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Crystal structure of PppA from Pseudomonas aeruginosa, a key regulatory component of type VI secretion systems
Biochemical and Biophysical Research Communications 516 (2019) 196e201
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Crystal structure of PppA from Pseudomonas aeruginosa, a key regulatory component of type VI secretion systems Yujie Wu a, b, Junyuan Gong b, Shan Liu b, Dongyang Li b, d, Yulan Wu b, Xi Zhang b, Yan Ren c, Shaojian Xu c, Jingchuan Sun b, d, Tao Wang b, c, Qihui Lin c, **, Li Liu b, c, * a
School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, 518055, China Department of Biology, Southern University of Science and Technology, Shenzhen, 518055, China Joint Laboratory for Infectious Disease Prevention and Control, Hygienic Section of Longhua Center for Disease Control and Prevention, Longhua District, Shenzhen, 518109, China d The Academy for Advanced Interdisciplinary Studies, Southern University of Science and Technology, Shenzhen, 518055, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 May 2019 Accepted 3 June 2019 Available online 14 June 2019
The Type VI secretion system (T6SS) is a membrane protein complex related to inter-bacterial competitions and host-pathogen interactions in Pseudomonas aeruginosa. The T6SS is regulated by a great variety of regulatory mechanisms at multiple levels, including post-translational modification with threonine phosphorylation mediated by Ser/Thr protein kinase PpkA and phosphatase PppA. The T6SS is activated by PpkA via Thr phosphorylation of Fha, and PppA can antagonize PpkA. PppA is a PP2C-family protein phosphatase and plays a key role in the disassembly and reassembly of T6SS organelles. Herein, we report the first crystal structure of PppA from Pseudomonas aeruginosa, which was determined at a resolution of 2.10 Å. The overall structure consists of a bacteria PPM structural core and a flexible flap subdomain. PppA harbors a catalytic pocket containing two manganese ions which correspond to the canonical dinuclear metal center of Ser/Thr protein phosphatases including the bacterial PPM phosphatases and human PP2C. The flexibility and the diversity of the sequence of flap subdomain across the homologues might provide clues for substrates specific recognition of phosphatases. © 2019 Elsevier Inc. All rights reserved.
Keywords: T6SS Pseudomonas aeruginosa Phosphatase PppA Crystal structure
1. Introduction The Type VI secretion system (T6SS) is a secretory multi-protein complex that mediates inter-bacterial competitions and hostpathogen interactions. It is one of the most recent identified secretion systems as effector translocation apparatus with inverted phage tail-like structure [1]. The T6SS core-components are composed of at least 13 protein subunits (TssA-TssM) [2,3]. The T6SS has been found in about a quarter of all sequenced Gramnegative bacteria, including Pseudomonas aeruginosa, Serratia marcescens and some other pathogens [4]. In the opportunistic Gram-negative pathogen Pseudomonas aeruginosa, the T6SS can
* Corresponding author. Department of Biology, Southern University of Science and Technology, Shenzhen, 518055, China. ** Corresponding author. Joint Laboratory for Infectious Disease Prevention and Control, Hygienic Section of Longhua Center for Disease Control and Prevention, Longhua District, Shenzhen, 518109, China. E-mail addresses: [email protected] (Q. Lin), [email protected] (L. Liu). https://doi.org/10.1016/j.bbrc.2019.06.020 0006-291X/© 2019 Elsevier Inc. All rights reserved.
sense and respond to external signals, then inject effector proteins into external competitive bacteria or host cells. The T6SS is regulated in transcriptional, post-transcriptional, and post-translational levels, including quorum sensing (QS) regulation by LasR [5,6], negative regulation by RNA binding protein RsmA [7], threonine phosphorylation (TPP)-dependent and TPP-independent regulation pathways. In Pseudomonas aeruginosa, the T6SS is activated by the phosphorylation of a serine/threonine (Ser/Thr) kinase PpkA, and inhibited by a Ser/Thr phosphatase PppA, which can antagonize PpkA [8]. PppA is a PP2C-family protein phosphatase in the T6SS gene cluster. In this TPP pathway, the T6SS is activated via Thr phosphorylation of Fha, an FHA domain protein, which regulated positively by PpkA and negatively by PppA. Furthermore, the T6SS of P. aeruginosa can be repressed by TagF in TPP-independent pathway [9]. The T6SS is a dynamic organelle regulated by PpkA-Fha1-PppA cycle, which participates in suppressing random formation of T6SS organelles, inducing their assembly precisely at the point of an external signal, and targeting the disassembly of T6SS organelles if necessary. So, PppA plays a
Y. Wu et al. / Biochemical and Biophysical Research Communications 516 (2019) 196e201
key role in bacterial T6SS ‘‘tit-for-tat’’ evolutionary strategy [10]. Furthermore, PppA also regulates quorum sensing, c-di-GMP production, polysaccharide biosynthesis and transport, and it shows that PppA participates in controlling virulence factors and pleiotropic phenotypes besides T6SS [11]. Structures of T6SS core complex and some regulated subunits, such as TagF and PpkA, have been well determined [12e14]. Atomic resolution structural information of PppA is still unclear. Herein we report the crystal structure of PppA from Pseudomonas aeruginosa, which has a bacteria PPM structural core with two-metal active site and a flexible flap subdomain. 