Journal of Structural Biology 189 (2015) 276–280

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Structure Report

Crystal structure of the effector protein HopA1 from Pseudomonas syringae Yangshin Park a,1, Inchul Shin a,1, Sangkee Rhee a,b,⇑ a b

Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea Center for Fungal Pathogenesis, Seoul National University, Seoul, Republic of Korea

a r t i c l e

i n f o

Article history: Received 14 November 2014 Received in revised form 29 January 2015 Accepted 4 February 2015 Available online 11 February 2015

a b s t r a c t Plants have evolved to protect themselves against pathogen attack; in these competitions, many Gramnegative bacteria translocate pathogen-originated proteins known as effectors directly into plant cells to interfere with cellular processes. Effector-triggered immunity (ETI) is a plant defense mechanism in which plant resistance proteins recognize the presence of effectors and initiate immune responses. Enhanced disease susceptibility 1 (EDS1) in Arabidopsis thaliana serves as a central node protein for basal immune resistance and ETI by interacting dynamically with other immune regulatory or resistance proteins. Recently, the effector HopA1 from Pseudomonas syringae was shown to affect these EDS1 complexes by binding EDS1 directly and activating the immune response signaling pathway. Here, we report the crystal structure of the effector HopA1 from P. syringae pv. syringae strain 61 and tomato strain DC3000. HopA1, a sequence-unrelated protein to EDS1, has an a + b fold in which the central antiparallel b-sheet is flanked by helices. A similar structural domain, an a/b fold, is one of the two domains in both EDS1 and the EDS1-interacting protein SAG101, and plays a crucial role in forming the EDS1 complex. Further analyses suggest structural similarity and differences between HopA1 and the a/b fold of SAG101, as well as between two HopA1s from different pathovars. Our structural analysis provides a foundation for understanding the molecular basis of the effect of HopA1 on plant immunity. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction Effector-triggered immunity (ETI) is a plant immune response that causes a hypersensitive response, including localized programmed cell death when plants are attacked by phytopathogen-originated proteins called effectors (Coll et al., 2011). In particular, most Gram-negative bacteria have evolved to evade different levels of innate immune systems in plants. To promote pathogenesis against the first line of host defense on cell surfaces mediated by pathogen-associated molecular pattern-recognition receptors, bacteria deliver their effectors directly into the cytosol of host cells via secretion systems, interfering with various cellular events in host cells (Mudgett, 2005; Postel and Kemmerling, 2009). In ETI, plants counteract an effector’s actions using disease resistance proteins, which recognize the presence and protect against specific effectors. Enhanced disease susceptibility 1 (EDS1) is a central protein in ETI (Wiermer et al., 2005). EDS1, which was identified first in ⇑ Corresponding author at: Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea. E-mail address: [email protected] (S. Rhee). 1 These authors contributed equally to this work. 1047-8477/Ó 2015 Elsevier Inc. All rights reserved.

Arabidopsis thaliana and later in other plants, plays a role in regulating the immune response by interacting with other immune regulatory proteins such as phytoalexin deficient 4 (PAD4), senescence-associated gene 101 (SAG101), and resistance proteins including Toll-interleukin-1 receptor-nucleotide binding leucinerich repeat (TIR-NB-LRR) proteins. Although detailed downstream events remain largely at the early stage of investigation, the resulting EDS1 in complex with its associated protein(s) represents a crucial element in the signaling pathway of plant immunity in response to pathogen attack. Structural analysis of the EDS1– SAG101 complex has provided molecular details regarding the binding mode of EDS1 to a regulator (Wagner et al., 2013). Recent studies of Arabidopsis immunity identified EDS1 as a target of two different sequence-unrelated bacterial effectors: AvrRps4 and HopA1 (Bhattacharjee et al., 2011; Heidrich et al., 2011). The avirulent effectors AvrRps4 and HopA1 from Pseudomonas syringae are detected by RPS4 and RPS6, respectively, resistance proteins of the Toll/interleukin-1 receptor-nucleotide binding site-Leu-rich repeat (TIR-NBS-LRR) class (Kim et al., 2009). These two effectors disrupted the EDS1-resistance protein complexes in vitro (i.e., EDS1-RPS4 and -RPS6, respectively) by interacting with EDS1 directly. This disruption triggered the

