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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.


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ITC measurements between wtTDR and TDIF mutants, CLE46, CLE9, and CLE9-R1H peptides.Representative ITC measurements between wild-type ecdTDR protein and TDIF-H1A/E2A/V3A, TDIF-N8A/I10A/N12A, TDIF-H1R, CLE9, and CLE9-R1H peptides are shown on the top panel; significant differences (greater than 2.5 fold) in the binding energy (dissociation constant Kd) between the wild type TDIF peptide and the mutants are summarized in the table below. In parallel, the calculated fold changes in Kd between wild type TDIF peptide and TDIF mutants along with CLE9 peptides are shown in a separate column. In comparison with wild type CLE9 and and CLE9-R1H mutant an additional fold change was found and is shown is red with a *. The sequence of each peptide is shown in the table, with the substituted resides colored in red.
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pone.0175317.g003: ITC measurements between wtTDR and TDIF mutants, CLE46, CLE9, and CLE9-R1H peptides.Representative ITC measurements between wild-type ecdTDR protein and TDIF-H1A/E2A/V3A, TDIF-N8A/I10A/N12A, TDIF-H1R, CLE9, and CLE9-R1H peptides are shown on the top panel; significant differences (greater than 2.5 fold) in the binding energy (dissociation constant Kd) between the wild type TDIF peptide and the mutants are summarized in the table below. In parallel, the calculated fold changes in Kd between wild type TDIF peptide and TDIF mutants along with CLE9 peptides are shown in a separate column. In comparison with wild type CLE9 and and CLE9-R1H mutant an additional fold change was found and is shown is red with a *. The sequence of each peptide is shown in the table, with the substituted resides colored in red.

Mentions: The TDIF peptide docks on a shallow groove formed by the contacting residues of TDR (Figs 1C and 2A). The groove extends to the N-terminus of the peptide and is closed toward the C-terminus of TDIF. This structural arrangement is crucial for the peptide binding just fitting the groove of the interaction surface. Any extension in the C-terminus of the peptide might have an adverse effect on the peptide binding. In fact, TDIF-R or TDIF-H has been shown to have a weaker binding affinity than the wild type peptide [12]. Surface complementarity plays an important role in TDIF binding. Among the conserved interacting residues of TDR, F279 obstructs the peptide to make a turn after G6 of TDIF. G6 is conserved in most CLE peptides, and previous mutational studies with CLV3 have confirmed that G6 is critical for its interaction with TDR and in vivo function [27, 34, 35]. Our Isothermal Titration Calorimetric (ITC) measurement of a G6A TDIF mutant peptide has about 14 times weaker binding than the wild type peptide (Fig 3). The side chains of the three residues around G6, Hyp4, S5, and Hyp7 direct upwards and do not make significant contacts with TDR. Accordingly, alanine substitution mutations of these residues do not have a significant effect on TDR binding (Fig 3 and S10 Fig).


Differential CLE peptide perception by plant receptors implicated from structural and functional analyses of TDIF-TDR interactions
ITC measurements between wtTDR and TDIF mutants, CLE46, CLE9, and CLE9-R1H peptides.Representative ITC measurements between wild-type ecdTDR protein and TDIF-H1A/E2A/V3A, TDIF-N8A/I10A/N12A, TDIF-H1R, CLE9, and CLE9-R1H peptides are shown on the top panel; significant differences (greater than 2.5 fold) in the binding energy (dissociation constant Kd) between the wild type TDIF peptide and the mutants are summarized in the table below. In parallel, the calculated fold changes in Kd between wild type TDIF peptide and TDIF mutants along with CLE9 peptides are shown in a separate column. In comparison with wild type CLE9 and and CLE9-R1H mutant an additional fold change was found and is shown is red with a *. The sequence of each peptide is shown in the table, with the substituted resides colored in red.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5383425&req=5

pone.0175317.g003: ITC measurements between wtTDR and TDIF mutants, CLE46, CLE9, and CLE9-R1H peptides.Representative ITC measurements between wild-type ecdTDR protein and TDIF-H1A/E2A/V3A, TDIF-N8A/I10A/N12A, TDIF-H1R, CLE9, and CLE9-R1H peptides are shown on the top panel; significant differences (greater than 2.5 fold) in the binding energy (dissociation constant Kd) between the wild type TDIF peptide and the mutants are summarized in the table below. In parallel, the calculated fold changes in Kd between wild type TDIF peptide and TDIF mutants along with CLE9 peptides are shown in a separate column. In comparison with wild type CLE9 and and CLE9-R1H mutant an additional fold change was found and is shown is red with a *. The sequence of each peptide is shown in the table, with the substituted resides colored in red.
Mentions: The TDIF peptide docks on a shallow groove formed by the contacting residues of TDR (Figs 1C and 2A). The groove extends to the N-terminus of the peptide and is closed toward the C-terminus of TDIF. This structural arrangement is crucial for the peptide binding just fitting the groove of the interaction surface. Any extension in the C-terminus of the peptide might have an adverse effect on the peptide binding. In fact, TDIF-R or TDIF-H has been shown to have a weaker binding affinity than the wild type peptide [12]. Surface complementarity plays an important role in TDIF binding. Among the conserved interacting residues of TDR, F279 obstructs the peptide to make a turn after G6 of TDIF. G6 is conserved in most CLE peptides, and previous mutational studies with CLV3 have confirmed that G6 is critical for its interaction with TDR and in vivo function [27, 34, 35]. Our Isothermal Titration Calorimetric (ITC) measurement of a G6A TDIF mutant peptide has about 14 times weaker binding than the wild type peptide (Fig 3). The side chains of the three residues around G6, Hyp4, S5, and Hyp7 direct upwards and do not make significant contacts with TDR. Accordingly, alanine substitution mutations of these residues do not have a significant effect on TDR binding (Fig 3 and S10 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.


Related in: MedlinePlus