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Yersinia effector YopO uses actin as bait to phosphorylate proteins that regulate actin polymerization

Nature Structural & Molecular Biology volume 22, pages 248–255 (2015)Cite this article

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Abstract

Pathogenic Yersinia species evade host immune systems through the injection of Yersinia outer proteins (Yops) into phagocytic cells. One Yop, YopO, also known as YpkA, induces actin-filament disruption, impairing phagocytosis. Here we describe the X-ray structure of Yersinia enterocolitica YopO in complex with actin, which reveals that YopO binds to an actin monomer in a manner that blocks polymerization yet allows the bound actin to interact with host actin-regulating proteins. SILAC-MS and biochemical analyses confirm that actin-polymerization regulators such as VASP, EVL, WASP, gelsolin and the formin diaphanous 1 are directly sequestered and phosphorylated by YopO through formation of ternary complexes with actin. This leads to a model in which YopO at the membrane sequesters actin from polymerization while using the bound actin as bait to recruit, phosphorylate and misregulate host actin-regulating proteins to disrupt phagocytosis.

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Figure 1: Structure of the YopO–actin complex.
Figure 2: The YopO–actin complex is polymerization incompetent.
Figure 3: Structural basis of differential actin isoform preference of YopO.
Figure 4: YopO-bound actin interacts with other actin-binding proteins.
Figure 5: Phosphorylation of actin-binding proteins by YopO.
Figure 6: Model for disabling actin polymerization by YopO.

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Acknowledgements

We thank L. Burtnick and W. Burkholder for discussions and critical reading of the manuscript. W.L.L. and R.C.R. thank the A*STAR for support. We acknowledge the Joint Centre for Structural Biology, Singapore, which is supported by Nanyang Technological University and the Biomedical Research Council (BMRC) of A*STAR, for providing research facilities and P. Kaldis for providing help and facilities for the kinase assays. We thank the Diamond Light Source (proposal MX8423) for crystal screening and beamline BL13B1 at the National Synchrotron Radiation Research Center, Taiwan (NSRRC) for final data collection. The Wellcome Trust Centre for Human Genetics is supported by the Wellcome Trust Core award (090532/Z/09/Z). We thank L. Blanchoin (Institut de Recherches en Technologies et Sciences pour le Vivant) and M. Hernandez-Valladares (University of Liverpool) for reagents.

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Authors and Affiliations

  1. Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore

    Wei Lin Lee & Robert C Robinson

  2. Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK

    Wei Lin Lee & Jonathan M Grimes

  3. Diamond Light Source, Oxfordshire, UK

    Jonathan M Grimes

  4. Department of Biochemistry, National University of Singapore, Singapore

    Robert C Robinson

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W.L.L. carried out the experimental work. All authors analyzed the data and prepared the manuscript.

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Integrated supplementary information

Supplementary Figure 1 The sequence and secondary structure of YopO and its interaction with actin.

Sequence/structural analysis of YopO of Yersinia enterocolitica (89-729) (accession NP_783721) aligned against YpkA of Yersinia pestis (accession NP_395206) and Yersinia pseudotuberculosis (accession YP_068414). The secondary structure of the kinase and GDI domains are indicated in blue and red above the sequence alignment, respectively. Disordered regions are represented by disconnected lines. The putative phosphorylation sites Ser90 and Ser95 are marked with black circles. The surface entropy reduction mutations are indicated by black asterisks. Residues that constitute the αC helix, the catalytic loop and the activation segment are shown in magenta, red and green, respectively. The catalytic residue Asp267 is marked with a red circle and residues involved in interaction with actin and Rac1 are indicated with cyan and yellow squares, respectively. The alignment was performed using Clustal Omega and drawn with ESPript.

Supplementary Figure 2 Details on the structure of the YopO–actin complex.

(a) Analysis of the surface entropy reduction mutations in the final structure. One of the mutations stabilizes a crystal contact. Actin is shown in cyan, the GDI domain in red and the kinase domain in blue, following the color scheme in Fig. 1a. One mutation on YopO (E207Y) interacts with a residue (Y588) on the GDI domain of a symmetry-related molecular complex, and is likely to be basis of the improvement in diffraction quality of the mutant protein crystals.

(b) Conformational changes to the GDI domain. The isolated GDI domain is shown in fawn (PDB: 2H7O), the Rac-bound GDI domain (PDB: 2H7V) is shown in light blue with Rac1 in yellow, and the GDI domain taken from the YopO:actin complex in red. The three GDI domains are superposed, as aligned on the N-terminal GDI subdomain.

(c) Comparison of the structural elements of the kinase domain of YopO (blue) with human PAK4 (PDB code 4FIJ, yellow) and human OSR1 (PDB code 2VWI, gray) (from left to right). The structural elements are colored following the scheme in Supplementary Note Figure 1. The P-loop is disordered in the YopO structure.

(d) Snapshots of the 2Fo-Fc (2σ, gray) experimental electron density omit map for different segments of the kinase domain.

Supplementary Figure 3 YopO mutants.

(a) Disrupting the YopO:actin interface in YopO nAct. YopO is colored as in Fig. 1a. Panels show close up of the interfaces between YopO and actin at the mutation sites, which were used to disrupt the interactions with actin in YopO nAct. Mutations V374D and T376D introduce large charged groups into a hydrophobic YopO:actin interface while mutations R723A and E727A disrupt electrostatic interactions between YopO and actin.