2. Materials and methods 2.1. Protein expression and purification The PppA (Gene ID: 879341) gene was amplified from the genome of Pseudomonas aeruginosa PAO1. The PCR product was sub-cloned into the Nde I and Xho I sites of a pET28a(þ) vector with an N-terminal His-tag. E. coli strains containing the recombinant plasmid were grown in Luria-Bertani broth medium with 50 mg/L kanamycin at 37 C. When the OD600 reached 0.6, IPTG (isopropyl bD-1-thiogalacto-pyranoside) was added to the growth medium to a final concentration of 0.2 mM to induce the expression of recombinant proteins. The induced cultures were further grown at 16 C for 16 h. The cells were harvested by centrifugation at 5500g for 10 min at 4 C and resuspended in lysis buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole). The PppA protein was purified using HisTrap HP column (GE Healthcare) in lysis buffer, followed by imidazole elution and further purification using sizeexclusion chromatography (Superdex75 10/300 GL) in the buffer containing 10 mM Tris-HCl with pH 7.4 and 200 mM NaCl. The peak fractions were pooled and concentrated to 10 mg/ml for crystallization. The purity of the protein was assessed by 12% SDS-PAGE. 2.2. Protein crystallization and data collection Crystal screening for the PppA was performed by the hangingdrop vapor diffusion method at 20 C using Crystal Screen kits (Hampton Research). High diffraction quality crystals were obtained after one week from a reservoir containing 18% (w/v) Polyethylene glycol 8000, 0.08 M Calcium acetate hydrate, 0.1 M HEPES (pH 7.2). Diffraction dataset was processed using HKL3000 [15]. 2.3. Crystal structure determination and refinement The structure was solved by molecular replacement in PHENIX suit [16] using Phaser [17], with the PDB entry 1TXO as the initial search model. The final models were manually built in Coot [18] with automatic refinement in PHENIX with Cartesian simulated annealing and TLS refinement [19]. The protein structures presented in the figures were rendered with PyMol [20]. The atomic coordinates and structure factors (codes 6JKV) have been deposited in the Protein Data Bank. 3. Results and discussion 3.1. Overall structure of PppA The structure of PppA has been solved at 2.1 Å resolution, and the statistics for data collection, phasing and structure refinement are summarized in Table 1. The crystal belongs to the P1 21 1 space group and contains two monomers in the asymmetric unit. Almost all residues of PppA have well defined conformations except Nterminal three residues and residues from A148 to R156 in
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Table 1 X-ray Data collection and structure refinement statistics. Parameter A. Data collection Space group Cell dimensions a/b/c (Å) Wavelength (Å) Resolution (Å) Average I/s (I) Total reflections Unique reflections Redundancy Completeness (%) Rmerge(%) B. Structure refinement Resolution (Å) Average B-factor (Å) Rwork/Rfree (%) R.m.s.d. bond lengths (Å) R.m.s.d. bond angles ( ) C. Ramachandran plot Most favored (%) Allowed (%) Disallowed (%)
PppA P 1 21 1 50.54/80.25/59.08 1.5418 24.28e2.10 (2.18e2.10) 4.9 (2.3) 51259 25293 2.03 (1.86) 98.6 (99.5) 0.119 (0.279) 24.08e2.10 23.0 23.3/28.3 0.0057 1.1402 96.4 3.6 0
monomer A or from D146 to H157 in monomer B. Structure of PppA mentioned below generally refers to monomer A unless otherwise specified definition. The final model of PppA monomer contains two metal ions. The analysis of X-ray anomalous diffraction data just above and below the Mn K edge confirmed the existence of manganese. The core structure of PppA is very similar to other PPM phosphatases structures, which have abba sandwich structure consisting of two five-stranded, antiparallel b sheets surrounded by two pairs of antiparallel a helices on either side (Fig. 1). Antiparallel b sheets of PppA are formed by residues 7e16, 122e127, 130e133, 186e191 and 234e242 in b-sheet 1, and 26e31, 36e43, 100e109, 112e118 and 177e182 in b-sheet 2. b-sheet 1 is flanked by a pair of antiparallel a helices (residues 202e209 and 215e228) and a short helix (residues 194e197). b-sheet 2 is flanked by a pair of antiparallel a helices (residues 49e62 and 69e94) (Fig. 1). There is a flap subdomain (residues 138e166) adjacent to the active site, which is partially disordered and a short a helix (residues 140e146) included. The flap subdomain is poor conserved in PPM phosphatases, and no electron density is visible from residues 148 to 156. This suggests that the flap subdomain is flexible and maybe correlated to substrates specificity of PppA. 3.2. The active site of PppA PppA harbors a catalytic pocket containing two manganese ions (M1 and M2). The crystal structure of PppA revealed a similar arrangement of two hexa-coordinated manganese ions in the catalytic site as in other PPMs [21,22]. Two manganese ions site correspond to the canonical dinuclear metal center of Ser/Thr protein phosphatases including the bacterial PPM phosphatases and human PP2C, which are coordinated by a strictly invariant set of protein residues [23,24] (Fig. S1). The two manganese ions separated by 3.56 Å are strictly coordinated by sidechain carboxyl groups of the conserved residues Asp41, Asp194, and Asp232, the main-chain carbonyl group of Gly42, and six water molecules, one of which bridges the two manganese ions (Fig. 2). The bridging water molecule may act as a catalytic nucleophile [25]. M1 is coordinated by three water molecules and one side-chain oxygen each of Asp41, Asp194 and Asp232. M2 is coordinated by four water molecules, one main-chain oxygen of Gly42 and one side-chain oxygen (OD1) of Asp41. Asp41 coordinates two manganese ions
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Fig. 1. Crystal Structure of PppA. A) Cartoon representation of PppA. The a-helices, b-sheets and loop regions are shown as cartoon colored with deep salmon, teal and gray respectively. Gray dashed lines depict unstructured regions of the flap subdomain. Two manganese ions are shown as spheres colored with teal.