Y. Park et al. / Journal of Structural Biology 189 (2015) 276–280

activation of TIR-NBS-LRR resistance proteins and caused the relocalization of EDS1 and resistance proteins between the cytoplasm and nucleus (Bhattacharjee et al., 2011; Heidrich et al., 2011). In this report, we determined the crystal structure of two HopA1s from P. syringae, each from different pathovars including syringae strain 61 (HopA1-Pss61) and tomato strain DC3000 (HopA1-Pst3000). These two HopA1s have different pathogenic effects; specifically, HopA1-Pss61 causes resistance in Arabidopsis by inducing a programmed cell death response, while HopA1Pst3000 is pathogenic (Kim et al., 2009). These two HopA1s, which exhibit a sequence identity of 57%, show no sequence similarity with other known proteins, excluding the N-terminal region. The N-terminal region of HopA1 contains a secretion signal (residues 1–20), and additional regions (i.e., residues 21–99 for HopA1Pss61 and 21-102 for HopA1-Pst3000) contain an ShcA1 chaperone binding domain (Janjusevic et al., 2013). The crystal structures of the two HopA1s in the absence of the structurally known N-terminal region revealed that both HopA1s form an a + b fold and show different distributions of electrostatic surface potentials. 2. HopA1 purification Full-length genes for HopA1-Pss61 (GenBank Accession No. EF514224:706–1833) and HopA1-Pst3000 (AE016853:60862436087385) were synthesized by Bioneer (Daejeon, Korea). Among various constructs of HopA1 from both pathovars, the C-terminal domain of HopA1-Pss61 (cHopA1-Pss61; residues Arg120 to Asn375) and HopA1-Pst3000 (cHopA1-Pst3000; residues Lys123 to Lys380), excluding an ShcA1 chaperone binding domain (Janjusevic et al., 2013), was observed to produce a crystal suitable for structural analysis. Specifically, genes for cHopA1-Pss61 and cHopA1-Pst3000 were amplified by polymerase chain reaction with sequence-specific primers (Table S1) and ligated into the expression vector pET-28a (Merck), which was modified to contain a tobacco etch virus cleavage site between a His5-tag and the multiple cloning sites. For structural studies, seleno-L-methionine (SeMet)-substituted, N-terminal His-tagged cHopA1-Pss61 was expressed in Escherichia coli BL21 (DE3) cells (Merck) grown at 37 °C in M9 minimal medium supplemented with the required amino acids according to a published protocol (Van Duyne et al., 1993). The expression of SeMet-substituted cHopA1-Pss61 was induced by the addition of 0.5 mM isopropyl-L-thio-b-D-galactopyranoside and 0.25 mM SeMet, and incubated for 18 h at 20 °C. The cells were harvested and sonicated in buffer A (50 mM HEPES, pH 7.5, and 200 mM NaCl). cHopA1-Pss61 was purified using immobilized metal affinity chromatography, and its N-terminal His-tag was removed by treatment with tobacco etch virus protease for 16 h at 4 °C. Further purification was performed employing immobilized metal affinity chromatography and size-exclusion chromatography using a Superdex 200 column (GE Healthcare) equilibrated with buffer A. The purified protein was concentrated to 9.9 mg ml 1. For structural studies on cHopA1-Pst3000, expression was performed using the E. coli BL21 (DE3) codon plus-RIL strain (Agilent Technologies). Cells were grown at 37 °C in Luria–Bertani medium, and purification for cHopA1-Pst3000 was similar to the procedure described above. The protein was concentrated to 11 mg ml 1. 3. Crystallization and structural determination Crystals of SeMet-cHopA1-Pss61 were obtained in the presence of 5 mM dithiothreitol at 22 °C using a crystallization solution of 56.43% (v/v) Tacsimate (pH 7.0). In contrast, crystals for cHopA1Pst3000 were produced at 22 °C in the absence of dithiothreitol


with a crystallization solution containing 0.1 M sodium citrate/citric acid (pH 5.5), 10% (v/v) 2-propanol, and 20% (w/v) polyethylene glycol 3350. In both cases, 30% ethylene glycol was used as a cryoprotectant for data collection. Multiwavelength anomalous dispersion data (Hendrickson, 1991) and single-wavelength data using a crystal of SeMet-cHopA1-Pss61 were collected on Beamline 7A at the Pohang Accelerator Laboratory (Pohang, Korea). Subsequently, single-wavelength diffraction data to a resolution of 2.3 Å using a crystal of cHopA1-Pst3000 were collected at the Pohang Accelerator Laboratory. All data were collected at 100 K and processed using the program HKL2000 (Otwinowski and Minor, 1997). The crystal structure of SeMet-cHopA1-Pss61 was determined using the programs SOLVE (Terwilliger and Berendzen, 1999) and RESOLVE (Terwilliger, 2000, 2003) for phasing and density modification, respectively. The initial electron density map was sufficient to trace most residues. Manual model building and refinement were performed using COOT (Emsley and Cowtan, 2004) and PHENIX (Adams et al., 2010), respectively. When the structure of SeMet-cHopA1-Pss61 was refined to about 21.1% and 24.8% for Rwork and Rfree, respectively, its coordinates were used as a search model for molecular replacement of the crystal structure of cHopA1-Pst3000. The program PHENIX with AUTOMR option was used for molecular replacement. The space group of the cHopA1Pss61 crystal was P41, with two monomers in an asymmetric unit, while cHopA1-Pst3000 crystals belonged to the space group P65, with a monomer per asymmetric unit (Table S2). In the final refinement stages, TLS refinement was performed using multiple TLS groups identified automatically in PHENIX. During TLS refinement, water molecules, whose refined temperature factors were less than 50 Å2, were assigned based on the possible hydrogen bonds to the protein or nearby water molecules. Details on data collection and refinement statistics are described in Table S2. Structural comparisons and analyses were performed using the CCP4 suite program (Winn et al., 2011) and figures were prepared using PyMOL (DeLano, 2002).

