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Structure of a pathogen effector reveals the enzymatic mechanism of a novel acetyltransferase family

Nature Structural & Molecular Biology volume 23, pages 847–852 (2016)Cite this article

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Abstract

Effectors secreted by the type III secretion system are essential for bacterial pathogenesis. Members of the Yersinia outer-protein J (YopJ) family of effectors found in diverse plant and animal pathogens depend on a protease-like catalytic triad to acetylate host proteins and produce virulence. However, the structural basis for this noncanonical acetyltransferase activity remains unknown. Here, we report the crystal structures of the YopJ effector HopZ1a, produced by the phytopathogen Pseudomonas syringae, in complex with the eukaryote-specific cofactor inositol hexakisphosphate (IP6) and/or coenzyme A (CoA). Structural, computational and functional characterizations reveal a catalytic core with a fold resembling that of ubiquitin-like cysteine proteases and an acetyl-CoA-binding pocket formed after IP6-induced structural rearrangements. Modeling-guided mutagenesis further identified key IP6-interacting residues of Salmonella effector AvrA that are required for acetylating its substrate. Our study reveals the structural basis of a novel class of acetyltransferases and the conserved allosteric regulation of YopJ effectors by IP6.

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Figure 1: Crystal structure of HopZ1a in complex with IP6 and CoA.
Figure 2: IP6 binding is critical for the acetyltransferase activity of HopZ1a.
Figure 3: Identification of the AcCoA-binding pocket.
Figure 4: IP6-dependent AcCoA binding in YopJ family effectors.

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References

  1. Galán, J.E., Lara-Tejero, M., Marlovits, T.C. & Wagner, S. Bacterial type III secretion systems: specialized nanomachines for protein delivery into target cells. Annu. Rev. Microbiol. 68, 415–438 (2014).

    Article  Google Scholar 

  2. Dean, P. Functional domains and motifs of bacterial type III effector proteins and their roles in infection. FEMS Microbiol. Rev. 35, 1100–1125 (2011).

    Article  CAS  Google Scholar 

  3. Ma, W., Dong, F.F.T., Stavrinides, J. & Guttman, D.S. Type III effector diversification via both pathoadaptation and horizontal transfer in response to a coevolutionary arms race. PLoS Genet. 2, e209 (2006).

    Article  Google Scholar 

  4. Orth, K. et al. Inhibition of the mitogen-activated protein kinase kinase superfamily by a Yersinia effector. Science 285, 1920–1923 (1999).

    Article  CAS  Google Scholar 

  5. Mukherjee, S. et al. Yersinia YopJ acetylates and inhibits kinase activation by blocking phosphorylation. Science 312, 1211–1214 (2006).

    Article  CAS  Google Scholar 

  6. Trosky, J.E. et al. VopA inhibits ATP binding by acetylating the catalytic loop of MAPK kinases. J. Biol. Chem. 282, 34299–34305 (2007).

    Article  CAS  Google Scholar 

  7. Jones, R.M. et al. Salmonella AvrA coordinates suppression of host immune and apoptotic defenses via JNK pathway blockade. Cell Host Microbe 3, 233–244 (2008).

    Article  CAS  Google Scholar 

  8. Lee, A.H. et al. A bacterial acetyltransferase destroys plant microtubule networks and blocks secretion. PLoS Pathog. 8, e1002523 (2012).

    Article  CAS  Google Scholar 

  9. Jiang, S. et al. Bacterial effector activates jasmonate signaling by directly targeting JAZ transcriptional repressors. PLoS Pathog. 9, e1003715 (2013).

    Article  Google Scholar 

  10. Tasset, C. et al. Autoacetylation of the Ralstonia solanacearum effector PopP2 targets a lysine residue essential for RRS1-R-mediated immunity in Arabidopsis. PLoS Pathog. 6, e1001202 (2010).

    Article  Google Scholar 

  11. Le Roux, C. et al. A receptor pair with an integrated decoy converts pathogen disabling of transcription factors to immunity. Cell 161, 1074–1088 (2015).

    Article  CAS  Google Scholar 

  12. Sarris, P.F. et al. A plant immune receptor detects pathogen effectors that target WRKY transcription factors. Cell 161, 1089–1100 (2015).

    Article  CAS  Google Scholar 

  13. Lee, J. et al. Acetylation of an NB-LRR plant immune-effector complex suppresses immunity. Cell Rep. 13, 1670–1682 (2015).

    Article  CAS  Google Scholar 

  14. Cheong, M.S. et al. AvrBsT acetylates Arabidopsis ACIP1, a protein that associates with microtubules and is required for immunity. PLoS Pathog. 10, e1003952 (2014).