Fig. 2. Active site of PppA. A) Active site in the overall structure of PppA. Residues coordinating the two manganese ions in the active site are showed as sticks colored with green (carbon atoms) and red (oxygen atoms). Two manganese ions are shown as spheres colored with teal. Water molecules coordinating manganese ions are showed as spheres colored with red and magentas (the one bridging the two manganese ions). dashed lines depict coordination of manganese ions with PppA residues or water molecules. B and C are magnified views of active site at A. Backbone carbonyl oxygen atom of G41 and sidechains of D41, D194 and D232 are showed as sticks and labelled. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
via two oxygen atoms of sidechain carboxyl group respectively on the side opposite the bridging water molecule (Fig. 2). The PppA structure also shows that Asp232 is positioned to accept a proton from the bridging water molecule. Arg21, which is responsible for substrate phosphoryl group binding in the active site, has a slightly different position in both monomers of the crystal asymmetric unit. But no phosphate molecule could be observed in the electron density of PppA since protein crystal grew in Tris hydrochloric acid buffer without phosphate. Although there is a third metal ion called M3 (Mg or Mn ion) in many identified structures of PPM phosphatases, the PppA structure does not contain the third metal ion (M3) close to the active site. The equivalent residues coordinated potential M3 in PppA are Asp120 and Asp194, which are strictly conserved in PPMs (Fig. 3B) [26,27]. It suggests that a third metal ion can be coordinated at the equivalent area near the active site of PppA. We propose most of potential M3 ligands in PppA are water molecules, M3 in PppA may
be less tightly bound than the M3 in other PPMs. Furthermore, the flap subdomain of PppA is more flexible than equivalent segment of other PPMs, which may lead to the lack of M3 in PppA. 3.3. Flap subdomain of PppA The PppA flap subdomain (residues 138e166) contains an a helix followed by a long loop (residues 148e156 are not visible in the electron density). Although amino acid residues of flap subdomains are not conserved in bacteria PPMs, the number of flap residues in PPMs is almost consistent (Fig. 3C). A large, irregular flap joining two beta strands forms one side of a cleft containing the two-metal active site of PppA (Fig. 3A). Flap subdomain is much more flexible in the PppA structure than in the other PPMs. The structural flexibility of flap subdomain may be related to the lack of a third metal ion (M3) in the PppA structure. On the other hand, the difference in metal binding of PppA may be due to the difference in
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Fig. 3. Flap subdomain of PppA and potential M3 ion binding site. A) Cartoon and surface of PppA crystal structure. Flap subdomain (cyan color) is close to active site (blue color). B) Residues in active site are showed as sticks colored with green (carbon atoms) and red (oxygen atoms). Residues in potential M3 ion binding site are showed as sticks colored with slate (carbon atoms), blue (nitrogen atoms) and red (oxygen atoms). Water molecules coordinating manganese ions and coordinated water molecules are showed like Fig. 2. C) Sequence alignment of flap subdomain of PppA from Pseudomonas aeruginosa with its homologues, including STP1(Stp1 from Staphylococcus aureus), SaSTP(serine/threonine phosphatase from Streptococcus agalactiae), tPphA(PP2C phosphatase tPphA from Thermosynechococcus elongatus), PstP(serine/threonine phosphatase PstP from Mycobacterium tuberculosis), (PPM Ser-Thr phosphatase MspP from Mycobacterium smegmatis) and PP2Ca (The protein serine/threonine phosphatase 2C from Homo sapiens). The alignment was performed with Clustal Omega [29] and ESPript 3.0 [30]. Similar residues are written with red characters and boxed. Identified residues are boxed in red. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
the location of the flap comparing to other PPMs. Previous reported that the flap may be a versatile structural element that occupies distinct positions in different PPM phosphatases [28]. Alternatively, the flap may constitute a mobile segment coupled conformationally to the binding of M3. 3.4. Structural comparison of PppA with other PPM phosphatases The overall ternary structure of PppA is highly consistent with human PP2C and other bacterial PPM phosphatases including tPphA, Stp1, PstP, MspP and SaSTP. Similar to other bacterial PPM phosphatases, PppA lacks three C-terminal alpha-helices which are present in human PP2C structures [21]. The active sites of all seven PPM structures (PppA, Stp1, tPphA, MspP, STP, PstP and human PP2C) are highly conserved, with equivalent amino acid residues coordinating metal ions or phosphates, which are necessary for the function of PPM phosphatases (Fig. 4) (Fig. S1). The main difference between PppA and other bacterial phosphatases is the number of metal ions in the active site (Fig. S2). In PppA, only two metal ions in the active site are observed, whereas in the bacterial Stp1, tPphA, STP, PstP and MspP, the third metal ion binds to the active site. The two metal ions in the active site of PppA are at the homologous position to that of the metal ions M1 and M2 in the other PPM phosphatases. M3 is considered to be loosely coordinated to the active site of PPM, and a third metal ion is not visible in crystal structures of human PP2C due to the lower pH of the solution at the protein crystallization condition. Crystal structure of PppA lacking M3 may also be due to this reason and Mn2þfree buffer of crystallization. The third metal ion M3 is distinct coordinated at active sites of
