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Structural evidence suggests that antiactivator ExsD from Pseudomonas aeruginosa is a DNA binding protein

  1. Robert C. Bernhards1,
  2. Xing Jing1,
  3. Nancy J. Vogelaar1,
  4. Howard Robinson2,
  5. Florian D. Schubot1,*

Article first published online: 6 JAN 2009

DOI: 10.1002/pro.48

Issue

Protein Science

Protein Science

Volume 18, Issue 3, pages 503–513, March 2009

Additional Information

How to Cite

Bernhards, R. C., Jing, X., Vogelaar, N. J., Robinson, H. and Schubot, F. D. (2009), Structural evidence suggests that antiactivator ExsD from Pseudomonas aeruginosa is a DNA binding protein. Protein Science, 18: 503–513. doi: 10.1002/pro.48

Author Information

  1. 1

    Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24060

  2. 2

    Biology Department, Brookhaven National Laboratory, Upton, New York 11973-5000

*Department of Biological Sciences, Washington Street, Life Science I, Room 125

Publication History

  1. Issue published online: 23 FEB 2009
  2. Article first published online: 6 JAN 2009
  3. Accepted manuscript online: 6 JAN 2009 12:00AM EST
  4. Manuscript Accepted: 1 DEC 2008
  5. Manuscript Revised: 25 NOV 2008
  6. Manuscript Received: 7 OCT 2008

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  • Virginia Polytechnic Institute and State University

