scholarly journals Functional Analysis of the Mycoplasma genitalium MG312 Protein Reveals a Specific Requirement of the MG312 N-Terminal Domain for Gliding Motility

2007 ◽  
Vol 189 (19) ◽  
pp. 7014-7023 ◽  
Author(s):  
Raul Burgos ◽  
Oscar Q. Pich ◽  
Enrique Querol ◽  
Jaume Piñol
Keyword(s):  
Complex Structure ◽  
Mycoplasma Genitalium ◽  
Gliding Motility ◽  
Target Cells ◽  
Structural Protein ◽  
Atp Binding Site ◽  
Terminal Domain ◽  
Organelle Assembly ◽  
Mediate Cell ◽  
Gliding Mechanism

ABSTRACT The human pathogen Mycoplasma genitalium is known to mediate cell adhesion to target cells by the attachment organelle, a complex structure also implicated in gliding motility. The gliding mechanism of M. genitalium cells is completely unknown, but recent studies have begun to elucidate the components of the gliding machinery. We report the study of MG312, a cytadherence-related protein containing in the N terminus a box enriched in aromatic and glycine residues (EAGR), which is also exclusively found in MG200 and MG386 gliding motility proteins. Characterization of an MG_312 deletion mutant obtained by homologous recombination has revealed that the MG312 protein is required for the assembly of the M. genitalium terminal organelle. This finding is consistent with the intermediate-cytadherence phenotype and the complete absence of gliding motility exhibited by this mutant. Reintroduction of several MG_312 deletion derivatives into the MG_312 null mutant allowed us to identify two separate functional domains: an N-terminal domain implicated in gliding motility and a C-terminal domain involved in cytadherence and terminal organelle assembly functions. In addition, our results also provide evidence that the EAGR box has a specific contribution to mycoplasma cell motion. Finally, the presence of a conserved ATP binding site known as a Walker A box in the MG312 N-terminal region suggests that this structural protein could also play an active function in the gliding mechanism.

Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1

2019 ◽  
Vol 476 (21) ◽  
pp. 3227-3240 ◽  
Author(s):  
Shanshan Wang ◽  
Yanxiang Zhao ◽  
Long Yi ◽  
Minghe Shen ◽  
Chao Wang ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Magnaporthe Oryzae ◽  
Structural Information ◽  
Complex Structure ◽  
Critical Role ◽  
Catalytic Process ◽  
Structural Basis ◽  
Salt Bridges ◽  
Terminal Domain

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (β3–β4 loop to α0 helix) and movement of a ‘shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a ‘closed' state compared with its ‘open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

scholarly journals Proteins P24 and P41 Function in the Regulation of Terminal-Organelle Development and Gliding Motility in Mycoplasma pneumoniae

2007 ◽  
Vol 189 (20) ◽  
pp. 7442-7449 ◽  
Author(s):  
Benjamin M. Hasselbring ◽  
Duncan C. Krause
Keyword(s):  
Cell Division ◽  
Mycoplasma Pneumoniae ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Gliding Motility ◽  
Atypical Pneumonia ◽  
Reading Frame ◽  
Spatial Positioning ◽  
Time Lapse Imaging ◽  
Gliding Mechanism

ABSTRACT Mycoplasma pneumoniae is a major cause of bronchitis and atypical pneumonia in humans. This cell wall-less bacterium has a complex terminal organelle that functions in cytadherence and gliding motility. The gliding mechanism is unknown but is coordinated with terminal-organelle development during cell division. Disruption of M. pneumoniae open reading frame MPN311 results in loss of protein P41 and downstream gene product P24. P41 localizes to the base of the terminal organelle and is required to anchor the terminal organelle to the cell body, but during cell division, MPN311 insertion mutants also fail to properly regulate nascent terminal-organelle development spatially or gliding activity temporally. We measured gliding velocity and frequency and used fluorescent protein fusions and time-lapse imaging to assess the roles of P41 and P24 individually in terminal-organelle development and gliding function. P41 was necessary for normal gliding velocity and proper spatial positioning of new terminal organelles, while P24 was required for gliding frequency and new terminal-organelle formation at wild-type rates. However, P41 was essential for P24 function, and in the absence of P41, P24 exhibited a dynamic localization pattern. Finally, protein P28 requires P41 for stability, but analysis of a P28− mutant established that the MPN311 mutant phenotype was not a function of loss of P28.

