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Differential CLE peptide perception by plant receptors implicated from structural and functional analyses of TDIF-TDR interactions

View Article: PubMed Central - PubMed

ABSTRACT

Tracheary Element Differentiation Inhibitory Factor (TDIF) belongs to the family of post-translationally modified CLE (CLAVATA3/embryo surrounding region (ESR)-related) peptide hormones that control root growth and define the delicate balance between stem cell proliferation and differentiation in SAM (shoot apical meristem) or RAM (root apical meristem). In Arabidopsis, Tracheary Element Differentiation Inhibitory Factor Receptor (TDR) and its ligand TDIF signaling pathway is involved in the regulation of procambial cell proliferation and inhibiting its differentiation into xylem cells. Here we present the crystal structures of the extracellular domains (ECD) of TDR alone and in complex with its ligand TDIF resolved at 2.65 Ǻ and 2.75 Ǻ respectively. These structures provide insights about the ligand perception and specific interactions between the CLE peptides and their cognate receptors. Our in vitro biochemical studies indicate that the interactions between the ligands and the receptors at the C-terminal anchoring site provide conserved binding. While the binding interactions occurring at the N-terminal anchoring site dictate differential binding specificities between different ligands and receptors. Our studies will open different unknown avenues of TDR-TDIF signaling pathways that will enhance our knowledge in this field highlighting the receptor ligand interaction, receptor activation, signaling network, modes of action and will serve as a structure function relationship model between the ligand and the receptor for various similar leucine-rich repeat receptor-like kinases (LRR-RLKs).

No MeSH data available.


Binding interface between TDR and TDIF.(A) Surface residues of TDR that make contacts with TDIF peptide are depicted in blue, with their side chains shown in stick representation and residue numbered indicated. The rest of the TDR structure is colored in cyan, and the structure of TDIF peptide is shown in red and stick representation, with each residue number indicated. The sequence of the TDIF is shown at the bottom of the panel. (B) The N-anchoring site of TDIF on TDR. Hydrogen bonds are shown in green dotted lines, salt-bridge is depicted in a red dotted line, and hydrophobic interaction is shown in a solid black line. (C) C-anchoring site of TDIF on TDR. Hydrogen bonds are shown in green dotted lines and hydrophobic interaction is shown in a solid black line.
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pone.0175317.g002: Binding interface between TDR and TDIF.(A) Surface residues of TDR that make contacts with TDIF peptide are depicted in blue, with their side chains shown in stick representation and residue numbered indicated. The rest of the TDR structure is colored in cyan, and the structure of TDIF peptide is shown in red and stick representation, with each residue number indicated. The sequence of the TDIF is shown at the bottom of the panel. (B) The N-anchoring site of TDIF on TDR. Hydrogen bonds are shown in green dotted lines, salt-bridge is depicted in a red dotted line, and hydrophobic interaction is shown in a solid black line. (C) C-anchoring site of TDIF on TDR. Hydrogen bonds are shown in green dotted lines and hydrophobic interaction is shown in a solid black line.