    Article  Google Scholar 

  15. Mittal, R., Peak-Chew, S.Y., Sade, R.S., Vallis, Y. & McMahon, H.T. The acetyltransferase activity of the bacterial toxin YopJ of Yersinia is activated by eukaryotic host cell inositol hexakisphosphate. J. Biol. Chem. 285, 19927–19934 (2010).

    Article  CAS  Google Scholar 

  16. Ma, K.W. et al. Two serine residues in Pseudomonas syringae effector HopZ1a are required for acetyltransferase activity and association with the host co-factor. New Phytol. 208, 1157–1168 (2015).

    Article  CAS  Google Scholar 

  17. Choudhary, C., Weinert, B.T., Nishida, Y., Verdin, E. & Mann, M. The growing landscape of lysine acetylation links metabolism and cell signalling. Nat. Rev. Mol. Cell Biol. 15, 536–550 (2014).

    Article  CAS  Google Scholar 

  18. Orth, K. et al. Disruption of signaling by Yersinia effector YopJ, a ubiquitin-like protein protease. Science 290, 1594–1597 (2000).

    Article  CAS  Google Scholar 

  19. Mossessova, E. & Lima, C.D. Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast. Mol. Cell 5, 865–876 (2000).

    Article  CAS  Google Scholar 

  20. Holm, L. & Rosenström, P. Dali server: conservation mapping in 3D. Nucleic Acids Res. 38, W545–W549 (2010).

    Article  CAS  Google Scholar 

  21. Vetting, M.W. et al. Structure and functions of the GNAT superfamily of acetyltransferases. Arch. Biochem. Biophys. 433, 212–226 (2005).

    Article  CAS  Google Scholar 

  22. Utley, R.T. & Côté, J. The MYST family of histone acetyltransferases. Curr. Top. Microbiol. Immunol. 274, 203–236 (2003).

    CAS  PubMed  Google Scholar 

  23. Mukherjee, S., Hao, Y.H. & Orth, K. A newly discovered post-translational modification: the acetylation of serine and threonine residues. Trends Biochem. Sci. 32, 210–216 (2007).

    Article  CAS  Google Scholar 

  24. Lupardus, P.J., Shen, A., Bogyo, M. & Garcia, K.C. Small molecule-induced allosteric activation of the Vibrio cholerae RTX cysteine protease domain. Science 322, 265–268 (2008).

    Article  CAS  Google Scholar 

  25. Pruitt, R.N. et al. Structure-function analysis of inositol hexakisphosphate-induced autoprocessing in Clostridium difficile toxin A. J. Biol. Chem. 284, 21934–21940 (2009).

    Article  CAS  Google Scholar 

  26. Hari, S.B., Merritt, E.A. & Maly, D.J. Sequence determinants of a specific inactive protein kinase conformation. Chem. Biol. 20, 806–815 (2013).

    Article  CAS  Google Scholar 

  27. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  28. Adams, P.D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954 (2002).

    Article  Google Scholar 

  29. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  30. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  31. Cunnac, S. et al. Genetic disassembly and combinatorial reassembly identify a minimal functional repertoire of type III effectors in Pseudomonas syringae. Proc. Natl. Acad. Sci. USA 108, 2975–2980 (2011).

    Article  CAS  Google Scholar 

  32. Morgan, R.L. et al. Catalytic domain of the diversified Pseudomonas syringae type III effector HopZ1 determines the allelic specificity in plant hosts. Mol. Microbiol. 76, 437–455 (2010).

    Article  CAS  Google Scholar 

  33. Micsonai, A. et al. Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy. Proc. Natl. Acad. Sci. USA 112, E3095–E3103 (2015).

    Article  CAS  Google Scholar 

  34. Gille, C., Fähling, M., Weyand, B., Wieland, T. & Gille, A. Alignment-Annotator web server: rendering and annotating sequence alignments. Nucleic Acids Res. 42, W3–W6 (2014).

    Article  CAS  Google Scholar 

  35. Søndergaard, C.R., Olsson, M.H.M., Rostkowski, M. & Jensen, J.H. Improved treatment of ligands and coupling effects in empirical calculation and rationalization of pKa values. J. Chem. Theory Comput. 7, 2284–2295 (2011).

    Article  Google Scholar 

  36. Klauda, J.B. et al. Update of the CHARMM all-atom additive force field for lipids: validation on six lipid types. J. Phys. Chem. B 114, 7830–7843 (2010).

    Article  CAS  Google Scholar 

  37. Vanommeslaeghe, K., Raman, E.P. & MacKerell, A.D. Jr. Automation of the CHARMM General Force Field (CGenFF) II: assignment of bonded parameters and partial atomic charges. J. Chem. Inf. Model. 52, 3155–3168 (2012).

    Article  CAS  Google Scholar 

  38. Schmauder, R., Kosanic, D., Hovius, R. & Vogel, H. Correlated optical and electrical single-molecule measurements reveal conformational diffusion from ligand binding to channel gating in the nicotinic acetylcholine receptor. ChemBioChem 12, 2431–2434 (2011).