Crystal structure of Ssu72, an essential eukaryotic phosphatase specific for the C-terminal domain of RNA polymerase II, in complex with a transition state analogue

2011 ◽  
Vol 434 (3) ◽  
pp. 435-444 ◽  
Author(s):  
Yong Zhang ◽  
Mengmeng Zhang ◽  
Yan Zhang
Keyword(s):  
Rna Polymerase ◽  
Transition State ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Complex Structure ◽  
Protein Tyrosine Phosphatases ◽  
Cysteine Residue ◽  
Phosphoryl Transfer ◽  
Terminal Domain ◽  
Deep Groove

Reversible phosphorylation of the CTD (C-terminal domain) of the eukaryotic RNA polymerase II largest subunit represents a critical regulatory mechanism during the transcription cycle and mRNA processing. Ssu72 is an essential phosphatase conserved in eukaryotes that dephosphorylates phosphorylated Ser5 of the CTD heptapeptide. Its function is implicated in transcription initiation, elongation and termination, as well as RNA processing. In the present paper we report the high resolution X-ray crystal structures of Drosophila melanogaster Ssu72 phosphatase in the apo form and in complex with an inhibitor mimicking the transition state of phosphoryl transfer. Ssu72 facilitates dephosphorylation of the substrate through a phosphoryl-enzyme intermediate, as visualized in the complex structure of Ssu72 with the oxo-anion compound inhibitor vanadate at a 2.35 Å (1 Å=0.1 nm) resolution. The structure resembles the transition state of the phosphoryl transfer with vanadate exhibiting a trigonal bi-pyramidal geometry covalently bonded to the nucleophilic cysteine residue. Interestingly, the incorporation of oxo-anion compounds greatly stabilizes a flexible loop containing the general acid, as detected by an increase of melting temperature of Ssu72 detected by differential scanning fluorimetry. The Ssu72 structure exhibits a core fold with a similar topology to that of LMWPTPs [low-molecular-mass PTPs (protein tyrosine phosphatases)], but with an insertion of a unique ‘cap’ domain to shelter the active site from the solvent with a deep groove in between where the CTD substrates bind. Mutagenesis studies in this groove established the functional roles of five residues (Met17, Pro46, Asp51, Tyr77 and Met85) that are essential specifically for substrate recognition.

The C-terminal domain of Salmonid Alphavirus nonstructural protein 2 (nsP2) is essential and sufficient to block RIG-I pathway induction and interferon-mediated antiviral response.

2021 ◽  
Author(s):  
Raphaël Jami ◽  
Emilie Mérour ◽  
Julie Bernard ◽  
Annie Lamoureux ◽  
Jean K. Millet ◽  
...  
Keyword(s):  
Immune Response ◽  
Immune System ◽  
Innate Immune Response ◽  
Nuclear Localization ◽  
Innate Immune ◽  
Type I ◽  
Annual Growth Rate ◽  
Structural Protein ◽  
Terminal Domain ◽  
Nuclear Localization Sequences

Salmonid alphavirus (SAV) is an atypical alphavirus, which has a considerable impact on salmon and trout farms. Unlike other alphaviruses such as the chikungunya virus, SAV is transmitted without an arthropod vector, and does not cause cell shut-off during infection. The mechanisms by which SAV escapes the host immune system remain unknown. By studying the role of SAV proteins on the RIG-I signaling cascade, the first line of defense of the immune system during infection, we demonstrated that non-structural protein 2 (nsP2) effectively blocks the induction of type I interferon (IFN). This inhibition, independent of the protease activity carried by nsP2, occurs downstream of IRF3 which is the transcription factor allowing the activation of the IFN promoter and its expression. The inhibitory effect of nsP2 on the RIG-I pathway depends on the localization of nsP2 in the host cell nucleus which is linked to two nuclear localization sequences (NLS) located in its C-terminal part. The C-terminal domain of nsP2 by itself is sufficient and necessary to block IFN induction. Mutation of the NLS of nsP2 is deleterious to the virus. Finally, nsP2 does not interact with IRF3, indicating that its action is possible through a targeted interaction within discrete areas of chromatin, as suggested by its punctate distribution observed in the nucleus. These results therefore demonstrate a major role for nsP2 in the control by SAV of the host cell’s innate immune response. Importance The global consumption of fish continues to rise and the future demand cannot be met by capture fisheries alone due to limited stocks of wild fish. Aquaculture is currently the world’s fastest growing food production sector with an annual growth rate of 6-8 %. Recurrent outbreaks of SAV result in significant economic losses with serious environmental consequences on wild stocks. While the clinical and pathological signs of SAV infection are fairly well known, the molecular mechanisms involved are poorly described. In the present study, we focus on the non-structural protein nsP2 and characterize a specific domain containing nuclear localization sequences that are critical for the inhibition of the host innate immune response mediated by the RIG-I pathway.

