Shigella effector protein OspF inactivates MAPKs in both yeast7 and mammals7–9. Conserved in both animal and plant pathogenic bacteria, OspF belongs to a new family of enzymes, phosphothreonine lyases9,10, that remove the phosphate moiety of phosphothreonine in MAPKs by cleaving the C-O rather than the O-P bond, generating a double bond–containing product. To study the catalytic mechanism of this enzyme family, we determined the crystal structures of one phosphothreonine lyase, SpvC, a critical virulence determinant for the animal pathogen Salmonella11, in free, sulfate-bound and substrate-bound forms. The structure of free SpvC was determined by the selenomethionine SAD method and all the others by molecular replacement (see Supplementary Methods and Supplementary Table 1 online). A shallow but prominent groove on the surface of SpvC formed by loops L1, L2 and L3 and the twisted five-stranded (b3, b5, b6, b4 and b7) b-sheet was the substrate-binding site (Fig. 1a). The doubly phosphorylated human ERK2-derived peptide Gly-Glu-Ala-pThrVal-pTyr-Asn-Ala-Thr (referred to as peptide hereafter) was unambiguously identified from the electron density in the structure of the SpvC-peptide complex (Fig. 1b). The two phosphorylated residues, pThr183 and pTyr185, formed the main contacts between the peptide and SpvC (Fig. 1b–d), providing the structural basis for the preference of this family of enzymes for the evolutionarily conserved MAPKs7–10, as they all contain the pThr-X-pTyr motif in their activated forms.
Structural basis for the catalytic mechanism of phosphothreonine lyase Linjie Chen1,2,5, Huayi Wang1,2,5, Jie Zhang2, Lichuan Gu3, Niu Huang2,4, Jian-Min Zhou2 & Jijie Chai2 Salmonella SpvC belongs to a new enzyme family designated phosphothreonine lyases that irreversibly inactivate mitogenactivated protein kinases. The crystal structure of SpvC reported here reveals that the two phosphorylated residues in the substrate peptide predominantly mediate its recognition by SpvC. Substrate-induced conformational changes in SpvC sequester the phosphothreonine in a completely solvent-free environment, preventing the hydrolysis of the phosphate group and facilitating the elimination reaction. Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved and are vital in animal and plant innate immune responses1–4. Therefore, one of the common strategies used by pathogenic bacteria is to interfere with MAPK signaling5. YopJ of Yersinia covalently modifies MAPK kinases to prevent their phosphorylation6. The
f
W
e
d
T BS A K1 6 R 0E 90 F1 E 0 K1 0E 3 Y1 4E 5 Y1 8E 5 E2 8F 15 A
c
22 0A
b
/R
a
BS A W T F8 6 K1 D 0 H 4A 10 K1 6A 3 R 6A 14 V1 8A 4 R 9D 21 R 3A 22 0 R A 14 8A
© 2007 Nature Publishing Group http://www.nature.com/nsmb
B R I E F C O M M U N I C AT I O N S
Figure 1 Structures of free SpvC and its complex with a doubly BSA ERK2 phosphorylated peptide. (a) Overall structure of free SpvC in space group P212121. (b) Electron density (at 1.3 d) for the SpvC peptide. SpvC is shown in the same orientation as in a. Phosphorylated ERK2 (c) Specific recognition of the phosphothreonine. The side chains of those residues of SpvC interacting with the phosphothreonine are colored yellow. (d) Specific recognition of the phosphotyrosine. (e) Effects on enzymatic activity of mutations in the residues of SpvC recognizing the phosphothreonine (the residues shown in c). Phosphorylated ERK2 was used as the substrate for various SpvC mutants. The residual phosphorylated ERK2 was detected by anti-pERK antibody. (f) Effects on enzymatic activity of mutations in the residues of SpvC shown in d.
1Graduate Program in Chinese Academy of Medical Sciences and Beijing Union Medical College, Beijing 100730, China. 2National Institute of Biological Sciences, Beijing 102206, China. 3State Key Lab of Microbial Technology, Shandong University, Jinan 250100, China. 4Department of Pharmaceutical Chemistry, University of California San Francisco, QB3 Building, 1700 Fourth Street, Box 2550, San Francisco, California 94143-2550, USA. 5These authors contributed equally to this work. Correspondence should be addressed to J.C. (
[email protected]).