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References

  • 1
    Labrec EH, Schneider H, Magnani TJ, Formal SB ( 1964) Epithelial cell penetration as an essential step in the pathogenesis of bacillary dysentery. J Bacteriol 88: 1503–1518.
  • 2
    Crouch Brewer S, Wunderink RG, Jones CB, Leeper KV, Jr ( 1996) Ventilator-associated pneumonia due to Pseudomonas aeruginosa. Chest 109: 1019–1029.
  • 3
    Cheng KH, Leung SL, Hoekman HW, Beekhuis WH, Mulder PG, Geerards AJ, Kijlstra A ( 1999) Incidence of contact-lens-associated microbial keratitis and its related morbidity. Lancet 354: 181–185.
  • 4
    Richards MJ, Edwards JR, Culver DH, Gaynes RP ( 1999) Nosocomial infections in medical intensive care units in the United States. National Nosocomial Infections Surveillance System. Crit Care Med 27: 887–892.
  • 5
    Garau J, Gomez L ( 2003) Pseudomonas aeruginosa pneumonia. Curr Opin Infect Dis 16: 135–143.
  • 6
    Obritsch MD, Fish DN, MacLaren R, Jung R ( 2005) Nosocomial infections due to multidrug-resistant Pseudomonas aeruginosa: epidemiology and treatment options. Pharmacotherapy 25: 1353–1364.
  • 7
    Stein GE ( 2005) Antimicrobial resistance in the hospital setting: impact, trends, and infection control measures. Pharmacotherapy 25: 44S–54S.
  • 8
    DeRyke CA, Lee SY, Kuti JL, Nicolau DP ( 2006) Optimising dosing strategies of antibacterials utilising pharmacodynamic principles: impact on the development of resistance. Drugs 66: 1–14.
  • 9
    Finck-Barbanðcon V, Goranson J, Zhu L, Sawa T, Wiener-Kronish JP, Fleiszig SM, Wu C, Mende-Mueller L, Frank DW ( 1997) ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury. Mol Microbiol 25: 547–557.
    Direct Link:
  • 10
    Fraylick JE, La Rocque JR, Vincent TS, Olson JC ( 2001) Independent and coordinate effects of ADP-ribosyltransferase and GTPase-activating activities of exoenzyme S on HT-29 epithelial cell function. Infect Immun 69: 5318–5328.
  • 11
    Krall R, Sun J, Pederson KJ, Barbieri JT ( 2002) In vivo rho GTPase-activating protein activity of Pseudomonas aeruginosa cytotoxin ExoS. Infect Immun 70: 360–367.
  • 12
    Sun J, Barbieri JT ( 2003) Pseudomonas aeruginosa ExoT ADP-ribosylates CT10 regulator of kinase (Crk) proteins. J Biol Chem 278: 32794–32800.
  • 13
    Karavolos MH, Roe AJ, Wilson M, Henderson J, Lee JJ, Gally DL, Khan CM ( 2005) Type III secretion of the Salmonella effector protein SopE is mediated via an N-terminal amino acid signal and not an mRNA sequence. J Bacteriol 187: 1559–1567.
  • 14
    Shaver CM, Hauser AR ( 2004) Relative contributions of Pseudomonas aeruginosa ExoU, ExoS, and ExoT to virulence in the lung. Infect Immun 72: 6969–6977.
  • 15
    Sory MP, Boland A, Lambermont I, Cornelis GR ( 1995) Identification of the YopE and YopH domains required for secretion and internalization into the cytosol of macrophages, using the cyaA gene fusion approach. Proc Natl Acad Sci USA 92: 11998–12002.
  • 16
    Anderson DM, Schneewind O ( 1997) A mRNA signal for the type III secretion of Yop proteins by Yersinia enterocolitica. Science 278: 1140–1143.
  • 17
    Ramamurthi KS, Schneewind O ( 2005) A synonymous mutation in Yersinia enterocolitica yopE affects the function of the YopE type III secretion signal. J Bacteriol 187: 707–715.
  • 18
    Birtalan SC, Phillips RM, Ghosh P ( 2002) Three-dimensional secretion signals in chaperone-effector complexes of bacterial pathogens. Mol Cell 9: 971–980.
  • 19
    Stebbins CE, Galâan JE ( 2001) Maintenance of an unfolded polypeptide by a cognate chaperone in bacterial type III secretion. Nature 414: 77–81.
  • 20
    Frank DW, Nair G, Schweizer HP ( 1994) Construction and characterization of chromosomal insertional mutations of the Pseudomonas aeruginosa exoenzyme S trans-regulatory locus. Infect Immun 62: 554–563.
  • 21
    Yahr TL, Frank DW ( 1994) Transcriptional organization of the trans-regulatory locus which controls exoenzyme S synthesis in Pseudomonas aeruginosa. J Bacteriol 176: 3832–3838.
  • 22
    Yahr TL, Hovey AK, Kulich SM, Frank DW ( 1995) Transcriptional analysis of the Pseudomonas aeruginosa exoenzyme S structural gene. J Bacteriol 177: 1169–1178.
  • 23
    Lobell RB, Schleif RF ( 1990) DNA looping and unlooping by AraC protein. Science 250: 528–532.
  • 24
    Ha UH, Kim J, Badrane H, Jia J, Baker HV, Wu D, Jin S ( 2004) An in vivo inducible gene of Pseudomonas aeruginosa encodes an anti-ExsA to suppress the type III secretion system. Mol Microbiol 54: 307–320.
    Direct Link:
  • 25
    McCaw ML, Lykken GL, Singh PK, Yahr TL ( 2002) ExsD is a negative regulator of the Pseudomonas aeruginosa type III secretion regulon. Mol Microbiol 46: 1123–1133.
    Direct Link:
  • 26
    Dasgupta N, Lykken GL, Wolfgang MC, Yahr TL ( 2004) A novel anti-anti-activator mechanism regulates expression of the Pseudomonas aeruginosa type III secretion system. Mol Microbiol 53: 297–308.
    Direct Link:
  • 27
