of the EF‐hand motif has been known for a while, it is difficult to conclude how these structures are combined and how it affects the protein functionality. We have determined a unique 1.9 Å resolution crystal structure for human FKBP22, which can serve as a prototype for other EF hand‐containing FKBPs. The EF‐hand motifs of two FKBP22 molecules form a dimeric complex with an elongated and predominantly hydrophobic cavity that can potentially be occupied by an aliphatic ligand. The FKBP‐type domains are separated by a cleft and their putative active sites can catalyze isomerazation of two bonds within a polypeptide chain in extended conformation. These structural results are of prime interest for understanding biological functions of ER resident FKBPs containing EF‐hand motifs.  相似文献   

19.
Tethering proteins play a key role in vesicular transport, ensuring that cargo arrives at a specific destination. The bacterial effector protein SidC and its paralog SdcA have been described as tethering factors encoded by the intracellular pathogen Legionella pneumophila. Here, we demonstrate that SidC proteins are important for early events unique to maturation of vacuoles containing Legionella and discover monoubiquitination of Rab1 as a new SidC‐dependent activity. The crystal structure of the SidC N‐terminus revealed a novel fold that is important for function and could be involved in Legionella adaptations to evolutionarily divergent host cells it encounters in natural environments.   相似文献   

20.
LEM-domain proteins share a folded structure, the 'LEM-domain', which binds a conserved chromatin protein named BAF. Most LEM-domain proteins are found at the nuclear membrane, but some are nucleoplasmic. All characterized members of this family bind nuclear lamin filaments. We summarize the 'founding' LEM-domain proteins LAP2, emerin and MAN1 ('SANE' or 'XMAN' in Xenopus) and their emerging roles in gene regulation and nuclear assembly. These roles are placed in the context of human diseases ('laminopathies') caused by mutations in either emerin or A-type lamins. Other LEM-domain proteins might modify the phenotype or severity of human laminopathy, or cause new laminopathies. We summarize evidence that the human genome encodes at least four additional LEM-domain proteins, designated Lem2 (NET-25), Lem3, Lem4 and Lem5. Early adaptation of a consistent nomenclature, such as the "Lem" names proposed here, will facilitate rapid progress in this field. Further investigation of 'founder' and novel members of this family will be important to understand nuclear structure, and presents new opportunities to understand human disease.  相似文献   

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