Jump to ContentJump to Main Navigation
Show Summary Details
More options …

Cellular and Molecular Biology Letters

See all formats and pricing
More options …
Volume 18, Issue 4

Proteolytic activation of Chlamydia trachomatis HTRA is mediated by PDZ1 domain interactions with protease domain loops L3 and LC and beta strand β5

James Marsh / William Lott / Joel Tyndall / Wilhelmina Huston
Published Online: 2013-12-29 | DOI: https://doi.org/10.2478/s11658-013-0103-2


Chlamydia trachomatis is a bacterial pathogen responsible for one of the most prevalent sexually transmitted infections worldwide. Its unique development cycle has limited our understanding of its pathogenic mechanisms. However, CtHtrA has recently been identified as a potential C. trachomatis virulence factor. CtHtrA is a tightly regulated quality control protein with a monomeric structural unit comprised of a chymotrypsin-like protease domain and two PDZ domains. Activation of proteolytic activity relies on the C-terminus of the substrate allosterically binding to the PDZ1 domain, which triggers subsequent conformational change and oligomerization of the protein into 24-mers enabling proteolysis. This activation is mediated by a cascade of precise structural arrangements, but the specific CtHtrA residues and structural elements required to facilitate activation are unknown. Using in vitro analysis guided by homology modeling, we show that the mutation of residues Arg362 and Arg224, predicted to disrupt the interaction between the CtHtrA PDZ1 domain and loop L3, and between loop L3 and loop LD, respectively, are critical for the activation of proteolytic activity. We also demonstrate that mutation to residues Arg299 and Lys160, predicted to disrupt PDZ1 domain interactions with protease loop LC and strand β5, are also able to influence proteolysis, implying their involvement in the CtHtrA mechanism of activation. This is the first investigation of protease loop LC and strand β5 with respect to their potential interactions with the PDZ1 domain. Given their high level of conservation in bacterial HtrA, these structural elements may be equally significant in the activation mechanism of DegP and other HtrA family members.

Keywords: Chlamydia; HtrA; DegP; Protease; Oligomerization

  • [1] Stephens, R.S. The cellular paradigm of chlamydial pathogenesis. Trends Microbiol. 11 (2003) 44–51. http://dx.doi.org/10.1016/S0966-842X(02)00011-2CrossrefGoogle Scholar

  • [2] Low, N. Incidence of severe reproductive tract complications associated with diagnosed genital chlamydial infection: the Uppsala Women’s Cohort Study. Sex. Transm. Infect. 82 (2006) 212–218. http://dx.doi.org/10.1136/sti.2005.017186CrossrefGoogle Scholar

  • [3] Huston, W.M., Theodoropoulos, C., Mathews, S.A. and Timms, P. Chlamydia trachomatis responds to heat shock, penicillin-induced persistence, and IFNgamma persistence by altering levels of the extracytoplasmic stress response protease HtrA. BMC Microbiol. 8 (2008). Web of ScienceGoogle Scholar

  • [4] Pedersen, L.L., Radulic, M.M., Doric, M.M. and Kwaik, Y.Y.A. HtrA homologue of Legionella pneumophila: an indispensable element for intracellular infection of mammalian but not protozoan cells. Infect. Immun. 69 (2001) 2569–2579. http://dx.doi.org/10.1128/IAI.69.4.2569-2579.2001CrossrefGoogle Scholar

  • [5] Lewis, C., Skovierova, H., Rowley, G., Rezuchova, B., Homerova, D., Stevenson, A., Spencer, J., Farn, J., Kormanec, J. and Roberts, M. Salmonella enterica serovar Typhimurium HtrA: regulation of expression and role of the chaperone and protease activities during infection. Microbiology 155 (2009) 873–881. http://dx.doi.org/10.1099/mic.0.023754-0CrossrefGoogle Scholar