Received 23 July; accepted 4 October; published online 16 December 2007; doi:10.1038/nsmb1329
NATURE STRUCTURAL & MOLECULAR BIOLOGY
ADVANCE ONLINE PUBLICATION
1
B R I E F C O M M U N I C AT I O N S sulfate-induced conformational changes are likely to trap one water molecule around the phosphate group and thereby sequester the catalytic intermediate from nucleophiles other than the water molecule, thus ensuring the hydrolysis product12. The solvent-free environment of pThr183 seems to be critical for the SpvC-catalyzed reaction, because it not only prevents the hydrolysis of the phosphate group, excluding the possibility that the enzyme will function as a phosphatase, but also facilitates the elimination reaction9, as reactions involving nucleophilic attack often become faster in aprotic solvents Figure 2 Substrate-induced conformational changes in SpvC result in complete sequestration of phosphothreonine. (a) Structural alignment of the free and substrate-bound forms of SpvC. (b) Binding because there is less solvation of the nucleoof the substrate triggers conformational changes around the phosphothreonine binding site in SpvC. phile. Three polar residues, Lys136, Tyr158 The peptide is colored cyan. Residues from the free and substrate-bound SpvC are shown in pink and Glu215, are located close to pThr183 and orange, respectively. The solvent-accessible surface area of pThr183 in the SpvC-peptide complex (Fig. 2b), and one of these residues can 2 ˚ is 0.0 A . potentially act as the nucleophile to catalyze generation of the double bond–containing Four invariant residues (Supplementary Fig. 1 online), Lys104, product9,10. In contrast with Y158F and E215A (Fig. 1f), K136A Arg148, Arg213 and Arg220, formed four pairs of bifurcated completely abolished enzymatic activity (Fig. 1e), indicating that the salt bonds with the phosphate group of pThr183 (Fig. 1c) and invariant Lys136 in SpvC (Supplementary Fig. 1) is a catalytic residue were required for SpvC enzymatic activity (Fig. 1e). The four that is likely to act as the nucleophile for the elimination reaction9. In corresponding residues in HopAI1, another phosphothreonine lyase, support of this conclusion, previous studies have demonstrated that are important for its immune-suppressing function10 (Supplementary mutation of the corresponding residue in OspF (ref. 9) or HopAI1 Fig. 2 online). The phosphate group was further stabilized by (ref. 10) abolishes enzymatic activity but has no effect on substrate hydrogen bonding with the conserved residue His106 (Fig. 1c). binding. Additionally, His106 may act as a catalytic acid to promote Mutation of this residue in SpvC substantially compromised the elimination reaction by protonating the oxyanion of the scissile its in vitro enzymatic activity (Fig. 1e). Analogous mutations in C-O bond (Fig. 1c). OspF (ref. 9) and HopAI1 (ref. 10) had similar effects in vitro and in vivo. The conserved hydrophobic residues Phe86 and Val149 Accession codes. Protein Data Bank: Coordinates and structure factor were important in recognizing pThr183 (Fig. 1c,e), explaining the files have been deposited with the accession codes 2Z8O (free SpvC in preference for phosphothreonine over phosphoserine by phospho- P4122), 2Z8M (free SpvC in P212121), 2Z8N (sulfate-bound SpvC) threonine lyases9. and 2Z8P (peptide-bound SpvC). There were important electrostatic and hydrophobic interactions between pTyr185 and SpvC. The phosphate moiety of pTyr185 was Note: Supplementary information is available on the Nature Structural & Molecular Biology website. recognized by Arg90, Lys134 and Lys160 in SpvC (Fig. 1d). Mutation of these residues reduced the enzymatic activity (Fig. 1f). Interaction ACKNOWLEDGMENTS of pTyr185 with the hydrophobic pocket constructed by Phe100, We thank Y. Dong and P. Liu at the Beijing Synchrotron Radiation Facility Tyr158 and the aliphatic portion of Lys134 (Fig. 1d) was also for assistance with the data collection. This research is funded by Chinese Ministry of Science and Technology ‘863’ grants 2003-AA210090 to J.C. important for the activity of SpvC (Fig. 1f). and 2003-AA210080 to J.Z. Conformational changes in SpvC were triggered by binding of substrate (Fig. 2a) or sulfate group (Supplementary Fig. 3a Published online at http://www.nature.com/nsmb online). The most prominent structural change occurred in loop L3 Reprints and permissions information is available online at http://npg.nature.com/ surrounding Arg220, whose Ca atom moved 10.1 A˚ toward reprintsandpermissions pThr183 (Fig. 2b and Supplementary Fig. 3b). In addition, 1. Akira, S., Uematsu, S. & Takeuchi, O. Cell 124, 783–801 (2006). a5 became a random coil in the substrate- or sulfate-bound 2. Ashwell, J.D. Nat. Rev. Immunol. 6, 532–540 (2006). structure (Fig. 2a and Supplementary Fig. 3a), allowing Arg148 3. Ausubel, F.M. Nat. Immunol. 6, 973–979 (2005). to rotate and form salt bridges with the phosphate oxyanion, 4. Tena, G., Asai, T., Chiu, W.L. & Sheen, J. Curr. Opin. Plant Biol. 4, 392–400 (2001). 5. Hornef, M.W., Wick, M.J., Rhen, M. & Normark, S. Nat. Immunol. 3, 1033–1040 whereas Val149 moved approximately 2.5 A˚ toward pThr183 to (2002). make hydrophobic contact with its methyl group (Fig. 2b and 6. Mukherjee, S. et al. Science 312, 1211–1214 (2006). 7. Kramer, R.W. et al. PLoS Pathogens [online] 3, e21 (2007). Supplementary Fig. 3b). 8. Arbibe, L. et al. Nat. Immunol. 8, 47–56 (2007). These structural changes keep pThr183 in a solvent-free environ- 9. Li, H. et al. Science 315, 1000–1003 (2007). ment (Fig. 2b), blocking water molecules or other nucleophiles than 10. Zhang, J. et al. Cell Host & Microbe 1, 175–185 (2007). 11. Matsui, H. et al. J. Bacteriol. 183, 4652–4658 (2001). Lys136 of SpvC from accessing it. This is in contrast with the structure 12. Schubert, H.L., Fauman, E.B., Stuckey, J.A., Dixon, J.E. & Saper, M.A.A. Protein Sci. 4, 1904–1913 (1995). of the Yop51 protein tyrosine phosphatase from Yersinia, in which
© 2007 Nature Publishing Group http://www.nature.com/nsmb
a
2
Bound SpvC Free SpvC
b
ADVANCE ONLINE PUBLICATION
NATURE STRUCTURAL & MOLECULAR BIOLOGY