4. Overall structure of cHopA1-Pss61 and cHopA1-Pst3000 Both HopA1s from different pathovars exist in solution as a monomer, as indicated by size-exclusion chromatography. However, in a crystalline state, the two monomers of cHopA1-Pss61 were present in an asymmetric unit and related by a root mean-square deviation (RMSD) of 0.27 Å for 255 Ca atoms (from Arg120 to Arg374). In particular, two monomers were arranged in a perpendicular manner by the face-to-face orientation of the concave groove of the central b-sheet (see below). The structure of monomeric cHopA1-Pss61 was composed of nine a-helices, three 310-helices, and seven b-strands (Fig. 1A and C). From a topological perspective, cHopA1-Pss61 can be characterized as an a + b fold with a concave groove in the central b-sheet. Specifically, the seven b-strands constitute a central antiparallel bsheet in the order of b1, b2, b4, b5, b3, b7, and b6 strand (Fig. 1A), resulting in a highly curved b-sheet flanked by two segments of helices, each on both sides of the b-sheet. One segment of helices, which consists of the N-terminal two helices (a1, a2; blue in Fig. 1A) and the C-terminal two helices (a8, a9; red in Fig. 1A), is found at one end of the ventral side of the b-sheet near the b7 strand. These two groups of helices are almost in a perpendicular orientation, with two consecutive helices in each group being packed in an antiparallel manner (Fig. S1). The other segment of helices, including a3, a4, a5, a6, and a7, is on the dorsal side of the concave b-sheet. Due to these arrangements of secondary structures, an a + b fold characterized in


Y. Park et al. / Journal of Structural Biology 189 (2015) 276–280

Fig.1. Overall structure and sequence alignment of HopA1. (A) The overall structure of HopA1-Pss61 is shown in rainbow mode from the N-terminus (blue) to the C-terminus (red). The right panel presents a side view with an arrow indicating the concave groove. (B) The overall structure of HopA1-Pst3000 is shown in an orientation identical with A. (C) The amino acid sequence of HopA1-Pss61 was compared in pairwise alignments with HopA1-Pst3000. Highly conserved residues are shown in red and boxed in blue, whereas strictly conserved residues are displayed with a red background. This figure was prepared using ESPript (Gouet et al., 1999).

cHopA1-Pss61 represents a tweezer-like conformation, with a concave groove of 15–33 Å depth and 25 Å width. Almost identical structural features were also observed in the structure of cHopA1-Pst3000 (Fig. 1B), which has a monomer in an asymmetric unit. cHopA1-Pst3000 exhibits 57% sequence identity with HopA1-Pst3000 (Fig. 1C) and is related to cHopA1-Pss61 with an RMSD of 1.52 Å for 240 Ca atoms. In a crystalline state, cHopA1-Pst3000 residues from Lys123 to Thr379 contain nine ahelices, one 310-helix, and seven b-strands. Contrary to the ventral-to-ventral interactions identified between the two monomers in an asymmetric unit of cHopA1-Pss61, various crystallographic symmetry-related interactions occur among monomers of cHopA1-Pst3000, including ventral-to-dorsal and dorsal-to-dorsal interactions. 5. Structural comparisons Since the amino acid sequences of HopA1 are unique and its function remains unknown, we searched for protein structures homologous to HopA1 using the software DALI (Holm and Rosenstrom, 2010). No structures were significantly similar to HopA1, indicating that HopA1 is a structurally unique protein. The highest Z-scores in these comparisons were found for phosphothreonine lyase (PDB code 2Z8M; Z-score 8.5; sequence identity 16%; RMSD of the 122 Ca atoms 2.91 Å) (Chen et al., 2008) and eukaryotic translation initiation factor 4E (PDB code

4AXG; Z-score 8.4; sequence identity 12%; RMSD of the 122 Ca atoms 2.92 Å) (Kinkelin et al., 2012). Specifically, phosphothreonine lyase contains conserved residues Lys104, Arg148, Arg213, and Arg220 in the b-sheet groove for binding the substrate phosphate groups, and consequently for its activity (Chen et al., 2008) (Fig. S2), while HopA1 does not contain these residues at the structurally equivalent positions. Further experiments are required to identify any possible enzymatic activity of HopA1.