  • [6] Hoy, B., Lower, M., Weydig, C., Carra, G., Tegtmeyer, N., Geppert, T., Schroder, P., Sewald, N., Backert, S., Schneider, G. and Wessler, S. Helicobacter pylori HtrA is a new secreted virulence factor that cleaves E-cadherin to disrupt intercellular adhesion. EMBO Rep. 11 (2010) 798–804. http://dx.doi.org/10.1038/embor.2010.114Web of ScienceCrossrefGoogle Scholar

  • [7] Strauch, K.L. and Beckwith, J. An Escherichia coli mutation preventing degradation of abnormal periplasmic proteins. Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 1576–1580. http://dx.doi.org/10.1073/pnas.85.5.1576CrossrefGoogle Scholar

  • [8] Lipinska, B.B., Zylicz, M. and Georgopoulos, C.C. The HtrA (DegP) protein, essential for Escherichia coli survival at high temperatures, is an endopeptidase. J. Bacteriol. 172 (1990) 1791–1797. Google Scholar

  • [9] Spiess, C.C., Beil, A.A. and Ehrmann, M.M. A temperature-dependent switch from chaperone to protease in a widely conserved heat shock protein. Cell 97 (1999) 339–347. http://dx.doi.org/10.1016/S0092-8674(00)80743-6CrossrefGoogle Scholar

  • [10] Baldi, A., De Luca, A., Morini, M., Battista, T., Felsani, A., Baldi, F., Catricalà, C., Amantea, A., Noonan, D.M., Albini, A., Natali, P.G., Lombardi, D. and Paggi, M.G. The HtrA1 serine protease is down-regulated during human melanoma progression and represses growth of metastatic melanoma cells. Oncogene 21 (2002) 6684–6688. http://dx.doi.org/10.1038/sj.onc.1205911CrossrefGoogle Scholar

  • [11] Li, W., Srinivasula, S.M., Chai, J., Li, P., Wu, J., Zhang, Z., Alnemri, E.S. and Shi, Y. Structural insights into the pro-apoptotic function of mitochondrial serine protease HtrA2/Omi. Nat. Struct. Biol. 9 (2002) 436–441. http://dx.doi.org/10.1038/nsb795CrossrefGoogle Scholar

  • [12] Clausen, T., Southan, C. and Ehrmann, M.M. The HtrA family of proteases: implications for protein composition and cell fate. Mol. Cell 10 (2002) 443–455. http://dx.doi.org/10.1016/S1097-2765(02)00658-5CrossrefGoogle Scholar

  • [13] Grau, S., Baldi, A., Bussani, R., Tian, X., Stefanescu, R., Przybylski, M., Richards, P., Jones, S.A., Shridhar, V., Clausen, T. and Ehrmann, M.M. Implications of the serine protease HtrA1 in amyloid precursor protein processing. Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 6021–6026. http://dx.doi.org/10.1073/pnas.0501823102CrossrefGoogle Scholar

  • [14] Hansen, G. and Hilgenfeld, R. Architecture and regulation of HtrA-family proteins involved in protein quality control and stress response. Cell. Mol. Life Sci. 70 (2012) 761–775. http://dx.doi.org/10.1007/s00018-012-1076-4CrossrefGoogle Scholar

  • [15] Rawlings, N.D., Barrett, A.J. and Bateman, A. MEROPS: the peptidase database. Nucleic Acids Res. 38 (2009) 227–233. http://dx.doi.org/10.1093/nar/gkp971CrossrefGoogle Scholar

  • [16] Spiers, A. PDZ domains facilitate binding of high temperature requirement protease A (HtrA) and tail-specific protease (Tsp) to heterologous substrates through recognition of the small stable RNA A (ssrA)-encoded peptide. J. Biol. Chem. 277 (2002) 39443–39449. http://dx.doi.org/10.1074/jbc.M202790200CrossrefGoogle Scholar