the five above-mentioned bacterial phosphatases: In the case of Stp1 the metal ion M3 (Mn2þ) is coordinated by two amino acids Asp120 and Asp194, as well as four water molecules, two of which are coordinated by Asn162 and Asp198, respectively [22]. Only two residues coordinate M3 in the structures of tPphA and STP: Asp119 and Asp193 of tPphA [21], Asp118 and Asp192 of STP [28]. While there are three residues (Asp118, Asp191 and Ser160) coordinating the metal ion M3 (Mn2þ) in the case of PstP [27]. The third metal ion M3 is coordinated by Asp185 and His153 and the phosphate molecule in the active site of MspP [25]. In addition to two conserved residues Asp120 and Asp194 coordinating the potential M3 at the active site of PppA, a third amino acid for the coordination of potential M3 could be Asn162 in the flap region (corresponding to Asn162 in Stp1). The flexible structural feature of the flap subdomain in PppA supports that coordination of M3 from the flap subdomain is not necessary for M3 binding. In summary, the first crystal structure of PppA from Pseudomonas aeruginosa has been determined, which shows that PppA consists of a canonical PPM structural core with two manganese ions at the active site and a more flexible flap subdomain. Since the flap subdomain is a versatile structural element contributed to the enzymatic activities in different PPM phosphatases, the dynamics and flexibilities found in PppA suggested that flap subdomain may couple with conformational changes for the binding of M3, thus play important roles for the allostery of phosphatases. The highresolution crystal structure of PppA provides novel clues for understanding the molecular mechanism of PppA in T6SS regulation pathways.
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Fig. 4. Structure comparisons of PPM phosphatases. A) Structure superimposition of PppA (green), Stp1 (cyan, PDB: 5F1M, A chain) and SaSTP (magentas, PDB: 2PK0, A chain). B) Structure superimposition of PppA (green), tPphA (yellow, PDB: 5ITI, A chain) and MspP (gray, PDB: 2JFR, A chain). C) Structure superimposition of PppA (green), PstP (salmon, PDB: 1TXO, A chain) and PP2C (slate, PDB: 1A6Q, A chain). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Acknowledgements This work was supported by grants from the National Key R&D Program of China [2018YFA0507103], and [NSFC-31870719] to T.W.; Shenzhen STIC [JCYC20160331-115853521] to T.W., Shenzhen STIC [JCYJ20170307-110657570] to L.L.; Shenzhen San-Ming Project [SZSM201809085] to T.W.; Chinese Postdoctoral Science Foundation Project [2018M641077] to Y.W.; Ministry of Science and Technology of China [2018ZX09101005-003-004], and Shenzhen STIC [JCYJ20170817110434640] to D.L. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.06.020. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.06.020. References [1] S. Coulthurst, The Type VI secretion system: a versatile bacterial weapon, Microbiology 165 (2019) 503e515. https://doi.org/10.1099/mic.0.000789. [2] F. Boyer, G. Fichant, J. Berthod, Y. Vandenbrouck, I. Attree, Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources? BMC Genomics 10 (2009) 104. https://doi.org/10.1186/1471-2164-10-104. [3] J.M. Silverman, Y.R. Brunet, E. Cascales, J.D. Mougous, Structure and regulation of the type VI secretion system, Annu. Rev. Microbiol. 66 (2012) 453e472. https://doi.org/10.1146/annurev-micro-121809-151619. [4] L. Chen, Y. Zou, P. She, Y. Wu, Composition, function, and regulation of T6SS in Pseudomonas aeruginosa, Microbiol. Res. 172 (2015) 19e25. https://doi.org/ 10.1016/j.micres.2015.01.004. [5] B. Lesic, M. Starkey, J. He, R. Hazan, L.G. Rahme, Quorum sensing differentially regulates Pseudomonas aeruginosa type VI secretion locus I and homologous loci II and III, which are required for pathogenesis, Microbiology-SGM 155 (2009) 2845e2855. https://doi.org/10.1099/mic.0.029082-0. [6] T.G. Sana, A. Hachani, I. Bucior, C. Soscia, S. Garvis, E. Termine, J. Engel, A. Filloux, S. Bleves, The second type VI secretion system of Pseudomonas aeruginosa strain PAO1 is regulated by quorum sensing and Fur and modulates internalization in epithelial cells, J. Biol. Chem. 287 (2012) 27095e27105. https://doi.org/10.1074/jbc.M112.376368. [7] A. Brencic, S. Lory, Determination of the regulon and identification of novel mRNA targets of Pseudomonas aeruginosa RsmA, Mol. Microbiol. 72 (2009) 612e632. https://doi.org/10.1111/j.1365-2958.2009.06670.x. [8] J.D. Mougous, C.A. Gifford, T.L. Ramsdell, J.J. Mekalanos, Threonine phosphorylation post-translationally regulates protein secretion in Pseudomonas aeruginosa, Nat. Cell Biol. 9 (2007). 797-U121, https://doi.org/10.1038/ ncb1605. [9] J.M. Silverman, L.S. Austin, F. Hsu, K.G. Hicks, R.D. Hood, J.D. Mougous, Separate inputs modulate phosphorylation-dependent and -independent type VI secretion activation, Mol. Microbiol. 82 (2011) 1277e1290. https://doi.org/10.