    Article  CAS  Google Scholar 

  39. Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38, 27–28 (1996).

    Article  CAS  Google Scholar 

  40. Pronk, S. et al. GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics 29, 845–854 (2013).

    Article  CAS  Google Scholar 

  41. Skjærven, L., Yao, X.Q., Scarabelli, G. & Grant, B.J. Integrating protein structural dynamics and evolutionary analysis with Bio3D. BMC Bioinformatics 15, 399 (2014).

    Article  Google Scholar 

  42. Grant, B.J., Rodrigues, A.P., ElSawy, K.M., McCammon, J.A. & Caves, L.S. Bio3d: an R package for the comparative analysis of protein structures. Bioinformatics 22, 2695–2696 (2006).

    Article  CAS  Google Scholar 

  43. Scarabelli, G. & Grant, B.J. Kinesin-5 allosteric inhibitors uncouple the dynamics of nucleotide, microtubule, and neck-linker binding sites. Biophys. J. 107, 2204–2213 (2014).

    Article  CAS  Google Scholar 

  44. Van Wart, A.T., Durrant, J., Votapka, L. & Amaro, R.E. Weighted implementation of suboptimal paths (WISP): an optimized algorithm and tool for dynamical network analysis. J. Chem. Theory Comput. 10, 511–517 (2014).

    Article  CAS  Google Scholar 

  45. Bakan, A., Meireles, L.M. & Bahar, I. ProDy: protein dynamics inferred from theory and experiments. Bioinformatics 27, 1575–1577 (2011).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by funds from a Kimmel Scholar Award from the Sidney Kimmel Foundation for Cancer Research, a University of California Cancer Research Coordination Committee Award (CRC-15-380558) and the March of Dimes Foundation (1-FY15-345) to J.S., and grants from the US NSF (IOS no. 0847870) and the USDA Agriculture Experimental Station Funding (CA-R-PPA-5075-H) to W.M. We thank A. Collmer (Cornell University) for providing the P. syringae strain PtoD28E, D. Maly (University of Washington) for the MKK4 construct and D. Borchardt (UC Riverside) for technical support in NMR and CD spectroscopy.

Author information

Author notes

  1. Shushu Jiang

    Present address: Present address: Sainsbury Laboratory, Norwich, UK.,

  2. Zhi-Min Zhang and Ka-Wai Ma: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry, University of California, Riverside, Riverside, California, USA.,

    Zhi-Min Zhang & Jikui Song

  2. Department of Plant Pathology and Microbiology, University of California, Riverside, Riverside, California, USA.,

    Ka-Wai Ma, Shushu Jiang, Eva Hawara & Wenbo Ma

  3. Laboratory of Physical Chemistry of Polymers and Membranes, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

    Shuguang Yuan

  4. State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China

    Youfu Luo

  5. Center for Plant Cell Biology, University of California, Riverside, Riverside, California, USA.,

    Songqin Pan & Wenbo Ma

  6. Institute of Integrative Genome Biology, University of California, Riverside, Riverside, California, USA.,

    Wenbo Ma

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Contributions

Z.-M.Z. determined the crystal structures of HopZ1a complexes and conducted ITC assays. K.-W.M. characterized NMR, CD and fluorescence spectra of HopZ1a or AvrA proteins. K.-W.M., S.J. and E.H. performed in vitro acetylation assays and in vivo functional analyses. S.Y. performed computational analysis. Z.-M.Z. and Y.L. crystalized HopZ1a complexes. Z.-M.Z. and S.P. performed limited proteolysis and mass spectrometry analysis. W.M. and J.S. designed and organized the study, Z.-M.Z., K.-W.M., S.Y., W.M. and J.S. prepared the manuscript.

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Correspondence to Wenbo Ma or Jikui Song.

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Integrated supplementary information

Supplementary Figure 1 Binding of IP6 and CoA to HopZ1a.

(a) Crystal structure of the HopZ1a–IP6 complex. IP6 is shown in the ball-and-stick model. (b) A close-up view of the catalytic center of HopZ1a. CoA is shown in the ball-and-stick model. Hydrogen bonds are shown in red dashed line. (c) An alternative conformation of H150 in the HopZ1a–IP6–CoA structure due to crystal packing. Citrate in the packing buffer is shown in stick representation. (d) Surface electrostatic view of HopZ1a showing the binding pocket of IP6. IP6 is shown in stick representation. (e) Fo-Fc omit map of IP6 in the HopZ1a–IP6-CoA complex structure at a contour level of 1.5σ. (f) Surface electrostatic view of HopZ1a showing the binding pocket of CoA. CoA is shown in stick representation. (g) Fo-Fc omit map of CoA in the HopZ1a–IP6–CoA complex structure at a contour level of 1.5σ.