  • [17] Kolmar, H.H., Waller, P.R. and Sauer, R.T. The DegP and DegQ periplasmic endoproteases of Escherichia coli: specificity for cleavage sites and substrate conformation. J. Bacteriol. 178 (1996) 5925–5929. Google Scholar

  • [18] Kim, S.S. and Sauer, R.T. Cage assembly of DegP protease is not required for substrate-dependent regulation of proteolytic activity or high-temperature cell survival. Proc. Natl. Acad. Sci. U.S.A. 109 (2012) 7263–7268. http://dx.doi.org/10.1073/pnas.1204791109CrossrefWeb of ScienceGoogle Scholar

  • [19] Krojer, T., Garrido-Franco, M., Huber, R., Ehrmann, M.M. and Clausen, T. Crystal structure of DegP (HtrA) reveals a new protease-chaperone machine. Nature 416 (2002) 455–459. http://dx.doi.org/10.1038/416455aCrossrefGoogle Scholar

  • [20] Krojer, T., Sawa, J., Huber, R. and Clausen, T. HtrA proteases have a conserved activation mechanism that can be triggered by distinct molecular cues. Nat. Struct. Mol. Biol. 17 (2010) 844–852. http://dx.doi.org/10.1038/nsmb.1840CrossrefWeb of ScienceGoogle Scholar

  • [21] Clausen, T., Kaiser, M., Huber, R. and Ehrmann, M.M. HtrA proteases: regulated proteolysis in protein quality control. Nat. Rev. Mol. Cell Biol. 12 (2011) 152–162. http://dx.doi.org/10.1038/nrm3065Web of ScienceCrossrefGoogle Scholar

  • [22] Jiang, J., Zhang, X., Chen, Y., Wu, Y., Zhou, Z.H., Chang, Z. and Sui, S. Activation of DegP chaperone-protease via formation of large cage-like oligomers upon binding to substrate proteins. Proc. Natl. Acad. Sci. U.S.A. 105 (2008) 11939–11944. http://dx.doi.org/10.1073/pnas.0805464105Web of ScienceCrossrefGoogle Scholar

  • [23] Sohn, J., Grant, R.A. and Sauer, R.T. OMP peptides activate the DegS stresssensor protease by a relief of inhibition mechanism. Structure 17 (2009) 1411–1421. http://dx.doi.org/10.1016/j.str.2009.07.017Web of ScienceCrossrefGoogle Scholar

  • [24] Wilken, C., Kitzing, K., Kurzbauer, R., Ehrmann, M.M. and Clausen, T. Crystal structure of the DegS stress sensor: how a PDZ domain recognizes misfolded protein and activates a protease. Cell 117 (2004) 483–494. http://dx.doi.org/10.1016/S0092-8674(04)00454-4CrossrefGoogle Scholar

  • [25] MohamedMohaideen, N.N., Palaninathan, S.K., Morin, P.M., Williams, B.J., Braunstein, M., Tichy, S.E., Locker, J., Russell, D.H., Jacobs, W.R. and Sacchettini, J.C. Structure and function of the virulence-associated hightemperature requirement A of Mycobacterium tuberculosis. Biochemistry 47 (2008) 6092–6102. http://dx.doi.org/10.1021/bi701929mCrossrefGoogle Scholar

  • [26] Huston, W.M., Tyndall, J.D.A., Lott, W.B., Stansfield, S.H. and Timms, P. Unique residues involved in activation of the multitasking protease/chaperone HtrA from Chlamydia trachomatis. PLoS ONE 6 (2011) e24547. http://dx.doi.org/10.1371/journal.pone.0024547CrossrefGoogle Scholar

  • [27] Finn, R.D., Clements, J. and Eddy, S.R. HMMER web server: interactive sequence similarity searching. Nucleic Acids Res. 39 (2011) 29–37. http://dx.doi.org/10.1093/nar/gkr367CrossrefWeb of ScienceGoogle Scholar