scholarly journals Periodicity in Attachment Organelle Revealed by Electron Cryotomography Suggests Conformational Changes in Gliding Mechanism ofMycoplasma pneumoniae

mBio ◽  
2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Akihiro Kawamoto ◽  
Lisa Matsuo ◽  
Takayuki Kato ◽  
Hiroki Yamamoto ◽  
Keiichi Namba ◽  
...  
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Conformational Changes ◽  
Hexagonal Lattice ◽  
Gliding Motility ◽  
Influenza Viruses ◽  
Pathogenic Bacterium ◽  
Dimensional Structure ◽  
Internal Core ◽  
Electron Cryotomography ◽  
Gliding Mechanism

ABSTRACTMycoplasma pneumoniae, a pathogenic bacterium, glides on host surfaces using a unique mechanism. It forms an attachment organelle at a cell pole as a protrusion comprised of knoblike surface structures and an internal core. Here, we analyzed the three-dimensional structure of the organelle in detail by electron cryotomography. On the surface, knoblike particles formed a two-dimensional array, albeit with limited regularity. Analyses using a nonbinding mutant and an antibody showed that the knoblike particles correspond to a naplike structure that has been observed by negative-staining electron microscopy and is likely to be formed as a complex of P1 adhesin, the key protein for binding and gliding. The paired thin and thick plates feature a rigid hexagonal lattice and striations with highly variable repeat distances, respectively. The combination of variable and invariant structures in the internal core and the P1 adhesin array on the surface suggest a model in which axial extension and compression of the thick plate along a rigid thin plate is coupled with attachment to and detachment from the substrate during gliding.IMPORTANCEHuman mycoplasma pneumonia, epidemic all over the world in recent years, is caused by a pathogenic bacterium,Mycoplasma pneumoniae. This tiny bacterium, about 2 µm in cell body length, glides on the surface of the human trachea to infect the host by binding to sialylated oligosaccharides, which are also the binding targets of influenza viruses. The mechanism of mycoplasmal gliding motility is not related to any other well-studied motility systems, such as bacterial flagella and cytoplasmic motor proteins. Here, we visualized the attachment organelle, a cellular architecture for gliding, three dimensionally by using electron cryotomography and other conventional methods. A possible gliding mechanism has been suggested based on the architectural images.

scholarly journals Differences in Determinants Required for Complex Formation and Transactivation in Related VP16 Proteins

2000 ◽  
Vol 74 (21) ◽  
pp. 10112-10121 ◽  
Author(s):  
Matthew Grapes ◽  
Peter O'Hare
Keyword(s):  
Dna Binding ◽  
Complex Formation ◽  
Chimeric Protein ◽  
Domain Model ◽  
Structural Protein ◽  
Terminal Domain ◽  
C Terminus ◽  
Independent Functions ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT VP16-H is an essential structural protein of herpes simplex virus type 1 (HSV-1) and is also a potent activator of virus immediate-early (IE) gene expression. Current models of functional determinants within VP16-H indicate that it consists of two domains, an N-terminal domain involved in recruiting VP16-H to a multicomponent DNA binding complex with two host proteins, Oct-1 and host cell factor (HCF), and an acidic C-terminal domain exclusively involved in transactivation. VP16-E, from equine herpesvirus 1 (EHV-1), exhibits strong conservation with the N-terminal domain of VP16-H but, with the exception of a short segment at the extreme C terminus, lacks almost the entire acidic C-terminal domain. Studies of key activation determinants within the C terminus of VP16-H would predict that VP16-E may activate poorly, if at all. However, VP16-E is a potent activator of both EHV-1 and HSV-1 IE gene transcription. We show that VP16-E does not follow the simple two-domain model of VP16-H. Thus, despite the conservation in the N-terminal domains, this region in VP16-E is not sufficient for assembly into the DNA binding complex with Oct-1 and HCF. The short conserved determinant close to the C terminus is completely dispensable in VP16-H but is absolutely required in VP16-E. In activation studies, the potency of intact VP16-E was not recapitulated in chimeric proteins in which it was fused with a GAL4 DNA binding domain. Furthermore, a chimeric protein consisting of the C-terminal region of VP16-E fused to the N-terminal domain of VP16-H, while able to promote complex formation, nevertheless exhibited very weak activation. These results indicate that the mode of recruitment of the activation domain, i.e., through complex formation with Oct-1 and HCF, may be crucial for activation and that key determinants required for activation in VP16-E, and possibly VP16-H, may involve interactions between regions of the C terminus and the N terminus rather than discrete domains with independent functions.