Mentions: We have crystallized the extracellular domain of A. thaliana TDR (ecdTDR) alone and ecdTDR in complex with a synthetic TDIF peptide. Both crystals have the same space group p41 and almost identical unit cell dimensions and crystal packing (Table 1). We have determined the apo-TDR structure by molecular replacement using the FLS2-ECD structure (PDB ID 4MN8) as an initial search model, and the structure of the ecdTDR/TDIF complex structure was then solved by molecular replacement using the apo-TDR structure as the search model. The atomic coordinates and structure factors of TDR and TDR-TIDF complex have been deposited in the Protein Data Bank under accession codes 5JFK and 5JFI respectively. The overall architecture of ecdTDR adopts an “S” shaped superhelical structure consisting of 22 LRRs (Fig 1), which resembles the other known plant LRR-RLK structures [29–32]. Almost all the 22 LRRs (except LRR18) in the TDR structures have a unified length of 24 amino acids with no variable insertion sequences. The conserved motif is “LXXLXLXXNXL/FXGXΦPXXΦXXLXX”, in which “X” stands for any residue and “Φ” stands for a hydrophobic residue (S3 Fig). Two pairs of cysteine residues, C390-C416 and C511-C535 form two disulfide bonds that tighten the parallel packing between LRR13-LRR14 and LRR18-LRR19 (S3 and S4 Figs). Five asparagine residues, N111, N356, N378, N471 and N525, are found to be N-glycosylated in the TDR structures. However, only one GlcNAc sugar residue on each site is visible in the electron density maps of the structures (S3 and S4 Figs). The TDIF peptide is bound on the concave surface of the TDR receptor, which stays on the middle of the surface and covers from LRR3 to LRR15. Each of the 12 residues of TDIF is visible in the electron density map, and the peptide adopts a fully extended conformation while making a blunt turn at the sixth residue G6 (Figs 1, 2 and S2 Fig). Superposition of our TDR-TDIF complex structure with the recently reported structures (PDB ID: 5GIJ and 5GR9) have resulted in a root mean square deviation (RMSD) of 0.596 and 0.682 over 594 residues respectively (S5 Fig), showing that these three ligand-receptor complex structures strongly agree with each other. However, in both our TDR apo-structure and the TDIF-TDR complex structure, the N-terminal (residues 40–81) and the C-terminal portions (residues 617–625) of the structures have poor electron density, indicating that those parts of the structures are flexible in the crystals. The rest of the structures are identical to the two previously published structures. The three TDIF ligand structures are superimposable, except that the side chain of E2 in our structure has about 150 degree rotation of the γ-carbon bond away from the structure solved by Zhang, et al., 2016 (PDB ID: 5GIJ), while it agrees with the structure of Morita, et al., 2016 (PDB ID: 5GR9) (S5 Fig).


Differential CLE peptide perception by plant receptors implicated from structural and functional analyses of TDIF-TDR interactions
Binding interface between TDR and TDIF.(A) Surface residues of TDR that make contacts with TDIF peptide are depicted in blue, with their side chains shown in stick representation and residue numbered indicated. The rest of the TDR structure is colored in cyan, and the structure of TDIF peptide is shown in red and stick representation, with each residue number indicated. The sequence of the TDIF is shown at the bottom of the panel. (B) The N-anchoring site of TDIF on TDR. Hydrogen bonds are shown in green dotted lines, salt-bridge is depicted in a red dotted line, and hydrophobic interaction is shown in a solid black line. (C) C-anchoring site of TDIF on TDR. Hydrogen bonds are shown in green dotted lines and hydrophobic interaction is shown in a solid black line.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5383425&req=5