  • [28] Shi, J., Blundell, T.L. and Mizuguchi, K. FUGUE: sequence-structure homology recognition using environment-specific substitution tables and structure-dependent gap penalties. J. Mol. Biol. 310 (2001) 243–257. http://dx.doi.org/10.1006/jmbi.2001.4762CrossrefGoogle Scholar

  • [29] Sali, A. and Blundell, T.L. Comparative protein modeling by satisfaction of spatial restraints. J. Mol. Biol. 234 (1993) 779–815. http://dx.doi.org/10.1006/jmbi.1993.1626CrossrefGoogle Scholar

  • [30] Ko, J., Lee, D., Park, H., Coutsias, E.A., Lee, J. and Seok, C. The FALC-Loop web server for protein loop modeling. Nucleic Acids Res. 39 (2011) 210–214. http://dx.doi.org/10.1093/nar/gkr352CrossrefGoogle Scholar

  • [31] Laskowski, R.A., MacArthur, M.W., Moss, D.S. and Thornton, J.M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26 (1993) 283–291. http://dx.doi.org/10.1107/S0021889892009944CrossrefGoogle Scholar

  • [32] Huston, W.M., Swedberg, J.E., Harris, J.M., Walsh, T.P., Mathews, S.A. and Timms, P. The temperature activated HtrA protease from pathogen Chlamydia trachomatis acts as both a chaperone and protease at 37°C. FEBS Lett. 581 (2007) 3382–3386. http://dx.doi.org/10.1016/j.febslet.2007.06.039Web of ScienceCrossrefGoogle Scholar

  • [33] Berry, L.J., Hickey, D.K., Skelding, K.A., Bao, S., Rendina, A.M., Hansbro, P.M., Gockel, C.M. and Beagley, K.W. Transcutaneous immunisation with combined cholera toxin and CpG adjuvant protects against Chlamydia muridarum genital tract infection. Infect. Immun. 72 (2004) 1019–1028. http://dx.doi.org/10.1128/IAI.72.2.1019-1028.2004CrossrefGoogle Scholar

  • [34] Hauske, P., Meltzer, M., Ottmann, C., Krojer, T., Clausen, T., Ehrmann, M.M. and Kaiser, M. Selectivity profiling of DegP substrates and inhibitors. Bioorgan. Med. Chem. 17 (2009) 2920–2924. http://dx.doi.org/10.1016/j.bmc.2009.01.073CrossrefGoogle Scholar

About the article

Published Online: 2013-12-29

Published in Print: 2013-12-01

Citation Information: Cellular and Molecular Biology Letters, Volume 18, Issue 4, Pages 522–537, ISSN (Online) 1689-1392, DOI: https://doi.org/10.2478/s11658-013-0103-2.

Export Citation

© 2013 Versita Warsaw. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. BY-NC-ND 3.0

Citing Articles

Here you can find all Crossref-listed publications in which this article is cited. If you would like to receive automatic email messages as soon as this article is cited in other publications, simply activate the “Citation Alert” on the top of this page.

Urszula Zarzecka, Anna Modrak-Wojcik, Martyna Bayassi, Maciej Szewczyk, Artur Gieldon, Adam Lesner, Tomasz Koper, Agnieszka Bzowska, Maurizio Sanguinetti, Steffen Backert, Barbara Lipinska, and Joanna Skorko-Glonek
International Journal of Biological Macromolecules, 2017
James W Marsh, Vanissa A Ong, William B Lott, Peter Timms, Joel DA Tyndall, and Wilhelmina M Huston
Future Microbiology, 2017, Volume 12, Number 9, Page 817
James W. Marsh, Bryan A. Wee, Joel D.A. Tyndall, William B. Lott, Robert J. Bastidas, Harlan D. Caldwell, Raphael H. Valdivia, L. Kari, and Wilhelmina M. Huston
BMC Microbiology, 2015, Volume 15, Number 1

Comments (0)

Please log in or register to comment.
Log in