PMC 20140719 pmc.key 4848090 CC BY no 0 0 10.7554/eLife.15075 4848090 27058169 15075 e15075 membrane signaling receptor kinase peptide hormone floral abscission plant development protein complexes <i>A. thaliana</i> This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. surname:Santiago;given-names:Julia surname:Brandt;given-names:Benjamin surname:Hothorn;given-names:Michael surname:Butenko;given-names:Melinka A surname:Brandt;given-names:Benjamin surname:Santiago;given-names:Julia surname:Wildhagen;given-names:Mari surname:Hohmann;given-names:Ulrich surname:Hothorn;given-names:Ludwig A surname:Butenko;given-names:Melinka A surname:Hothorn;given-names:Michael surname:Zhang;given-names:Mingjie surname:Hothorn;given-names:Michael surname:Hothorn;given-names:Michael TITLE Author Keywords Research Organism front 5 2016 0 Mechanistic insight into a peptide hormone signaling complex mediating floral organ abscission protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T08:29:24Z peptide hormone ABSTRACT abstract 95 Plants constantly renew during their life cycle and thus require to shed senescent and damaged organs. Floral abscission is controlled by the leucine-rich repeat receptor kinase (LRR-RK) HAESA and the peptide hormone IDA. It is unknown how expression of IDA in the abscission zone leads to HAESA activation. Here we show that IDA is sensed directly by the HAESA ectodomain. Crystal structures of HAESA in complex with IDA reveal a hormone binding pocket that accommodates an active dodecamer peptide. A central hydroxyproline residue anchors IDA to the receptor. The HAESA co-receptor SERK1, a positive regulator of the floral abscission pathway, allows for high-affinity sensing of the peptide hormone by binding to an Arg-His-Asn motif in IDA. This sequence pattern is conserved among diverse plant peptides, suggesting that plant peptide hormone receptors may share a common ligand binding mode and activation mechanism. taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:33:49Z Plants protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T08:34:57Z leucine-rich repeat receptor kinase protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T08:35:06Z LRR-RK protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:05Z HAESA protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T08:29:24Z peptide hormone protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:42Z IDA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:42Z IDA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:05Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:42Z IDA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:05Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:34:33Z ectodomain evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:25Z Crystal structures protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:05Z HAESA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:35:29Z in complex with protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:42Z IDA site SO: melaniev@ebi.ac.uk 2023-03-17T09:30:04Z hormone binding pocket protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:12Z active structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:34:45Z dodecamer chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T16:28:58Z peptide residue_name SO: melaniev@ebi.ac.uk 2023-03-17T08:36:09Z hydroxyproline protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:42Z IDA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:05Z HAESA protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T08:36:27Z co-receptor protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:29Z SERK1 protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T08:29:24Z peptide hormone structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:01:51Z Arg-His-Asn motif protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:42Z IDA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:36:42Z conserved taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:01Z plant chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T16:29:02Z peptides taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:01Z plant protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T08:36:36Z peptide hormone receptors ABSTRACT abstract 1019 DOI: http://dx.doi.org/10.7554/eLife.15075.001 ABSTRACT abstract_title_1 1066 eLife digest ABSTRACT abstract 1079 Plants can shed their leaves, flowers or other organs when they no longer need them. But how does a leaf or a flower know when to let go? A receptor protein called HAESA is found on the surface of the cells that surround a future break point on the plant. When its time to shed an organ, a hormone called IDA instructs HAESA to trigger the shedding process. However, the molecular details of how IDA triggers organ shedding are not clear. taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:33:49Z Plants protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T08:38:22Z receptor protein protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:05Z HAESA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T08:38:42Z hormone protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:42Z IDA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:05Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:42Z IDA ABSTRACT abstract 1518 The shedding of floral organs (or leaves) can be easily studied in a model plant called Arabidopsis. Santiago et al. used protein biochemistry, structural biology and genetics to uncover how the IDA hormone activates HAESA. The experiments show that IDA binds directly to a canyon shaped pocket in HAESA that extends out from the surface of the cell. IDA binding to HAESA allows another receptor protein called SERK1 to bind to HAESA, which results in the release of signals inside the cell that trigger the shedding of organs. taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:01Z plant taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:40:57Z Arabidopsis experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T08:41:04Z protein biochemistry experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T08:41:10Z structural biology experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T08:41:13Z genetics protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:42Z IDA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T08:41:17Z hormone protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:05Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:42Z IDA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:42:02Z binds directly to protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:41:22Z canyon shaped site SO: melaniev@ebi.ac.uk 2023-03-17T08:41:25Z pocket protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:05Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-22T10:16:55Z IDA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:05Z HAESA protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T08:41:40Z receptor protein protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:29Z SERK1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:42:20Z to bind to protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:05Z HAESA ABSTRACT abstract 2046 The next step following on from this work is to understand what signals are produced when IDA activates HAESA. Another challenge will be to find out where IDA is produced in the plant and what causes it to accumulate in specific places in preparation for organ shedding. protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:42Z IDA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:05Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:42Z IDA taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:01Z plant ABSTRACT abstract 2317 DOI: http://dx.doi.org/10.7554/eLife.15075.002 INTRO title_1 2364 Introduction elife-15075-fig1-figsupp1.jpg fig1s1 FIG fig_title_caption 2377 The HAESA ectodomain folds into a superhelical assembly of 21 leucine-rich repeats. protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:05Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:34:33Z ectodomain structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:43:20Z superhelical assembly structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:44:11Z leucine-rich repeats elife-15075-fig1-figsupp1.jpg fig1s1 FIG fig_caption 2461 (A) SDS PAGE analysis of the purified Arabidopsis thaliana HAESA ectodomain (residues 20–620) obtained by secreted expression in insect cells. The calculated molecular mass is 65.7 kDa, the actual molecular mass obtained by mass spectrometry is 74,896 Da, accounting for the N-glycans. (B) Ribbon diagrams showing front (left panel) and side views (right panel) of the isolated HAESA LRR domain. The N- (residues 20–88) and C-terminal (residues 593–615) capping domains are shown in yellow, the central 21 LRR motifs are in blue and disulphide bonds are highlighted in green (in bonds representation). (C) Structure based sequence alignment of the 21 leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison. Conserved hydrophobic residues are shaded in gray, N-glycosylation sites visible in our structures are highlighted in blue, cysteine residues involved in disulphide bridge formation in green. (D) Asn-linked glycans mask the N-terminal portion of the HAESA ectodomain. Oligomannose core structures (containing two N-actylglucosamines and three terminal mannose units) as found in Trichoplusia ni cells and in plants were modeled onto the seven glycosylation sites observed in our HAESA structures, to visualize the surface areas potentially not masked by carbohydrate. The HAESA ectodomain is shown in blue (in surface representation), the glycan structures are shown in yellow. Molecular surfaces were calculated with the program MSMS, with a probe radius of 1.5 Å. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T09:45:35Z SDS PAGE species MESH: melaniev@ebi.ac.uk 2023-03-17T08:47:13Z Arabidopsis thaliana protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:06Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:34:33Z ectodomain residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:47:22Z 20–620 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T08:47:18Z secreted expression in insect cells experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T08:47:25Z mass spectrometry chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T08:47:39Z N-glycans protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:06Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:47:45Z LRR domain residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:47:57Z 20–88 residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:48:04Z 593–615 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:48:11Z capping domains structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:48:52Z LRR motifs ptm MESH: melaniev@ebi.ac.uk 2023-03-17T08:49:02Z disulphide bonds experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T08:49:09Z Structure based sequence alignment structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:44:13Z leucine-rich repeats protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:06Z HAESA taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:01Z plant structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:49:42Z LRR protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:49:46Z Conserved protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:49:56Z hydrophobic structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:50:01Z residues site SO: melaniev@ebi.ac.uk 2023-03-17T08:50:06Z N-glycosylation sites evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:50:12Z structures residue_name SO: melaniev@ebi.ac.uk 2023-03-17T08:50:25Z cysteine ptm MESH: melaniev@ebi.ac.uk 2023-03-17T08:50:33Z disulphide bridge ptm MESH: melaniev@ebi.ac.uk 2023-03-17T08:50:57Z Asn-linked glycans protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:06Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:34:33Z ectodomain chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T08:51:14Z Oligomannose chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T08:51:20Z N-actylglucosamines chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T08:51:29Z mannose species MESH: melaniev@ebi.ac.uk 2023-03-17T08:51:37Z Trichoplusia ni taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:33:49Z plants site SO: melaniev@ebi.ac.uk 2023-03-17T08:52:19Z glycosylation sites protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:06Z