pone.0175317.g002: Binding interface between TDR and TDIF.(A) Surface residues of TDR that make contacts with TDIF peptide are depicted in blue, with their side chains shown in stick representation and residue numbered indicated. The rest of the TDR structure is colored in cyan, and the structure of TDIF peptide is shown in red and stick representation, with each residue number indicated. The sequence of the TDIF is shown at the bottom of the panel. (B) The N-anchoring site of TDIF on TDR. Hydrogen bonds are shown in green dotted lines, salt-bridge is depicted in a red dotted line, and hydrophobic interaction is shown in a solid black line. (C) C-anchoring site of TDIF on TDR. Hydrogen bonds are shown in green dotted lines and hydrophobic interaction is shown in a solid black line.
Mentions: We have crystallized the extracellular domain of A. thaliana TDR (ecdTDR) alone and ecdTDR in complex with a synthetic TDIF peptide. Both crystals have the same space group p41 and almost identical unit cell dimensions and crystal packing (Table 1). We have determined the apo-TDR structure by molecular replacement using the FLS2-ECD structure (PDB ID 4MN8) as an initial search model, and the structure of the ecdTDR/TDIF complex structure was then solved by molecular replacement using the apo-TDR structure as the search model. The atomic coordinates and structure factors of TDR and TDR-TIDF complex have been deposited in the Protein Data Bank under accession codes 5JFK and 5JFI respectively. The overall architecture of ecdTDR adopts an “S” shaped superhelical structure consisting of 22 LRRs (Fig 1), which resembles the other known plant LRR-RLK structures [29–32]. Almost all the 22 LRRs (except LRR18) in the TDR structures have a unified length of 24 amino acids with no variable insertion sequences. The conserved motif is “LXXLXLXXNXL/FXGXΦPXXΦXXLXX”, in which “X” stands for any residue and “Φ” stands for a hydrophobic residue (S3 Fig). Two pairs of cysteine residues, C390-C416 and C511-C535 form two disulfide bonds that tighten the parallel packing between LRR13-LRR14 and LRR18-LRR19 (S3 and S4 Figs). Five asparagine residues, N111, N356, N378, N471 and N525, are found to be N-glycosylated in the TDR structures. However, only one GlcNAc sugar residue on each site is visible in the electron density maps of the structures (S3 and S4 Figs). The TDIF peptide is bound on the concave surface of the TDR receptor, which stays on the middle of the surface and covers from LRR3 to LRR15. Each of the 12 residues of TDIF is visible in the electron density map, and the peptide adopts a fully extended conformation while making a blunt turn at the sixth residue G6 (Figs 1, 2 and S2 Fig). Superposition of our TDR-TDIF complex structure with the recently reported structures (PDB ID: 5GIJ and 5GR9) have resulted in a root mean square deviation (RMSD) of 0.596 and 0.682 over 594 residues respectively (S5 Fig), showing that these three ligand-receptor complex structures strongly agree with each other. However, in both our TDR apo-structure and the TDIF-TDR complex structure, the N-terminal (residues 40–81) and the C-terminal portions (residues 617–625) of the structures have poor electron density, indicating that those parts of the structures are flexible in the crystals. The rest of the structures are identical to the two previously published structures. The three TDIF ligand structures are superimposable, except that the side chain of E2 in our structure has about 150 degree rotation of the γ-carbon bond away from the structure solved by Zhang, et al., 2016 (PDB ID: 5GIJ), while it agrees with the structure of Morita, et al., 2016 (PDB ID: 5GR9) (S5 Fig).

View Article: PubMed Central - PubMed

ABSTRACT

Tracheary Element Differentiation Inhibitory Factor (TDIF) belongs to the family of post-translationally modified CLE (CLAVATA3/embryo surrounding region (ESR)-related) peptide hormones that control root growth and define the delicate balance between stem cell proliferation and differentiation in SAM (shoot apical meristem) or RAM (root apical meristem). In Arabidopsis, Tracheary Element Differentiation Inhibitory Factor Receptor (TDR) and its ligand TDIF signaling pathway is involved in the regulation of procambial cell proliferation and inhibiting its differentiation into xylem cells. Here we present the crystal structures of the extracellular domains (ECD) of TDR alone and in complex with its ligand TDIF resolved at 2.65 Ǻ and 2.75 Ǻ respectively. These structures provide insights about the ligand perception and specific interactions between the CLE peptides and their cognate receptors. Our in vitro biochemical studies indicate that the interactions between the ligands and the receptors at the C-terminal anchoring site provide conserved binding. While the binding interactions occurring at the N-terminal anchoring site dictate differential binding specificities between different ligands and receptors. Our studies will open different unknown avenues of TDR-TDIF signaling pathways that will enhance our knowledge in this field highlighting the receptor ligand interaction, receptor activation, signaling network, modes of action and will serve as a structure function relationship model between the ligand and the receptor for various similar leucine-rich repeat receptor-like kinases (LRR-RLKs).

No MeSH data available.