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Allosteric receptor activation by the plant peptide hormone phytosulfokine

Abstract

Phytosulfokine (PSK) is a disulfated pentapeptide that has a ubiquitous role in plant growth and development1,2. PSK is perceived by its receptor PSKR3,4, a leucine-rich repeat receptor kinase (LRR-RK). The mechanisms underlying the recognition of PSK, the activation of PSKR and the identity of the components downstream of the initial binding remain elusive. Here we report the crystal structures of the extracellular LRR domain of PSKR in free, PSK- and co-receptor-bound forms. The structures reveal that PSK interacts mainly with a β-strand from the island domain of PSKR, forming an anti-β-sheet. The two sulfate moieties of PSK interact directly with PSKR, sensitizing PSKR recognition of PSK. Supported by biochemical, structural and genetic evidence, PSK binding enhances PSKR heterodimerization with the somatic embryogenesis receptor-like kinases (SERKs). However, PSK is not directly involved in PSKR–SERK interaction but stabilizes PSKR island domain for recruitment of a SERK. Our data reveal the structural basis for PSKR recognition of PSK and allosteric activation of PSKR by PSK, opening up new avenues for the design of PSKR-specific small molecules.

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Figure 1: Recognition mechanism of PSK by PSKR1LRR.
Figure 2: PSK promotes PSKR–SERK heterodimerization.
Figure 3: PSK stabilizes the PSKRID for interaction with SERKsLRR.
Figure 4: Mutagenesis analysis of PSKR–SERK interaction.

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Acknowledgements

We thank S. Huang and J. He for assistance with data collection, J. Li for the serk1, serk2, bak1 single and triple mutant seeds and W. Li and W. Chu for providing facility support. This research was funded by Projects of International Cooperation and Exchanges NSFC (31420103906), Chinese Ministry of Science and Technology (2015CB910200) and State Key Program of National Natural Science of China (31130063) to J.C.; Chinese Natural Science Foundation (31330053) to W.Y. and Ministry of Science and Technology of China (2015CB910202) to H.L.

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Authors and Affiliations

Authors

Contributions

J.C., W.Y., J.W., H.L. and Z.H. designed the experiments. J.W., H.L., H.Z., T.W. and G.L. performed the experiments. Data were analysed by J.C., W.Y., J.W., H.L. and J.C.; J.C., W.Y., J.W., H.L. and Z.H. contributed to manuscript preparation. J.C. wrote the manuscript.

Corresponding authors

Correspondence to Weicai Yang or Jijie Chai.

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Competing interests

The authors declare no competing financial interests.

Additional information

The atomic coordinates and structure factors have been deposited in the Protein Data Bank. The PDB code of free DcPSKRLRR is 4Z62. The PDB codes of PSK–PSKR1LRR and PSK–DcPSKRLRR are 4Z63 and 4Z5W, respectively. The PDB codes of PSK–PSKR1LRR–SERK1LRR and PSK–DcPSKRLRR–SERK2LRR are 4Z64 and 4Z61, respectively.

Extended data figures and tables

Extended Data Figure 1 Recognition mechanism of PSK by PSKRsLRR is highly conserved.

a, Overall structure of PSK–DcPSKRLRR complex. The sulfated tyrosines of PSK are shown in stick. Colour codes are indicated. ID, island domain; N, N terminus; C, C terminus. b, Detailed interactions between PSK (purple) and the island domain (salmon) of DcPSKRLRR. Dashed lines indicate polar interactions. c, Detailed interactions between PSK and the inner side (cyan) of DcPSKRLRR. d, PSKRs are conserved in PSK perception and interaction with SERKs. Sequence alignment of the ectodomains of carrot DcPSKR and Arabidopsis PSKR1/2. Conserved and similar residues are boxed with red ground and red font, respectively. Residues involved in recognition of PSK and interaction with a SERK member are indicated with blue solid circles and squares at the bottom, respectively.

Extended Data Figure 2 Mutagenesis analysis of PSKR recognition of PSK and PSKR–SERK interaction.

a, Sulfation enhances PSK interaction with DcPSKRLRR. Quantification of binding affinity between DcPSKRLRR and PSK or the desulfated peptide (dPSK) by MST (MicroScale Thermophoresis). Data points indicate the difference in normalized fluorescence (‰) generated by PSK or dPSK binding DcPSKRLRR protein, and curves indicate the calculated fits. Error bars represent standard error of 3 independent measurements. b, Mutagenesis analysis of DcPSKRLRR by MST. Quantification of binding affinity between WT DcPSKRLRR or various mutants as indicated and PSK by MST. Error bars represent standard error of 3 independent measurements. c, pskr1-3 plants transformed with mutated PSKR1 which compromised PSK or SERKs binding are less responsive to PSK than wild type or pskr1-3 transformed with PSKR1. The line was the same as that used in Fig. 1e and 4d. Average (±s.e.m.) primary root lengths of seedlings were determined in three independent experiments with 30 seedlings analysed per genotype in the presence or absence of 1.0 µM PSK.

Extended Data Figure 3 PSK binding induces no oligomerization of PSKRLRR.