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Crystal structure of PppA from Pseudomonas aeruginosa, a key regulatory component of type VI secretion systems Yujie Wu a, b, Junyuan Gong b, Shan Liu b, Dongyang Li b, d, Yulan Wu b, Xi Zhang b, Yan Ren c, Shaojian Xu c, Jingchuan Sun b, d, Tao Wang b, c, Qihui Lin c, **, Li Liu b, c, * a
School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, 518055, China Department of Biology, Southern University of Science and Technology, Shenzhen, 518055, China Joint Laboratory for Infectious Disease Prevention and Control, Hygienic Section of Longhua Center for Disease Control and Prevention, Longhua District, Shenzhen, 518109, China d The Academy for Advanced Interdisciplinary Studies, Southern University of Science and Technology, Shenzhen, 518055, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 May 2019 Accepted 3 June 2019 Available online 14 June 2019
The Type VI secretion system (T6SS) is a membrane protein complex related to inter-bacterial competitions and host-pathogen interactions in Pseudomonas aeruginosa. The T6SS is regulated by a great variety of regulatory mechanisms at multiple levels, including post-translational modification with threonine phosphorylation mediated by Ser/Thr protein kinase PpkA and phosphatase PppA. The T6SS is activated by PpkA via Thr phosphorylation of Fha, and PppA can antagonize PpkA. PppA is a PP2C-family protein phosphatase and plays a key role in the disassembly and reassembly of T6SS organelles. Herein, we report the first crystal structure of PppA from Pseudomonas aeruginosa, which was determined at a resolution of 2.10 Å. The overall structure consists of a bacteria PPM structural core and a flexible flap subdomain. PppA harbors a catalytic pocket containing two manganese ions which correspond to the canonical dinuclear metal center of Ser/Thr protein phosphatases including the bacterial PPM phosphatases and human PP2C. The flexibility and the diversity of the sequence of flap subdomain across the homologues might provide clues for substrates specific recognition of phosphatases. © 2019 Elsevier Inc. All rights reserved.
Keywords: T6SS Pseudomonas aeruginosa Phosphatase PppA Crystal structure
1. Introduction The Type VI secretion system (T6SS) is a secretory multi-protein complex that mediates inter-bacterial competitions and hostpathogen interactions. It is one of the most recent identified secretion systems as effector translocation apparatus with inverted phage tail-like structure [1]. The T6SS core-components are composed of at least 13 protein subunits (TssA-TssM) [2,3]. The T6SS has been found in about a quarter of all sequenced Gramnegative bacteria, including Pseudomonas aeruginosa, Serratia marcescens and some other pathogens [4]. In the opportunistic Gram-negative pathogen Pseudomonas aeruginosa, the T6SS can
* Corresponding author. Department of Biology, Southern University of Science and Technology, Shenzhen, 518055, China. ** Corresponding author. Joint Laboratory for Infectious Disease Prevention and Control, Hygienic Section of Longhua Center for Disease Control and Prevention, Longhua District, Shenzhen, 518109, China. E-mail addresses: [email protected] (Q. Lin), [email protected] (L. Liu). https://doi.org/10.1016/j.bbrc.2019.06.020 0006-291X/© 2019 Elsevier Inc. All rights reserved.
sense and respond to external signals, then inject effector proteins into external competitive bacteria or host cells. The T6SS is regulated in transcriptional, post-transcriptional, and post-translational levels, including quorum sensing (QS) regulation by LasR [5,6], negative regulation by RNA binding protein RsmA [7], threonine phosphorylation (TPP)-dependent and TPP-independent regulation pathways. In Pseudomonas aeruginosa, the T6SS is activated by the phosphorylation of a serine/threonine (Ser/Thr) kinase PpkA, and inhibited by a Ser/Thr phosphatase PppA, which can antagonize PpkA [8]. PppA is a PP2C-family protein phosphatase in the T6SS gene cluster. In this TPP pathway, the T6SS is activated via Thr phosphorylation of Fha, an FHA domain protein, which regulated positively by PpkA and negatively by PppA. Furthermore, the T6SS of P. aeruginosa can be repressed by TagF in TPP-independent pathway [9]. The T6SS is a dynamic organelle regulated by PpkA-Fha1-PppA cycle, which participates in suppressing random formation of T6SS organelles, inducing their assembly precisely at the point of an external signal, and targeting the disassembly of T6SS organelles if necessary. So, PppA plays a
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key role in bacterial T6SS ‘‘tit-for-tat’’ evolutionary strategy [10]. Furthermore, PppA also regulates quorum sensing, c-di-GMP production, polysaccharide biosynthesis and transport, and it shows that PppA participates in controlling virulence factors and pleiotropic phenotypes besides T6SS [11]. Structures of T6SS core complex and some regulated subunits, such as TagF and PpkA, have been well determined [12e14]. Atomic resolution structural information of PppA is still unclear. Herein we report the crystal structure of PppA from Pseudomonas aeruginosa, which has a bacteria PPM structural core with two-metal active site and a flexible flap subdomain. 