HAESA evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:51:47Z structures chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T08:51:55Z carbohydrate protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:06Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:34:33Z ectodomain chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T08:52:03Z glycan elife-15075-fig1-figsupp1.jpg fig1s1 FIG fig_caption 3975 DOI: http://dx.doi.org/10.7554/eLife.15075.004 elife-15075-fig1-figsupp2.jpg fig1s2 FIG fig_title_caption 4022 Hydrophobic contacts and a hydrogen-bond network mediate the interaction between HAESA and the peptide hormone IDA. site SO: melaniev@ebi.ac.uk 2023-06-15T08:44:48Z hydrogen-bond network protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:06Z HAESA protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T08:29:24Z peptide hormone protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:42Z IDA elife-15075-fig1-figsupp2.jpg fig1s2 FIG fig_caption 4138 (A) Details of the IDA binding pocket. HAESA is shown in blue (ribbon diagram), the C-terminal Arg-His-Asn motif (left panel), the central Hyp anchor (center) and the N-terminal Pro-rich motif in IDA (right panel) are shown in yellow (in bonds representation). HAESA interface residues are shown as sticks, selected hydrogen bond interactions are denoted as dotted lines (in magenta). (B) View of the complete IDA (in bonds representation, in yellow) binding pocket in HAESA (surface view, in blue). Orientation as in (A). (C) Structure based sequence alignment of leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison. Residues mediating hydrophobic interactions with the IDA peptide are highlighted in blue, residues contributing to hydrogen bond interactions and/or salt bridges are shown in red. The IDA binding pocket covers LRRs 2–14 and all residues originate from the inner surface of the HAESA superhelix. site SO: melaniev@ebi.ac.uk 2023-03-17T09:01:38Z IDA binding pocket protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:06Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:01:51Z Arg-His-Asn motif structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:02:42Z Hyp anchor structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:02:59Z Pro-rich motif protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:42Z IDA site SO: melaniev@ebi.ac.uk 2023-03-17T09:03:09Z HAESA interface residues protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:42Z IDA site SO: melaniev@ebi.ac.uk 2023-03-17T09:03:27Z binding pocket protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:06Z HAESA experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T09:03:30Z Structure based sequence alignment structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:44:13Z leucine-rich repeats protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:06Z HAESA taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:01Z plant structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:03:37Z LRR evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:03:40Z consensus sequence chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T16:08:50Z IDA peptide site SO: melaniev@ebi.ac.uk 2023-03-17T09:01:38Z IDA binding pocket structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:04:34Z LRRs 2–14 protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:06Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:04:41Z superhelix elife-15075-fig1-figsupp2.jpg fig1s2 FIG fig_caption 5090 DOI: http://dx.doi.org/10.7554/eLife.15075.005 elife-15075-fig1-figsupp3.jpg fig1s3 FIG fig_title_caption 5137 The IDA-HAESA and SERK1-HAESA complex interfaces are conserved among HAESA and HAESA-like proteins from different plant species. complex_assembly GO: melaniev@ebi.ac.uk 2023-03-17T09:05:48Z IDA-HAESA complex_assembly GO: melaniev@ebi.ac.uk 2023-03-17T09:05:55Z SERK1-HAESA site SO: melaniev@ebi.ac.uk 2023-03-17T09:06:00Z interfaces protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:06:03Z conserved protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:06Z HAESA protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T09:06:10Z HAESA-like proteins taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:01Z plant elife-15075-fig1-figsupp3.jpg fig1s3 FIG fig_caption 5267 Structure-based sequence alignment of the HAESA family members: Arabidopsis thaliana HAESA (Uniprot (http://www.uniprot.org) ID P47735), Arabidopsis thaliana HSL2 (Uniprot ID C0LGX3), Capsella rubella HAESA (Uniprot ID R0F2U6), Citrus clementina HSL2 (Uniprot ID V4U227), Vitis vinifera HAESA (Uniprot ID F6HM39). The alignment includes a secondary structure assignment calculated with the program DSSP and colored according to Figure 1, with the N- and C-terminal caps and the 21 LRR motifs indicated in orange and blue, respectively. Cysteine residues engaged in disulphide bonds are depicted in green. HAESA residues interacting with the IDA peptide and/or the SERK1 co-receptor kinase ectodomain are highlighted in blue and orange, respectively. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T09:07:34Z Structure-based sequence alignment protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T09:07:37Z HAESA family members species MESH: melaniev@ebi.ac.uk 2023-03-17T08:47:13Z Arabidopsis thaliana protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:07Z HAESA species MESH: melaniev@ebi.ac.uk 2023-03-17T08:47:13Z Arabidopsis thaliana protein PR: melaniev@ebi.ac.uk 2023-03-17T09:07:45Z HSL2 species MESH: melaniev@ebi.ac.uk 2023-03-17T09:07:54Z Capsella rubella protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:07Z HAESA species MESH: melaniev@ebi.ac.uk 2023-03-17T09:08:02Z Citrus clementina protein PR: melaniev@ebi.ac.uk 2023-03-17T09:07:45Z HSL2 species MESH: melaniev@ebi.ac.uk 2023-03-17T09:08:11Z Vitis vinifera protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:07Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:08:27Z caps structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:09:03Z LRR motifs residue_name SO: melaniev@ebi.ac.uk 2023-03-17T08:50:25Z Cysteine ptm MESH: melaniev@ebi.ac.uk 2023-03-17T08:49:02Z disulphide bonds protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:07Z