Supplementary Figure 2 Sequence alignment of YopJ family effectors and ULP1.

Sequence alignment of YopJ family effectors with demonstrated acetyltransferase activity (HopZ1a and HopZ3 from Pseudomonas syringae, AvrBsT from Xanthomonas campestris, PopP2 from Ralstonia solanacearum, VopA from Vibrio parahaemolyticus, AvrA from Salmonella enterica, and YopJ from Yersinia pestis) and ULP1. Secondary structure elements of HopZ1a are shown above the aligned sequences. The catalytic triads are marked by red triangles; residues involved in IP6 binding are marked by green diamonds; residues involved in CoA binding are marked by black triangles; and the region in ULP1 that is involved in SUMO binding is underlined.

Supplementary Figure 3 In planta activity analyses of HopZ1a mutants.

(a) IP6- and CoA-binding mutants of HopZ1a-HA were expressed in PtoD28E and the transformants were used to inoculate leaves of 5-week-old Arabidopsis eco. Col-0. Electrolyte leakage of the inoculated leaf disks was measured during a time course. Error bars, S.D., n=3 leaf disks. (b) Immunoblot showing the expression of wild-type and mutant HopZ1a-HA in PtoD28E. HopZ1a proteins were induced by growing P. syringae in M9 minimal medium and detected by anti-HA antibody (Roche Diagnostics). PtoD28E carrying the empty vector (EV) was used as a negative control. (c) Effect of single mutations of the three AcCoA-binding residues on the HR-triggering ability of HopZ1a in Arabidopsis eco. Col-0. The catalytic mutant C216A was used as a control. Leaves showing HR were labeled with an asterisk. Ratio: number of leaves with indicated phenotype/total number of leaves inoculated in each treatment.

Supplementary Figure 4 1D 1H NMR and CD spectra of wild-type HopZ1a, the catalytic mutant and the IP6-binding-deficient mutants.

(a-e, left and middle) Selected regions of NMR spectra and CD spectra in the absence (black) or presence (red) of IP6 for WT. Note that the NMR signal with 1H chemical shift of -0.7ppm (marked with asterisk) only appears in the NMR spectra of wild-type and C216A HopZ1a when mixed with IP6. (a-e, right) Far-UV CD spectra in the presence of various amount of IP6 are overlaid. A slight but notable decrease in ellipticities at 208 nm and 222 nm of wild-type HopZ1a or the catalytic mutant C216A upon addition of IP6 indicated increases of helical content.

Supplementary Figure 5 Elastase cleavage assay of HopZ1a in the presence or absence of IP6 and/or CoA.

Thirty µg of HopZ1a was digested in a 30 µL reaction mixture incubated on ice for 1 hour. SDS-PAGE was used to separate the elastase cleavage products of HopZ1a.

Supplementary Figure 6 Molecular dynamics simulation of HopZ1a in IP6 and/or CoA binding states.

(a) The B-factor from MD simulations of HopZ1a, its IP6 bound form and/or IP6–CoA bound form. Regions with high B-factor are indicated by red putty. Low B-factor regions are depicted by blue putty. (b) Simultaneous view of community residue interaction network and 3D structure of HopZ1a (left), HopZ1a–IP6 (middle) and HopZ1a–IP6–CoA (right). The correlation network representation nodes correspond to protein residues connected by edges and weighted by the strength of their respective correlation values. Variations in the connectivity of the network give rise to modules or local communities in the network. Nodes belonging to the same community are more strongly and densely interconnected to one another with weaker connections to other nodes in the network43.

Supplementary Figure 7 Conserved enzymatic regulation of YopJ effectors.

(a) Three-dimensional sequence alignment of IP6–binding (left) and CoA-binding residues (right) in YopJ family effectors. (b) Structural modeling of AvrA based on the structure of HopZ1a–IP6. The IP6-binding pocket is boxed and amplified. (c) Conformational changes of wild-type and mutant AvrA upon the addition of IP6 (protein:IP6 molar ratio = 1:2) were determined by tryptophan fluorescence analysis over time.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Tables 1–3 (PDF 1902 kb)

Supplementary Data Set 1

Uncropped images for Figs. 2–4 (PDF 495 kb)

Normal mode analysis (NMA) of HopZ1a upon IP6 binding

The regulatory and catalytic domains are colored in pink and cyan, respectively. The NMA analysis indicates that the movements loopαL-αM and loopβ7-β8 correlate with each other after the binding of IP6 molecule. The length of arrow correlates with the motion of each domain. (AVI 4967 kb)

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Zhang, ZM., Ma, KW., Yuan, S. et al. Structure of a pathogen effector reveals the enzymatic mechanism of a novel acetyltransferase family. Nat Struct Mol Biol 23, 847–852 (2016). https://doi.org/10.1038/nsmb.3279

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