Shown on the top is superposition of the gel filtration chromatograms of the PSKR1LRR (left) or DcPSKRLRR (right) protein in the absence (grey) and presence (red) of PSK. The vertical and horizontal axes represent ultraviolet absorbance (λ = 280 nm) and elution volume (ml), respectively. Bottom, Coomassie blue staining of the peak fractions shown on the top following SDS–PAGE. M, molecular weight ladder (kDa).

Extended Data Figure 4 PSK induces PSKR1LRR or DcPSKRLRR interaction with SERK members in gel filtration.

a, PSK induces PSKR1LRR–SERK2LRR heterodimerization. Right, Coomassie blue staining of the peak fractions shown on the left following SDS–PAGE. M, molecular weight ladder (kDa). b, PSK induces PSKR1LRR heterodimerization with BAK1LRR. The assay was performed as described in a. c, PSK induces DcPSKRLRR heterodimerization with SERK1LRR. The assays were performed as described in a. The red and black arrows indicate the elution position of PSK–DcPSKRLRR–SERK1LRR and the retention volumes of molecular weight markers, respectively. d, PSK induces DcPSKRLRR heterodimerization with SERK2LRR. The assay was performed as described in a.

Extended Data Figure 5 PSK induces PSKR1LRR interaction with SERK members in sedimentation-velocity analytical ultracentrifugation.

PSK induces PSKR1LRR–SERK2LRR (left panel) or PSKR1LRR–BAK1LRR (right panel) interaction in sedimentation-velocity analytical ultracentrifugation assays. The assays were performed as described in Fig. 2b. The glycoprotein nature of PSKR1LRR may confer to the slight difference of calculated molecular weights. PSK induced the formation of a monomeric PSK–PSKR1LRR–SERK2LRR or PSK–PSKR1LRR–BAK1LRR complex, leading to the shift of PSKR1LRR to a higher S.

Extended Data Figure 6 SERK members function redundantly in PSK-induced plant growth.

ac, Average (±s.e.m.) primary root lengths of seedlings were determined for the wild-type or SERK knockout Arabidopsis plants grown for 10 days on plates with (red) or without (blue) 1.0 µM PSK. Three independent experiments per genotype with 30 seedlings were performed. The statistics are shown in a, b and c. All the genotypes are compared in the absence of PSK in a and in the presence of PSK in b. The single or double SERK knockout plants only showed slightly shortened roots compared to the triple mutants. Asterisks within the bars indicate significant difference between the wild type and SERK knockout mutants and those above the bars indicate significant difference between different SERK knockout mutants. Each genotype in the presence and absence of PSK is compared in c. Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001. NS, non-significant (P > 0.05).

Extended Data Figure 7 Different mechanism of PSK induced PSKR–SERK interaction compared to BRI1– BAK1 or FLS2–BAK1 complex.

a, Overall structure of PSK–DcPSKRLRR–SERK2LRR complex. b, Structural comparison of PSK–PSKR1LRR–SERK1LRR and brassinosteroid–BRI1LRR–BAK1LRR. The structure of PSKR1LRR (residues 77–634) was used as the template for alignment with that of BRI1 (residues 174–766; PDB code 4M7E) with a r.m.s.d. of 2.43 Å. c, Structural comparison of PSK–PSKR1LRR–SERK1LRR and flg22–FLS2LRR–BAK1LRR. The structure of PSKR1LRR (residues 82–554) was used as the template for alignment with that of FLS2 (residues 79–509; PDB code 4MN8) with a r.m.s.d. of 4.4 Å. SERK1LRR bound by PSKR1LRR rotates about 30 degrees and shifts about 20 Å relative to the BAK1LRR-bound FLS2LRR. d, Electron density around the island domain of DcPSKRLRR and PSK-bound DcPSKRLRR in the finally refined structures. Top panel, electron density 2FoFc (left) and FoFc (right) contoured at 1.30 sigma and 2.7 sigma, respectively, for the finally refined free DcPSKRLRR structure. Bottom panel: electron density 2FoFc (left) and FoFc (right) omitted around the island domain in the structure of PSK-bound DcPSKRLRR. The island domain (residues 511–535) and the β–sheet (residues 474–480, 450–456, 427–432, 402–408, 376–381 and 352–357) interacting with the ID were not included in refinement and electron density calculation. All the deleted residues are shown in pink. The marker residue proline 536 is shown in red.

Extended Data Figure 8 Mutagenesis analysis of DcPSKRLRR–SERK2LRR interaction.

a, Superposition of the gel filtration chromatograms of the mutant DcPSKRLRR and SERK2LRR proteins in the presence of PSK. The assays were performed as described in Extended Data Fig. 4a. b, Coomassie blue staining of the peak fractions shown on the left chromatograms following SDS–PAGE. M, molecular weight ladder (kDa). c, The amino acids of SERKs involved in PSKRs interaction are conserved. Sequence alignment of the ectodomains of SERK family proteins. Conserved and similar residues are boxed with red ground and red font, respectively. Residues involved in interaction with PSKR are indicated with blue solid squares at the bottom. The sequence of SERK3 is 100% identical to BAK1.

Extended Data Table 1 Data collection and refinement statistics

Supplementary information

Supplementary Data

This file contains the full blots for figures 2c, 2d, 4c and 4e. (PDF 1037 kb)

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Wang, J., Li, H., Han, Z. et al. Allosteric receptor activation by the plant peptide hormone phytosulfokine. Nature 525, 265–268 (2015). https://doi.org/10.1038/nature14858

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