2. Materials and methods 2.1. Protein expression and purification The PppA (Gene ID: 879341) gene was amplified from the genome of Pseudomonas aeruginosa PAO1. The PCR product was sub-cloned into the Nde I and Xho I sites of a pET28a(þ) vector with an N-terminal His-tag. E. coli strains containing the recombinant plasmid were grown in Luria-Bertani broth medium with 50 mg/L kanamycin at 37 C. When the OD600 reached 0.6, IPTG (isopropyl bD-1-thiogalacto-pyranoside) was added to the growth medium to a final concentration of 0.2 mM to induce the expression of recombinant proteins. The induced cultures were further grown at 16 C for 16 h. The cells were harvested by centrifugation at 5500g for 10 min at 4 C and resuspended in lysis buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole). The PppA protein was purified using HisTrap HP column (GE Healthcare) in lysis buffer, followed by imidazole elution and further purification using sizeexclusion chromatography (Superdex75 10/300 GL) in the buffer containing 10 mM Tris-HCl with pH 7.4 and 200 mM NaCl. The peak fractions were pooled and concentrated to 10 mg/ml for crystallization. The purity of the protein was assessed by 12% SDS-PAGE. 2.2. Protein crystallization and data collection Crystal screening for the PppA was performed by the hangingdrop vapor diffusion method at 20 C using Crystal Screen kits (Hampton Research). High diffraction quality crystals were obtained after one week from a reservoir containing 18% (w/v) Polyethylene glycol 8000, 0.08 M Calcium acetate hydrate, 0.1 M HEPES (pH 7.2). Diffraction dataset was processed using HKL3000 [15]. 2.3. Crystal structure determination and refinement The structure was solved by molecular replacement in PHENIX suit [16] using Phaser [17], with the PDB entry 1TXO as the initial search model. The final models were manually built in Coot [18] with automatic refinement in PHENIX with Cartesian simulated annealing and TLS refinement [19]. The protein structures presented in the figures were rendered with PyMol [20]. The atomic coordinates and structure factors (codes 6JKV) have been deposited in the Protein Data Bank. 3. Results and discussion 3.1. Overall structure of PppA The structure of PppA has been solved at 2.1 Å resolution, and the statistics for data collection, phasing and structure refinement are summarized in Table 1. The crystal belongs to the P1 21 1 space group and contains two monomers in the asymmetric unit. Almost all residues of PppA have well defined conformations except Nterminal three residues and residues from A148 to R156 in
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Table 1 X-ray Data collection and structure refinement statistics. Parameter A. Data collection Space group Cell dimensions a/b/c (Å) Wavelength (Å) Resolution (Å) Average I/s (I) Total reflections Unique reflections Redundancy Completeness (%) Rmerge(%) B. Structure refinement Resolution (Å) Average B-factor (Å) Rwork/Rfree (%) R.m.s.d. bond lengths (Å) R.m.s.d. bond angles ( ) C. Ramachandran plot Most favored (%) Allowed (%) Disallowed (%)
PppA P 1 21 1 50.54/80.25/59.08 1.5418 24.28e2.10 (2.18e2.10) 4.9 (2.3) 51259 25293 2.03 (1.86) 98.6 (99.5) 0.119 (0.279) 24.08e2.10 23.0 23.3/28.3 0.0057 1.1402 96.4 3.6 0
monomer A or from D146 to H157 in monomer B. Structure of PppA mentioned below generally refers to monomer A unless otherwise specified definition. The final model of PppA monomer contains two metal ions. The analysis of X-ray anomalous diffraction data just above and below the Mn K edge confirmed the existence of manganese. The core structure of PppA is very similar to other PPM phosphatases structures, which have abba sandwich structure consisting of two five-stranded, antiparallel b sheets surrounded by two pairs of antiparallel a helices on either side (Fig. 1). Antiparallel b sheets of PppA are formed by residues 7e16, 122e127, 130e133, 186e191 and 234e242 in b-sheet 1, and 26e31, 36e43, 100e109, 112e118 and 177e182 in b-sheet 2. b-sheet 1 is flanked by a pair of antiparallel a helices (residues 202e209 and 215e228) and a short helix (residues 194e197). b-sheet 2 is flanked by a pair of antiparallel a helices (residues 49e62 and 69e94) (Fig. 1). There is a flap subdomain (residues 138e166) adjacent to the active site, which is partially disordered and a short a helix (residues 140e146) included. The flap subdomain is poor conserved in PPM phosphatases, and no electron density is visible from residues 148 to 156. This suggests that the flap subdomain is flexible and maybe correlated to substrates specificity of PppA. 3.2. The active site of PppA PppA harbors a catalytic pocket containing two manganese ions (M1 and M2). The crystal structure of PppA revealed a similar arrangement of two hexa-coordinated manganese ions in the catalytic site as in other PPMs [21,22]. Two manganese ions site correspond to the canonical dinuclear metal center of Ser/Thr protein phosphatases including the bacterial PPM phosphatases and human PP2C, which are coordinated by a strictly invariant set of protein residues [23,24] (Fig. S1). The two manganese ions separated by 3.56 Å are strictly coordinated by sidechain carboxyl groups of the conserved residues Asp41, Asp194, and Asp232, the main-chain carbonyl group of Gly42, and six water molecules, one of which bridges the two manganese ions (Fig. 2). The bridging water molecule may act as a catalytic nucleophile [25]. M1 is coordinated by three water molecules and one side-chain oxygen each of Asp41, Asp194 and Asp232. M2 is coordinated by four water molecules, one main-chain oxygen of Gly42 and one side-chain oxygen (OD1) of Asp41. Asp41 coordinates two manganese ions
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Fig. 1. Crystal Structure of PppA. A) Cartoon representation of PppA. The a-helices, b-sheets and loop regions are shown as cartoon colored with deep salmon, teal and gray respectively. Gray dashed lines depict unstructured regions of the flap subdomain. Two manganese ions are shown as spheres colored with teal.