scholarly journals Genetic and Biochemical Analysis of Phosphatase Activity of Escherichia coli NRII (NtrB) and Its Regulation by the PII Signal Transduction Protein

2003 ◽  
Vol 185 (4) ◽  
pp. 1299-1315 ◽  
Author(s):  
Augen A. Pioszak ◽  
Alexander J. Ninfa
Keyword(s):  
Escherichia Coli ◽  
Signal Transduction ◽  
Binding Site ◽  
Phosphatase Activity ◽  
Atp Binding ◽  
Wild Type ◽  
Small Surface ◽  
Central Domain ◽  
Atp Binding Site ◽  
Terminal Domain

ABSTRACT Mutant forms of Escherichia coli NRII (NtrB) were isolated that retained wild-type NRII kinase activity but were defective in the PII-activated phosphatase activity of NRII. Mutant strains were selected as mimicking the phenotype of a strain (strain BK) that lacks both of the related PII and GlnK signal transduction proteins and thus has no mechanism for activation of the NRII phosphatase activity. The selection and screening procedure resulted in the isolation of numerous mutants that phenotypically resembled strain BK to various extents. Mutations mapped to the glnL (ntrB) gene encoding NRII and were obtained in all three domains of NRII. Two distinct regions of the C-terminal, ATP-binding domain were identified by clusters of mutations. One cluster, including the Y302N mutation, altered a lid that sits over the ATP-binding site of NRII. The other cluster, including the S227R mutation, defined a small surface on the “back” or opposite side of this domain. The S227R and Y302N proteins were purified, along with the A129T (NRII2302) protein, which has reduced phosphatase activity due to a mutation in the central domain of NRII, and the L16R protein, which has a mutation in the N-terminal domain of NRII. The S227R, Y302N, and L16R proteins were specifically defective in the PII-activated phosphatase activity of NRII. Wild-type NRII, Y302N, A129T, and L16R proteins bound to PII, while the S227R protein was defective in binding PII. This suggests that the PII-binding site maps to the “back” of the C-terminal domain and that mutation of the ATP-lid, central domain, and N-terminal domain altered functions necessary for the phosphatase activity after PII binding.

scholarly journals Mycoplasma genitalium mg200 and mg386 genes are involved in gliding motility but not in cytadherence

2006 ◽  
Vol 60 (6) ◽  
pp. 1509-1519 ◽  
Author(s):  
Oscar Q. Pich ◽  
Raul Burgos ◽  
Mario Ferrer-Navarro ◽  
Enrique Querol ◽  
Jaume Pinol
Keyword(s):  
Mycoplasma Genitalium ◽  
Gliding Motility

scholarly journals Insights into the function of Mycoplasma pneumoniae protein P30 from orthologous gene replacement

Microbiology ◽  
2011 ◽  
Vol 157 (10) ◽  
pp. 2862-2870 ◽  
Author(s):  
Ryan F. Relich ◽  
Mitchell F. Balish
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Molecular Mechanisms ◽  
Bacterial Species ◽  
Mycoplasma Genitalium ◽  
Orthologous Gene ◽  
Gene Replacement ◽  
Gliding Motility ◽  
Mycoplasma Species ◽  
Phylogenetic Cluster ◽  
Species Specific

The attachment organelles of bacterial species belonging to the Mycoplasma pneumoniae phylogenetic cluster are required for host cytadherence, gliding motility and virulence. Despite being closely related, these bacteria possess distinct cellular morphologies and gliding characteristics. The molecular mechanisms for most attachment organelle phenotypes, including shape and ability to power motility, are obscure. The attachment organelle-associated P30 protein of M. pneumoniae is implicated in both adherence and motility, with mutations negatively impacting cell morphology, adherence, gliding and virulence. To test whether the P30 alleles of different mycoplasma species confer species-specific attachment organelle properties, we created an M. pneumoniae strain in which the Mycoplasma genitalium P30 orthologue, P32, was substituted for the native P30. Selected clones were visualized by scanning electron microscopy to assess morphology and by indirect immunofluorescence microscopy to localize P32. Cytadherence ability and gliding motility were assessed by haemadsorption assay and phase-contrast microcinematography, respectively. Cell and attachment organelle morphologies were indistinguishable from wild-type M. pneumoniae as well as M. pneumoniae II-3 expressing a C-terminally 6×His-tagged P30 construct. P32 was localized to the tip of the attachment organelle of transformant cells. Although a specific role for P30 in species-specific phenotypes was not identified, this first test of orthologous gene replacement in different mycoplasma species demonstrates that the differences in the M. pneumoniae and M. genitalium proteins contribute little if anything to the different attachment organelle phenotypes between these species.

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