6. EDS1 as a target of HopA1 The EDS1–SAG101 heterodimer is a complex required for basallevel immune resistance. Its structural features included an a/b fold as one of two domains in both EDS1 and SAG101, and it serves as an interaction motif in forming the EDS1–SAG101 heterodimer (Wagner et al., 2013). Further sequence and mutational analysis showed that the a/b fold of EDS1 is homologous with that of a hydrolase, but it does not exhibit the expected hydrolase activity (Wagner et al., 2013). Therefore, the a/b fold in EDS1 and SAG101 was proposed to be a structural domain that plays a role in protein–protein interactions. Although the DALI search failed to identify the full-length EDS1 and SAG101 as structurally close proteins to HopA1, the isolated a/b fold of EDS1 and SAG101 is similar to the a + b fold of HopA1 and has respective RMSDs of 2.3 Å for the 227 Ca atoms and 5.32 Å for the 102 Ca atoms.

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In the EDS1–SAG101 complex, a helix protruding from the a/b fold of EDS1 is inserted in the hydrophobic pocket of the a/b fold in SAG101, corresponding to the b-sheet ventral side of HopA1 (Wagner et al., 2013) (Figs. 2 and S3). In fact, this binding mode might not be applicable to HopA1, in which the presence of two loops at the entrance to the ventral side of HopA1 precludes the possible binding of the protruding helix of EDS1 in the ventral side of b-sheet (Fig. 2). These two loops in HopA1 (one between a2 and a3 and the other between g3 and b6) are structural features unique to HopA1. Currently, it is not known whether the helix protruding from the a/b fold of EDS1 can function as a HopA1-interacting motif. Our structural comparisons of cHopA1-Pss61 and cHopA1Pst3000 indicate that these two structures show high similarities, including conserved residues and hydrophobicity on both the ventral and dorsal sides (Fig. S4). The only notable differences are changes in the distributions of the electrostatic surface potentials between HopA1-Pss61 and HopA1-Pst3000 (Fig. 3).


Given that only HopA1-Pss61 causes programmed-cell death and ETI is mediated by forming a complex with EDS1, the two HopA1s likely have different binding affinities to EDS1. It would be interesting to investigate whether differences in the surface electrostatic charges contribute to the binding affinity of HopA1 to EDS1. Notably, electronegative surface patches on AvrRPS4, another effector interacting with EDS1, play a crucial role in RPS4-dependent immunity (Sohn et al., 2012); e.g., the surface charge of AvrRPS4 is used to mediate interactions with other proteins. Our structural analysis constitutes the first step in clarifying the molecular basis of HopA1 in plant immunity. Further studies are required to identify residues or other factor(s) required for the interactions between HopA1 and EDS1. 7. Conclusions In this study, we determined the crystal structure of P. syringae effector HopA1 from two different pathovars: syringae strain 61 and tomato strain DC3000. HopA1 has a unique sequence and is known to interact with EDS1 protein, which is an essential element in plant immunity. HopA1 is folded into a well-known a + b fold. DALI searches found no structures homologous to HopA1. Further analyses suggest structural differences between HopA1 and the a/b fold of EDS1 and SAG101, as well as between two HopA1 proteins from different pathovars. Accession numbers The atomic coordinates and structure factors of cHopA1-Pss61 and cHopA1-Pst3000 have been deposited in the Protein Data Bank with accession code 4RSW and 4RSX, respectively. Acknowledgments

Fig.2. Structural comparison of HopA1 with SAG101. The structure of HopA1-Pss61 is superimposed with an a/b fold of SAG101 (residues 1–290) in the EDS1–SAG101 complex (Wagner et al., 2013) using the programs in CCP4mg (McNicholas et al., 2011). The protruding helix from EDS1 is shown in red, and SAG101 and HopA1 are indicated in blue and magenta, respectively. Note that presence of two loops in HopA1 blocks the binding of the protruding helix from EDS1.

This work was supported by a grant from National Research Foundation (2013R1A1A2060285), Ministry of Education, and from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center No. PJ011019), Rural Development Administration, Republic of Korea. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at References

Fig.3. Electrostatic surface potential of HopA1. The dorsal and ventral surface potentials of HopA1-Pss61 and HopA1-Pst3000 are displayed in scale from 81 kTV (red) to +81 kTV (blue). The regions with negative and positive charge are presented in red and blue, respectively.

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Crystal structure of the effector protein HopA1 from Pseudomonas syringae.

Plants have evolved to protect themselves against pathogen attack; in these competitions, many Gram-negative bacteria translocate pathogen-originated ...
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