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Protein & Peptide Letters

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ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

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Research Article

Crystal Structure of the Type VI Secretion System Accessory Protein TagF from Pseudomonas Aeruginosa

Author(s): Chang-Kyu Ok and Jeong Ho Chang*

Volume 26, Issue 3, 2019

Page: [204 - 214] Pages: 11

DOI: 10.2174/0929866526666190119121859

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Abstract

Background: Type VI Secretion System (T6SS) has been found in approximately onequarter of the gram-negative bacterial species, and its structural characteristics appear to slightly differ from species to species. The genes encoding T6SS are designated as type six secretion A–M (tssA–M). The expression of the tss gene cluster is regulated by various accessory genes, designated as type VI-associated genes A–P (tagA–P). Tag family proteins have been commonly found in bacteria expressing T6SS but not in all bacterial species. For instance, the tag gene cluster is well-conserved in Pseudomonas aeruginosa (Pa). The PaTagF protein has large homology with ImpM in Rhizobium leguminosarum and SciT in Salmonella enterica. The overexpression of PaTagF represses T6SS complex accumulation and suppresses T6SS antibacterial activity. Thus, the functions of TagF are mediated through direct interactions with the forkhead-associated protein Fha, as evident from the results of the yeast-two hybrid assays. Fha is involved in recruiting a membrane-associated complex either in threonine phosphorylation pathway-dependent or - independent manner. However, functional reports of the tag gene cluster are still limited.

Objective: In this article, our motivation is to understand the molecular mechanism underlying the regulation of expression of the type VI secretion system complex.

Methods: In this article, we start with obtaining the gene encoding PaTagF protein by polymerase chain reaction (PCR). Subsequently, the cloned gene is applied to overexpress of PaTagF protein in Escherichia coli, then purify the recombinant PaTagF protein. Thereafter, the protein is crystallized in a condition of 2.5 M NaCl, 0.1 M imidazole (pH 8.0), 3.2 M NaCl, 0.1 M BIS-TRIS propane (pH 7.0) and diffraction datasets of the PaTagF crystals are collected at the Pohang Accelerator Laboratory (PAL). The molecular structure of PaTagF protein is determined by molecular replacement using the uncharacterized protein PA0076 (PDB code:2QNU) as an initial search model by PHENIX crystallographic software package. Model building of PaTagF structure is performed using Coot program. Finally, the structural model is validated using phenix.refine program.

Results: PaTagF exists as a tetramer in the asymmetric unit, and the overall fold of each monomer is composed of continuous beta-sheets wrapped by alpha-helices. Each monomer has variable conformations and lengths of both the N- and C-termini. Twelve residues, including the His6 tag from the N-terminus of a symmetry-related molecule, have been found in two of the tetrameric PaTagF structures. A structural homology search revealed that PaTagF was similar to the α-β-α sandwichlike structure of the longin domain on the differentially expressed in normal and neoplastic (DENN) superfamily, which is commonly found in proteins related to trafficking.

Conclusion: The tetrameric structure of PaTagF comprises varied N- and C-terminal regions in each subunit and may be stabilized by a symmetry-related molecule. This feature was also shown in the TssL structure from V. cholerae. Furthermore, our study showed that the overall fold of PaTagF is homologous to the longin domain of the DENN family. Therefore, further studies are warranted to elucidate the structure-based evolutionary relationship between protein transport systems from the bacteria and eukaryotic cells.

Keywords: Type VI secretion system, TagF, crystallography, Pseudomonas aeruginosa, longin domain, DENN family.