(b) Size exclusion chromatograms of YopO WT and mutants. YopO WT and mutants have a similar rate of migration on size exclusion chromatography, suggesting that the mutations do not disrupt the normal folding of YopO. WT, KD, nAct and nRac denote wild type, kinase-dead, non-actin binding and non-Rac binding respectively.

Supplementary Figure 4 Sequence alignment of actin protein sequences.

Sequences include S.frugiperda actin (SfACT, accession: AEK82131), D.discoideum actin (DdACT, AAA33145), human β-actin (hACTB, accession: NP_001092.1), mouse β-actin (mACTB, accession: AAA37164) and rabbit α-actin (aACTA, accession: CAA43139.1). The corresponding position for Thr202 of S.frugiperda is marked with a yellow box. Red and blue squares beneath the alignment represent interactions with the YopO GDI and kinase domains, respectively. The alignment was performed using Clustal Omega and presented using ESPript.

Supplementary Figure 5 YopO uses actin as bait for phosphorylation.

(a) Model of the ternary complex of YopO:actin:gelsolin (G4–G6), following the color scheme in Fig. 1a, with gelsolin in green (PDB code 1H1V). Note that gelsolin comprises six domains, two of which bind G-actin similarly, G1 and G4. CapG consists of three gelsolin domains. Models of YopO–actin–G1–3 (Fig. 4b) and YopO–actin–G4–6 represent potential models of the YopO–actin–CapG structure.

(b) Model of the ternary complex of YopO–actin–ADFH, with the ADFH of twinfilin in magenta (PDB code 3DAW). Note that this represents a model of the YopO–actin–cofilin structure. Full length twinfilin contains a second ADFH domain. CapG consists of three gelsolin domains and Twf1, of two ADF-H domains. Both CapG and Twf1 are not sufficiently elongated to span from the actin-binding site to the kinase catalytic cleft in order to be phosphorylated by YopO–actin.

(c) In vitro phosphorylation assay of YopO and mutants. YopO WT or mutants (4.7 μM) were incubated with or without Sf9 actin (4.7 μM), VASP as substrate (14.1 μM) or profilin (14.1 μM) as indicated, supplemented with 5μCi [γ-32P]ATP. Lane 1: YopO does not phosphorylate itself in the absence of actin. Lane 2: YopO autophosphorylates in the presence of actin. Lane 3: VASP is phosphorylated in the presence of actin and YopO; which is not impeded by the presence of profilin (lane 4). Lane 5: Kinase-dead (KD) YopO does not phosphorylate itself or VASP, and neither does nAct the reduced actin binding YopO mutant (lane 6). Lane 7: The non-Rac binding YopO mutant (nRac) phosphorylates itself and VASP.

(d) SDS-PAGE analysis of the proteins used in the in vitro phosphorylation assay in Fig. 5. YopO, VASP, EVL, gelsolin, CapG, twinfilin1 are without fusion tags. mDia1 (583–1262) and WASP (150–502) each contain both an N-terminal GST and a C-terminal His6 tag. Due to overlap in the molecular weights of YopO and GST-WASP, in Fig. 5, the phosphorylated GST-WASP was cleaved with thrombin prior to SDS-PAGE. In this figure, GST-WASP was not cleaved with thrombin. Red stars indicate the molecular weight of the substrates. Invitrogen Benchmark protein ladder is loaded in the first lane, with the molecular weights of bands in the ladder indicated. The gel was stained with Coomassie blue.

Supplementary Figure 6 Structural comparison of actin-binding proteins from pathogens and hypothetical models for structural activation of YopO.

(a) Superposition of toxofilin (PDB:2Q97) on the structure of YopO:actin, following the colour scheme in Fig. 1a, with toxofilin in yellow. The N- and C- terminal residues of the toxofilin construct are shown as yellow and magenta spheres, respectively. The C-terminus of toxofilin wraps around actin subdomain 4 in a manner similar to that of YopO, although the topologies and the exact interaction interfaces differ.

(b) Superposition of Iota toxin component Ia (PDB:3BUZ) on the structure of YopO:actin, following the colour scheme in Fig. 1a, with Iota toxin Ia in magenta.

(c) Hypothetical model of YopO activation. The GDI domain is shown in red and the kinase domain is shown in blue, with actin shown as a surface representation in marine. In this hypothetical model of the inactive structure, the backbone helix of the GDI domain would be straight and the kinase active site would likely be inaccessible. In the transition from the inactive YopO to YopO in the YopO:actin complex, two potential “intermediate” complexes are possible. Actin could first bind to either the kinase domain or the GDI domain, though the latter is more plausible due to a greater solvation free energy gain for the formation of this interface as compared to binding of actin to the kinase domain. The series of conformational changes that lead to sandwiching of actin between the two domains would lead to the bending of the backbone helix of the GDI domain and the kinase substrate binding groove being exposed as in the YopO:actin structure.

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Lee, W., Grimes, J. & Robinson, R. Yersinia effector YopO uses actin as bait to phosphorylate proteins that regulate actin polymerization. Nat Struct Mol Biol 22, 248–255 (2015). https://doi.org/10.1038/nsmb.2964

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