Abstract
Chlamydia trachomatis is an obligate intracellular bacterium that causes the most common sexually transmitted bacterial diseases in the world. With a biphasic developmental cycle, the bacteria utilize a type III secretion system (T3SS) to invade host cells as infectious elemental bodies, which then differentiate into actively dividing reticulate bodies. The regulation of the developmental cycle and the T3SS are linked by the bi-functional protein, specific Chlamydia chaperone 4 (Scc4). Scc4 is a class I T3SS chaperone forming a heterodimer with specific Chlamydia chaperone 1 (Scc1) to chaperone the essential virulence effector, Chlamydia outer membrane protein N. Scc4 also functions as a transcription factor by binding to the RNA polymerase holoenzyme between the flap region of the β subunit and region 4 of σ66. In order to investigate the mechanism behind Scc4’s dual functions and target its protein-protein interactions as a route for drug development, the structure and dynamics of Scc4 are being pursued. In the course of this effort, we assigned 89.2% of the backbone and sidechain 1H, 15N, and 13C resonances of full-length Scc4. The assigned chemical shifts were used to predict the secondary structure and dynamic properties. The type and order of Scc4’s determined secondary structure are consistent with the X-ray crystal structures of other bacterial T3SS chaperones.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
The raw NMR data and assignments are available through the Biological Magnetic Resonance Data Bank (http://www.bmrb.wisc.edu/), the accession number 28101.
Notes
Specific Chlamydia chaperone 4, formerly CT663, UniprotKB O84670.
RpoB, UniprotKB P0CE09.
σ66 or RpoD, UniprotKB P18333.
Specific Chlamydia chaperone 1 or CT088, UniprotKB O84090.
Low calcium response E or Chlamydia outer membrane protein N (CopN) or CT089, UniprotKB O84091.
Escherichia coli CesT (PDB id 1K3E chain A, Uniprot P58233), Yersinia pestis SycH (PDB id 1TTW chain A, Uniprot Q7BTX0), Salmonella enterica SicP (PDB id 1JYO chain A, Uniprot P0CL16), Pseudomonas syringae Shc (PDB id 4G6T chain A, Uniprot Q87UE6), and Salmonella enterica STM2138 (PDB id 3EPU chain B, Uniprot Q8ZNP3).
References
Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE (2000) The protein data bank. Nucleic Acids Res 28:235–242. https://doi.org/10.1093/nar/28.1.235
Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293. https://doi.org/10.1007/bf00197809
Eghbalnia HR, Wang L, Bahrami A, Assadi A, Markley JL (2005) Protein energetic conformational analysis from NMR chemical shifts (PECAN) and its use in determining secondary structural elements. J Biomol NMR 32:71–81. https://doi.org/10.1007/s10858-005-5705-1
Gao L, Cong Y, Plano GV, Rao X, Gisclair LN, Schesser Bartra S, Macnaughtan M, Shen L (2020) Context-dependent action of Scc4 reinforces control of the type III secretion system. J Bacteriol. https://doi.org/10.1128/JB.00132-20
Huang J, Lesser CF, Lory S (2008) The essential role of the CopN protein in Chlamydia pneumoniae intracellular growth. Nature 456:112–115. https://doi.org/10.1038/nature07355
Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10:845–858. https://doi.org/10.1038/nprot.2015.053
Krzywinski M (2020) Designing for color blindness: color choices and transformations for deuteranopia and other afflictions. http://mkweb.bcgsc.ca/colorblind/index.mhtml
Lee W, Tonelli M, Markley JL (2015) NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy. Bioinformatics 31:1325–1327. https://doi.org/10.1093/bioinformatics/btu830
Lescop E, Schanda P, Brutscher B (2007) A set of BEST triple-resonance experiments for time-optimized protein resonance assignment. J Magn Reson 187:163–169. https://doi.org/10.1016/j.jmr.2007.04.002
Page AL, Parsot C (2002) Chaperones of the type III secretion pathway: Jacks of all trades. Mol Microbiol 46:1–11. https://doi.org/10.1046/j.1365-2958.2002.03138.x