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[1]
Gerlach, R.G.; Hensel, M. Protein secretion systems and adhesins: The molecular armory of Gram-negative pathogens. Int. J. Med. Microbiol., 2007, 297(6), 401-415.
[2]
Desvaux, M.; Hebraud, M.; Talon, R.; Henderson, I.R. Secretion and subcellular localizations of bacterial proteins: A semantic awareness issue. Trends Microbiol., 2009, 17(4), 139-145.
[3]
Desvaux, M.; Dumas, E.; Chafsey, I.; Hebraud, M. Protein cell surface display in Gram-positive bacteria: From single protein to macromolecular protein structure. FEMS Microbiol. Lett., 2006, 256(1), 1-15.
[4]
Rego, A.T.; Chandran, V.; Waksman, G. Two-step and one-step secretion mechanisms in Gram-negative bacteria: Contrasting the type IV secretion system and the chaperone-usher pathway of pilus biogenesis. Biochem. J., 2010, 425(3), 475-488.
[5]
Costa, T.R.; Felisberto-Rodrigues, C.; Meir, A.; Prevost, M.S.; Redzej, A.; Trokter, M.; Waksman, G. Secretion systems in Gram-negative bacteria: Structural and mechanistic insights. Nat. Rev. Microbiol., 2015, 13(6), 343-359.
[6]
Rapisarda, C.; Tassinari, M.; Gubellini, F.; Fronzes, R. Using Cryo-EM to investigate bacterial secretion systems. Annu. Rev. Microbiol., 2018, 72, 231-254.
[7]
Papanikou, E.; Karamanou, S.; Economou, A. Bacterial protein secretion through the translocase nanomachine. Nat. Rev. Microbiol., 2007, 5(11), 839-851.
[8]
Pukatzki, S.; Ma, A.T.; Sturtevant, D.; Krastins, B.; Sarracino, D.; Nelson, W.C.; Heidelberg, J.F.; Mekalanos, J.J. Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc. Natl. Acad. Sci. USA, 2006, 103(5), 1528-1533.
[9]
Mougous, J.D.; Cuff, M.E.; Raunser, S.; Shen, A.; Zhou, M.; Gifford, C.A.; Goodman, A.L.; Joachimiak, G.; Ordonez, C.L.; Lory, S.; Walz, T.; Joachimiak, A.; Mekalanos, J.J. A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science, 2006, 312(5779), 1526-1530.
[10]
Shalom, G.; Shaw, J.G.; Thomas, M.S. In vivo expression technology identifies a type VI secretion system locus in Burkholderia pseudomallei that is induced upon invasion of macrophages. Microbiology, 2007, 153(Pt 8), 2689-2699.
[11]
Zoued, A.; Brunet, Y.R.; Durand, E.; Aschtgen, M.S.; Logger, L.; Douzi, B.; Journet, L.; Cambillau, C.; Cascales, E. Architecture and assembly of the Type VI secretion system. Biochim. Biophys. Acta, 2014, 1843(8), 1664-1673.
[12]
Durand, E.; Nguyen, V.S.; Zoued, A.; Logger, L.; Pehau-Arnaudet, G.; Aschtgen, M.S.; Spinelli, S.; Desmyter, A.; Bardiaux, B.; Dujeancourt, A.; Roussel, A.; Cambillau, C.; Cascales, E.; Fronzes, R. Biogenesis and structure of a type VI secretion membrane core complex. Nature, 2015, 523(7562), 555-560.
[13]
Cherrak, Y.; Rapisarda, C.; Pellarin, R.; Bouvier, G.; Bardiaux, B.; Allain, F.; Malosse, C.; Rey, M.; Chamot-Rooke, J.; Cascales, E.; Fronzes, R.; Durand, E. Biogenesis and structure of a type VI secretion baseplate. Nat. Microbiol., 2018, 3(12), 1404-1416.
[14]
Nazarov, S.; Schneider, J.P.; Brackmann, M.; Goldie, K.N.; Stahlberg, H.; Basler, M. Cryo-EM reconstruction of Type VI secretion system baseplate and sheath distal end. EMBO J., 2018, 37(4), pii: e97103.
[15]
English, G.; Byron, O.; Cianfanelli, F.R.; Prescott, A.R.; Coulthurst, S.J. Biochemical analysis of TssK, a core component of the bacterial Type VI secretion system, reveals distinct oligomeric states of TssK and identifies a TssK-TssFG subcomplex. Biochem. J., 2014, 461(2), 291-304.
[16]
Dix, S.R.; Owen, H.J.; Sun, R.; Ahmad, A.; Shastri, S.; Spiewak, H.L.; Mosby, D.J.; Harris, M.J.; Batters, S.L.; Brooker, T.A.; Tzokov, S.B.; Sedelnikova, S.E.; Baker, P.J.; Bullough, P.A.; Rice, D.W.; Thomas, M.S. Structural insights into the function of type VI secretion system TssA subunits. Nat. Commun., 2018, 9(1), 4765.
[17]
Basler, M.; Pilhofer, M.; Henderson, G.P.; Jensen, G.J.; Mekalanos, J.J. Type VI secretion requires a dynamic contractile phage tail-like structure. Nature, 2012, 483(7388), 182-186.
[18]
Kudryashev, M.; Wang, R.Y.; Brackmann, M.; Scherer, S.; Maier, T.; Baker, D.; DiMaio, F.; Stahlberg, H.; Egelman, E.H.; Basler, M. Structure of the type VI secretion system contractile sheath. Cell, 2015, 160(5), 952-962.
[19]