Fig. 2. Active site of PppA. A) Active site in the overall structure of PppA. Residues coordinating the two manganese ions in the active site are showed as sticks colored with green (carbon atoms) and red (oxygen atoms). Two manganese ions are shown as spheres colored with teal. Water molecules coordinating manganese ions are showed as spheres colored with red and magentas (the one bridging the two manganese ions). dashed lines depict coordination of manganese ions with PppA residues or water molecules. B and C are magnified views of active site at A. Backbone carbonyl oxygen atom of G41 and sidechains of D41, D194 and D232 are showed as sticks and labelled. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
via two oxygen atoms of sidechain carboxyl group respectively on the side opposite the bridging water molecule (Fig. 2). The PppA structure also shows that Asp232 is positioned to accept a proton from the bridging water molecule. Arg21, which is responsible for substrate phosphoryl group binding in the active site, has a slightly different position in both monomers of the crystal asymmetric unit. But no phosphate molecule could be observed in the electron density of PppA since protein crystal grew in Tris hydrochloric acid buffer without phosphate. Although there is a third metal ion called M3 (Mg or Mn ion) in many identified structures of PPM phosphatases, the PppA structure does not contain the third metal ion (M3) close to the active site. The equivalent residues coordinated potential M3 in PppA are Asp120 and Asp194, which are strictly conserved in PPMs (Fig. 3B) [26,27]. It suggests that a third metal ion can be coordinated at the equivalent area near the active site of PppA. We propose most of potential M3 ligands in PppA are water molecules, M3 in PppA may
be less tightly bound than the M3 in other PPMs. Furthermore, the flap subdomain of PppA is more flexible than equivalent segment of other PPMs, which may lead to the lack of M3 in PppA. 3.3. Flap subdomain of PppA The PppA flap subdomain (residues 138e166) contains an a helix followed by a long loop (residues 148e156 are not visible in the electron density). Although amino acid residues of flap subdomains are not conserved in bacteria PPMs, the number of flap residues in PPMs is almost consistent (Fig. 3C). A large, irregular flap joining two beta strands forms one side of a cleft containing the two-metal active site of PppA (Fig. 3A). Flap subdomain is much more flexible in the PppA structure than in the other PPMs. The structural flexibility of flap subdomain may be related to the lack of a third metal ion (M3) in the PppA structure. On the other hand, the difference in metal binding of PppA may be due to the difference in
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Fig. 3. Flap subdomain of PppA and potential M3 ion binding site. A) Cartoon and surface of PppA crystal structure. Flap subdomain (cyan color) is close to active site (blue color). B) Residues in active site are showed as sticks colored with green (carbon atoms) and red (oxygen atoms). Residues in potential M3 ion binding site are showed as sticks colored with slate (carbon atoms), blue (nitrogen atoms) and red (oxygen atoms). Water molecules coordinating manganese ions and coordinated water molecules are showed like Fig. 2. C) Sequence alignment of flap subdomain of PppA from Pseudomonas aeruginosa with its homologues, including STP1(Stp1 from Staphylococcus aureus), SaSTP(serine/threonine phosphatase from Streptococcus agalactiae), tPphA(PP2C phosphatase tPphA from Thermosynechococcus elongatus), PstP(serine/threonine phosphatase PstP from Mycobacterium tuberculosis), (PPM Ser-Thr phosphatase MspP from Mycobacterium smegmatis) and PP2Ca (The protein serine/threonine phosphatase 2C from Homo sapiens). The alignment was performed with Clustal Omega [29] and ESPript 3.0 [30]. Similar residues are written with red characters and boxed. Identified residues are boxed in red. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
the location of the flap comparing to other PPMs. Previous reported that the flap may be a versatile structural element that occupies distinct positions in different PPM phosphatases [28]. Alternatively, the flap may constitute a mobile segment coupled conformationally to the binding of M3. 3.4. Structural comparison of PppA with other PPM phosphatases The overall ternary structure of PppA is highly consistent with human PP2C and other bacterial PPM phosphatases including tPphA, Stp1, PstP, MspP and SaSTP. Similar to other bacterial PPM phosphatases, PppA lacks three C-terminal alpha-helices which are present in human PP2C structures [21]. The active sites of all seven PPM structures (PppA, Stp1, tPphA, MspP, STP, PstP and human PP2C) are highly conserved, with equivalent amino acid residues coordinating metal ions or phosphates, which are necessary for the function of PPM phosphatases (Fig. 4) (Fig. S1). The main difference between PppA and other bacterial phosphatases is the number of metal ions in the active site (Fig. S2). In PppA, only two metal ions in the active site are observed, whereas in the bacterial Stp1, tPphA, STP, PstP and MspP, the third metal ion binds to the active site. The two metal ions in the active site of PppA are at the homologous position to that of the metal ions M1 and M2 in the other PPM phosphatases. M3 is considered to be loosely coordinated to the active site of PPM, and a third metal ion is not visible in crystal structures of human PP2C due to the lower pH of the solution at the protein crystallization condition. Crystal structure of PppA lacking M3 may also be due to this reason and Mn2þfree buffer of crystallization. The third metal ion M3 is distinct coordinated at active sites of