    Urbanowski ML, Lykken GL, Yahr TL ( 2005) A secreted regulatory protein couples transcription to the secretory activity of the Pseudomonas aeruginosa type III secretion system. Proc Natl Acad Sci USA 102: 9930–9935.
  • 28
    Lykken GL, Chen G, Brutinel ED, Chen L, Yahr TL ( 2006) Characterization of ExsC and ExsD self-association and heterocomplex formation. J Bacteriol 188: 6832–6840.
  • 29
    Zheng Z, Chen G, Joshi S, Brutinel ED, Yahr TL, Chen L ( 2007) Biochemical characterization of a regulatory cascade controlling transcription of the Pseudomonas aeruginosa type III secretion system. J Biol Chem 282: 6136–6142.
  • 30
    Rayment I ( 1997) Reductive alkylation of lysine residues to alter crystallization properties of proteins. Methods Enzymol 276: 171–179.
  • 31
    Brunger AT ( 1992) Free R value: a novel statistical quantity for assessing the accuracy of crystal structures. Nature 355: 472–475.
  • 32
    DeLano WL ( 2001) The PyMOL molecular graphics system. San Carlos, CA: DeLano Scientific LLC. Available at: http://www.pymol.org.
  • 33
    Holm L, Sander C ( 1995) Dali: a network tool for protein structure comparison. Trends Biochem Sci 20: 478–480.
  • 34
    Abola EE, Bernstein FC, Bryant SH, Koetzel TF, Weng J Protein Data Bank. In AllenFH, BergerhoffG, SieversR, Eds. ( 1987) Crystallographic databases—information content, software systems, scientific applications. Bonn: Data Commission of the International Union of Crystallography, pp 107–132.
  • 35
    De Silva RS, Kovacikova G, Lin W, Taylor RK, Skorupski K, Kull FJ ( 2005) Crystal structure of the virulence gene activator AphA from Vibrio cholerae reveals it is a novel member of the winged helix transcription factor superfamily. J Biol Chem 280: 13779–13783.
  • 36
    Khare D, Ziegelin G, Lanka E, Heinemann U ( 2004) Sequence-specific DNA binding determined by contacts outside the helix-turn-helix motif of the ParB homolog KorB. Nat Struct Mol Biol 11: 656–663.
  • 37
    Coulocheri SA, Pigis DG, Papavassiliou KA, Papavassiliou AG ( 2007) Hydrogen bonds in protein-DNA complexes: where geometry meets plasticity. Biochimie 89: 1291–1303.
  • 38
    Jen-Jacobson L ( 1997) Protein-DNA recognition complexes: conservation of structure and binding energy in the transition state. Biopolymers 44: 153–180.
    Direct Link:
  • 39
    Matulis D, Kranz JK, Salemme FR, Todd MJ ( 2005) Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor. Biochemistry 44: 5258–5266.
  • 40
    Niesen FH, Berglund H, Vedadi M ( 2007) The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc 2: 2212–2221.
  • 41
    Korkhin Y, Kalb AJ, Peretz M, Bogin O, Burstein Y, Frolow F ( 1999) Oligomeric integrity—the structural key to thermal stability in bacterial alcohol dehydrogenases. Protein Sci 8: 1241–1249.
    Direct Link:
  • 42
    Tanaka Y, Tsumoto K, Yasutake Y, Umetsu M, Yao M, Fukada H, Tanaka I, Kumagai I ( 2004) How oligomerization contributes to the thermostability of an archaeon protein. Protein L-isoaspartyl-O-methyltransferase from Sulfolobus tokodaii. J Biol Chem 279: 32957–32967.
  • 43
    Natalello A, Doglia SM, Carey J, Grandori R ( 2007) Role of flavin mononucleotide in the thermostability and oligomerization of Escherichia coli stress-defense protein WrbA. Biochemistry 46: 543–553.
  • 44
    Rietsch A, Vallet-Gely I, Dove SL, Mekalanos JJ ( 2005) ExsE, a secreted regulator of type III secretion genes in Pseudomonas aeruginosa. Proc Natl Acad Sci USA 102: 8006–8011.
  • 45
    Bingle LE, Macartney DP, Fantozzi A, Manzoor SE, Thomas CM ( 2005) Flexibility in repression and cooperativity by KorB of broad host range IncP-1 plasmid RK2. J Mol Biol 349: 302–316.
  • 46
    Chugani SA, Parsek MR, Chakrabarty AM ( 1998) Transcriptional repression mediated by LysR-type regulator CatR bound at multiple binding sites. J Bacteriol 180: 2367–2372.
  • 47
    Nallamsetty S, Austin BP, Penrose KJ, Waugh DS ( 2005) Gateway vectors for the production of combinatorially-tagged His6-MBP fusion proteins in the cytoplasm and periplasm of Escherichia coli. Protein Sci 14: 2964–2971.
    Direct Link:
  • 48
    Kapust RB, Tozser J, Fox JD, Anderson DE, Cherry S, Copeland TD, Waugh DS ( 2001) Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng 14: 993–1000.
  • 49
    Doubliâe S ( 1997) Preparation of selenomethionyl proteins for phase determination. Methods Enzymol 276: 523–530.
  • 50
    Otwinowski ZM, Minor W ( 1997) HKL2000. Methods Enzymol 276: 307–326.
  • 51
    Zwart PH, Afonine PV, Grosse-Kunstleve RW, Hung LW, Ioerger TR, McCoy AJ, McKee E, Moriarty NW, Read RJ, Sacchettini JC, Sauter NK, Storoni LC, Terwilliger TC, Adams PD ( 2008) Automated structure solution with the PHENIX suite. Methods Mol Biol 426: 419–435.
  • 52
    Jones TA, Zou J-Y, Cowan SW, Kjeldgaard M ( 1991) Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Cryst A 47: 110–119.
    Direct Link:
  • 53
    Laskowski RA, MacArthur MW, Moss DS, Thornton JM ( 1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Cryst 26: 283–291.
    Direct Link:
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