Shneider, M.M.; Buth, S.A.; Ho, B.T.; Basler, M.; Mekalanos, J.J.; Leiman, P.G. PAAR-repeat proteins sharpen and diversify the type VI secretion system spike. Nature, 2013, 500(7462), 350-353.
[20]
Chang, Y.W.; Rettberg, L.A.; Ortega, D.R.; Jensen, G.J. In vivo structures of an intact type VI secretion system revealed by electron cryotomography. EMBO Rep., 2017, 18(7), 1090-1099.
[21]
Mougous, J.D.; Gifford, C.A.; Ramsdell, T.L.; Mekalanos, J.J. Threonine phosphorylation post-translationally regulates protein secretion in Pseudomonas aeruginosa. Nat. Cell Biol., 2007, 9(7), 797-803.
[22]
Hsu, F.; Schwarz, S.; Mougous, J.D. TagR promotes PpkA-catalysed type VI secretion activation in Pseudomonas aeruginosa. Mol. Microbiol., 2009, 72(5), 1111-1125.
[23]
Silverman, J.M.; Austin, L.S.; Hsu, F.; Hicks, K.G.; Hood, R.D.; Mougous, J.D. Separate inputs modulate phosphorylation-dependent and -independent type VI secretion activation. Mol. Microbiol., 2011, 82(5), 1277-1290.
[24]
Boyer, F.; Fichant, G.; Berthod, J.; Vandenbrouck, Y.; Attree, I. Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources? BMC Genomics, 2009, 10, 104.
[25]
Lin, J.S.; Pissaridou, P.; Wu, H.H.; Tsai, M.D.; Filloux, A.; Lai, E.M. TagF-mediated repression of bacterial type VI secretion systems involves a direct interaction with the cytoplasmic protein Fha. J. Biol. Chem., 2018, 293(23), 8829-8842.
[26]
Ostrowski, A.; Cianfanelli, F.R.; Porter, M.; Mariano, G.; Peltier, J.; Wong, J.J.; Swedlow, J.R.; Trost, M.; Coulthurst, S.J. Killing with proficiency: Integrated post-translational regulation of an offensive Type VI secretion system. PLoS Pathog., 2018, 14(7), e1007230.
[27]
Ok, C.K.; Chang, J.H. Purification, crystallization and X-ray crystallographic analysis of the type VI secretion system accessory protein TagF from Pseudomonas aeruginosa. Biodesign, 2017, 5(3), 118-121.
[28]
Adams, P.D.; Afonine, P.V.; Bunkoczi, G.; Chen, V.B.; Davis, I.W.; Echols, N.; Headd, J.J.; Hung, L.W.; Kapral, G.J.; Grosse-Kunstleve, R.W.; McCoy, A.J.; Moriarty, N.W.; Oeffner, R.; Read, R.J.; Richardson, D.C.; Richardson, J.S.; Terwilliger, T.C.; Zwart, P.H. PHENIX: A comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr., 2010, 66(Pt 2), 213-221.
[29]
Emsley, P.; Cowtan, K. Model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr, 2004, 60(Pt 12 Pt1), 2126-2132.
[30]
Holm, L.; Rosenstrom, P. Dali server: Conservation mapping in 3D. Nucleic Acids Res, 2010, 38, (Web Server issue), W545-549.
[31]
Chang, J.H.; Kim, Y.G. Crystal structure of the bacterial type VI secretion system component TssL from Vibrio cholerae. J. Microbiol., 2015, 53(1), 32-37.
[32]
Pacitto, A.; Ascher, D.B.; Wong, L.H.; Blaszczyk, B.K.; Nookala, R.K.; Zhang, N.; Dokudovskaya, S.; Levine, T.P.; Blundell, T.L. Lst4, the yeast Fnip1/2 orthologue, is a DENN-family protein. Open Biol., 2015, 5(12), 150174.
[33]
Menko, F.H.; van Steensel, M.A.; Giraud, S.; Friis-Hansen, L.; Richard, S.; Ungari, S.; Nordenskjold, M.; Hansen, T.V.; Solly, J.; Maher, E.R.; European, B.H.D.C. Birt-Hogg-Dube syndrome: Diagnosis and management. Lancet Oncol., 2009, 10(12), 1199-1206.
[34]
Baba, M.; Hong, S.B.; Sharma, N.; Warren, M.B.; Nickerson, M.L.; Iwamatsu, A.; Esposito, D.; Gillette, W.K.; Hopkins, R.F., 3rd; Hartley, J.L.; Furihata, M.; Oishi, S.; Zhen, W.; Burke, T.R., Jr; Linehan, W.M.; Schmidt, L.S.; Zbar, B. Folliculin encoded by the BHD gene interacts with a binding protein, FNIP1, and AMPK, and is involved in AMPK and mTOR signaling. Proc. Natl. Acad. Sci. USA, 2006, 103(42), 15552-15557.
[35]
Marat, A.L.; Dokainish, H.; McPherson, P.S. DENN domain proteins: Regulators of Rab GTPases. J. Biol. Chem., 2011, 286(16), 13791-13800.
[36]
De Franceschi, N.; Wild, K.; Schlacht, A.; Dacks, J.B.; Sinning, I.; Filippini, F.T. Longin and GAF domains: Structural evolution and adaptation to the subcellular trafficking machinery. Traffic, 2014, 15(1), 104-121.
[37]
Zhang, D.; Iyer, L.M.; He, F.; Aravind, L. Discovery of novel DENN proteins: Implications for the evolution of eukaryotic intracellular membrane structures and human disease. Front. Genet., 2012, 3, 283.

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