Rao X, Deighan P, Hua Z, Hu X, Wang J, Luo M, Wang J, Liang Y, Zhong G, Hochschild A, Shen L (2009) A regulator from Chlamydia trachomatis modulates the activity of RNA polymerase through direct interaction with the beta subunit and the primary sigma subunit. Genes Dev 23:1818–1829. https://doi.org/10.1101/gad.1784009
Schubot FD, Jackson MW, Penrose KJ, Cherry S, Tropea JE, Plano GV, Waugh DS (2005) Three-dimensional structure of a macromolecular assembly that regulates type III secretion in Yersinia pestis. J Mol Biol 346:1147–1161. https://doi.org/10.1016/j.jmb.2004.12.036
Shen Y, Bax A (2010) Prediction of Xaa-Pro peptide bond conformation from sequence and chemical shifts. J Biomol NMR 46:199–204. https://doi.org/10.1007/s10858-009-9395-y
Shen Y, Bax A (2013) Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks. J Biomol NMR 56:227–241. https://doi.org/10.1007/s10858-013-9741-y
Shen L, Macnaughtan MA, Frohlich KM, Cong YG, Goodwin OY, Chou CW, LeCour L, Krup K, Luo M, Worthylake DK (2015) Multipart chaperone-effector recognition in the type III secretion system of Chlamydia trachomatis. J Biol Chem 290:28141–28155. https://doi.org/10.1074/jbc.M115.670232
Shin J, Lee W, Lee W (2008) Structural proteomics by NMR spectroscopy. Expert Rev Proteomics 5:589–601. https://doi.org/10.1586/14789450.5.4.589
Spaeth KE, Chen Y-S, Valdivia RH (2009) The Chlamydia type III secretion system C-ring engages a chaperone-effector protein complex. PLoS Pathog 5:1–11. https://doi.org/10.1371/journal.ppat.1000579
Ukwaththage TO, Goodwin OY, Songok AC, Tafaro AM, Shen L, Macnaughtan MA (2019) Purification of tag-free Chlamydia trachomatis Scc4 for structural studies using sarkosyl-assisted on-column complex dissociation. Biochemistry 58:4284–4292. https://doi.org/10.1021/acs.biochem.9b00665
Ulrich EL, Akutsu H, Doreleijers JF, Harano Y, Ioannidis YE, Lin J, Livny M, Mading S, Maziuk D, Miller Z, Nakatani E, Schulte CF, Tolmie DE, Kent Wenger R, Yao H, Markley JL (2008) BioMagResBank. Nucleic Acids Res 36:D402–D408. https://doi.org/10.1093/nar/gkm957
Ying J, Delaglio F, Torchia DA, Bax A (2017) Sparse multidimensional iterative lineshape-enhanced (SMILE) reconstruction of both non-uniformly sampled and conventional NMR data. J Biomol NMR 68:101–118. https://doi.org/10.1007/s10858-016-0072-7
Acknowledgements
The authors thank Dr. Woonghee Lee for software assistance with NMRFAM-SPARKY and Dr. Ronnie Frederick for assistance in concentrating the NMR samples prior to data acquisition.
Funding
This work was supported by funding from the National Institutes of Health Grant R15GM109413 (Megan A. Macnaughtan). Thilini O. Ukwaththage thanks the Louisiana State University Department of Chemistry for providing support through teaching assistantships. This study made use of the National Magnetic Resonance Facility at Madison, which is supported by NIH grant P41GM103399 (NIGMS), old number: P41RR002301. Equipment was purchased with funds from the University of Wisconsin-Madison, the NIH P41GM103399, S10RR02781, S10RR08438, S10RR023438, S10RR025062, S10RR029220), the NSF (DMB-8415048, OIA-9977486, BIR-9214394), and the USDA.
Author information
Authors and Affiliations
Contributions
TOU prepared materials and contributed to the study conception and design with MAM. MT collected and processed the NMR data. The first draft of the manuscript was written by TOU and edited by MAM and MT. All authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical standard
The experiments comply with the current laws of the United States, the country in which they were performed.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Ukwaththage, T.O., Tonelli, M. & Macnaughtan, M.A. Backbone and sidechain resonance assignments and secondary structure of Scc4 from Chlamydia trachomatis. Biomol NMR Assign 14, 301–307 (2020). https://doi.org/10.1007/s12104-020-09965-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12104-020-09965-4