- Research article
- Open Access
Proteolytic activation of Chlamydia trachomatis HTRA is mediated by PDZ1 domain interactions with protease domain loops L3 and LC and beta strand β5
Cellular & Molecular Biology Letters volume 18, pages522–537(2013)
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.
Chlamydia trachomatis high temperature requirement A
major outer membrane protein
mean residue weight
outer membrane protein A
polymorphic membrane protein C
sodium dodecyl sulphate polyacrylamide gel electrophoresis
Stephens, R.S. The cellular paradigm of chlamydial pathogenesis. Trends Microbiol. 11 (2003) 44–51.
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.
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Rawlings, N.D., Barrett, A.J. and Bateman, A. MEROPS: the peptidase database. Nucleic Acids Res. 38 (2009) 227–233.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Finn, R.D., Clements, J. and Eddy, S.R. HMMER web server: interactive sequence similarity searching. Nucleic Acids Res. 39 (2011) 29–37.
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.
Sali, A. and Blundell, T.L. Comparative protein modeling by satisfaction of spatial restraints. J. Mol. Biol. 234 (1993) 779–815.
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.
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.
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.
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.
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.
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Marsh, J.W., Lott, W.B., Tyndall, J.D.A. et al. Proteolytic activation of Chlamydia trachomatis HTRA is mediated by PDZ1 domain interactions with protease domain loops L3 and LC and beta strand β5. Cell Mol Biol Lett 18, 522–537 (2013). https://doi.org/10.2478/s11658-013-0103-2