the five above-mentioned bacterial phosphatases: In the case of Stp1 the metal ion M3 (Mn2þ) is coordinated by two amino acids Asp120 and Asp194, as well as four water molecules, two of which are coordinated by Asn162 and Asp198, respectively [22]. Only two residues coordinate M3 in the structures of tPphA and STP: Asp119 and Asp193 of tPphA [21], Asp118 and Asp192 of STP [28]. While there are three residues (Asp118, Asp191 and Ser160) coordinating the metal ion M3 (Mn2þ) in the case of PstP [27]. The third metal ion M3 is coordinated by Asp185 and His153 and the phosphate molecule in the active site of MspP [25]. In addition to two conserved residues Asp120 and Asp194 coordinating the potential M3 at the active site of PppA, a third amino acid for the coordination of potential M3 could be Asn162 in the flap region (corresponding to Asn162 in Stp1). The flexible structural feature of the flap subdomain in PppA supports that coordination of M3 from the flap subdomain is not necessary for M3 binding. In summary, the first crystal structure of PppA from Pseudomonas aeruginosa has been determined, which shows that PppA consists of a canonical PPM structural core with two manganese ions at the active site and a more flexible flap subdomain. Since the flap subdomain is a versatile structural element contributed to the enzymatic activities in different PPM phosphatases, the dynamics and flexibilities found in PppA suggested that flap subdomain may couple with conformational changes for the binding of M3, thus play important roles for the allostery of phosphatases. The highresolution crystal structure of PppA provides novel clues for understanding the molecular mechanism of PppA in T6SS regulation pathways.
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Fig. 4. Structure comparisons of PPM phosphatases. A) Structure superimposition of PppA (green), Stp1 (cyan, PDB: 5F1M, A chain) and SaSTP (magentas, PDB: 2PK0, A chain). B) Structure superimposition of PppA (green), tPphA (yellow, PDB: 5ITI, A chain) and MspP (gray, PDB: 2JFR, A chain). C) Structure superimposition of PppA (green), PstP (salmon, PDB: 1TXO, A chain) and PP2C (slate, PDB: 1A6Q, A chain). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Acknowledgements This work was supported by grants from the National Key R&D Program of China [2018YFA0507103], and [NSFC-31870719] to T.W.; Shenzhen STIC [JCYC20160331-115853521] to T.W., Shenzhen STIC [JCYJ20170307-110657570] to L.L.; Shenzhen San-Ming Project [SZSM201809085] to T.W.; Chinese Postdoctoral Science Foundation Project [2018M641077] to Y.W.; Ministry of Science and Technology of China [2018ZX09101005-003-004], and Shenzhen STIC [JCYJ20170817110434640] to D.L. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.06.020. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.06.020. References [1] S. Coulthurst, The Type VI secretion system: a versatile bacterial weapon, Microbiology 165 (2019) 503e515. https://doi.org/10.1099/mic.0.000789. [2] F. Boyer, G. Fichant, J. Berthod, Y. Vandenbrouck, I. Attree, Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources? BMC Genomics 10 (2009) 104. https://doi.org/10.1186/1471-2164-10-104. [3] J.M. Silverman, Y.R. Brunet, E. Cascales, J.D. Mougous, Structure and regulation of the type VI secretion system, Annu. Rev. Microbiol. 66 (2012) 453e472. https://doi.org/10.1146/annurev-micro-121809-151619. [4] L. Chen, Y. Zou, P. She, Y. Wu, Composition, function, and regulation of T6SS in Pseudomonas aeruginosa, Microbiol. Res. 172 (2015) 19e25. https://doi.org/ 10.1016/j.micres.2015.01.004. [5] B. Lesic, M. Starkey, J. He, R. Hazan, L.G. Rahme, Quorum sensing differentially regulates Pseudomonas aeruginosa type VI secretion locus I and homologous loci II and III, which are required for pathogenesis, Microbiology-SGM 155 (2009) 2845e2855. https://doi.org/10.1099/mic.0.029082-0. [6] T.G. Sana, A. Hachani, I. Bucior, C. Soscia, S. Garvis, E. Termine, J. Engel, A. Filloux, S. Bleves, The second type VI secretion system of Pseudomonas aeruginosa strain PAO1 is regulated by quorum sensing and Fur and modulates internalization in epithelial cells, J. Biol. Chem. 287 (2012) 27095e27105. https://doi.org/10.1074/jbc.M112.376368. [7] A. Brencic, S. Lory, Determination of the regulon and identification of novel mRNA targets of Pseudomonas aeruginosa RsmA, Mol. Microbiol. 72 (2009) 612e632. https://doi.org/10.1111/j.1365-2958.2009.06670.x. [8] J.D. Mougous, C.A. Gifford, T.L. Ramsdell, J.J. Mekalanos, Threonine phosphorylation post-translationally regulates protein secretion in Pseudomonas aeruginosa, Nat. Cell Biol. 9 (2007). 797-U121, https://doi.org/10.1038/ ncb1605. [9] J.M. Silverman, L.S. Austin, F. Hsu, K.G. Hicks, R.D. Hood, J.D. Mougous, Separate inputs modulate phosphorylation-dependent and -independent type VI secretion activation, Mol. Microbiol. 82 (2011) 1277e1290. https://doi.org/10.
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