HAESA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T16:08:59Z IDA peptide protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:29Z SERK1 protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T09:09:39Z co-receptor kinase structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:34:33Z ectodomain elife-15075-fig1-figsupp3.jpg fig1s3 FIG fig_caption 6017 DOI: http://dx.doi.org/10.7554/eLife.15075.006 elife-15075-fig1.jpg fig1 FIG fig_title_caption 6064 The peptide hormone IDA binds to the HAESA LRR ectodomain. protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T08:29:24Z peptide hormone protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:43Z IDA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:07Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:10:10Z LRR ectodomain elife-15075-fig1.jpg fig1 FIG fig_caption 6123 (A) Multiple sequence alignment of selected IDA family members. The conserved PIP motif is highlighted in yellow, the central Hyp in blue. The PKGV motif present in our N-terminally extended IDA peptide is highlighted in red. (B) Isothermal titration calorimetry of the HAESA ectodomain vs. IDA and including the synthetic peptide sequence. (C) Structure of the HAESA – IDA complex with HAESA shown in blue (ribbon diagram). IDA (in bonds representation, surface view included) is depicted in yellow. The peptide binding pocket covers HAESA LRRs 2–14. (D) Close-up view of the entire IDA (in yellow) peptide binding site in HAESA (in blue). Details of the interactions between the central Hyp anchor in IDA and the C-terminal Arg-His-Asn motif with HAESA are highlighted in (E) and (F), respectively. Hydrogren bonds are depicted as dotted lines (in magenta), a water molecule is shown as a red sphere. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T09:12:20Z Multiple sequence alignment protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T10:19:03Z IDA family members protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:12:51Z conserved structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:12:59Z PIP motif residue_name SO: melaniev@ebi.ac.uk 2023-03-17T09:13:21Z Hyp structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:13:30Z PKGV motif protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:28:24Z N-terminally extended chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T16:09:00Z IDA peptide experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T09:14:02Z Isothermal titration calorimetry protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:07Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:34:33Z ectodomain protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:43Z IDA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:14:06Z synthetic chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T09:14:10Z peptide complex_assembly GO: melaniev@ebi.ac.uk 2023-03-17T16:29:32Z HAESA – IDA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:07Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:43Z IDA site SO: melaniev@ebi.ac.uk 2023-03-17T09:14:20Z peptide binding pocket protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:07Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:04:34Z LRRs 2–14 protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:43Z IDA site SO: melaniev@ebi.ac.uk 2023-03-17T09:14:28Z peptide binding site protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:07Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:02:42Z Hyp anchor protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:43Z IDA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:01:51Z Arg-His-Asn motif protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:07Z HAESA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T09:15:02Z water elife-15075-fig1.jpg fig1 FIG fig_caption 7031 DOI: http://dx.doi.org/10.7554/eLife.15075.003 INTRO paragraph 7078 During their growth, development and reproduction plants use cell separation processes to detach no-longer required, damaged or senescent organs. Abscission of floral organs in Arabidopsis is a model system to study these cell separation processes in molecular detail. The LRR-RKs HAESA (greek: to adhere to) and HAESA-LIKE 2 (HSL2) redundantly control floral abscission. Loss-of-function of the secreted small protein INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) causes floral organs to remain attached while its over-expression leads to premature shedding. Full-length IDA is proteolytically processed and a conserved stretch of 20 amino-acids (termed EPIP) can rescue the IDA loss-of-function phenotype (Figure 1A). It has been demonstrated that a dodecamer peptide within EPIP is able to activate HAESA and HSL2 in transient assays in tobacco cells. This sequence motif is highly conserved among IDA family members (IDA-LIKE PROTEINS, IDLs) and contains a central Pro residue, presumed to be post-translationally modified to hydroxyproline (Hyp; Figure 1A). The available genetic and biochemical evidence suggests that IDA and HAESA together control floral abscission, but it is poorly understood if IDA is directly sensed by the receptor kinase HAESA and how IDA binding at the cell surface would activate the receptor. taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:33:49Z plants taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:19:27Z Arabidopsis structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:19:47Z LRR-RKs protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:07Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T09:19:35Z HAESA-LIKE 2 protein PR: melaniev@ebi.ac.uk 2023-03-17T09:07:45Z HSL2 protein PR: melaniev@ebi.ac.uk 2023-03-17T09:20:13Z INFLORESCENCE DEFICIENT IN ABSCISSION protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:43Z IDA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:19:56Z Full-length protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:43Z IDA ptm MESH: melaniev@ebi.ac.uk 2023-03-17T09:20:35Z proteolytically processed protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:20:38Z conserved residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:20:41Z stretch of 20 amino-acids structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:21:35Z EPIP protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:43Z IDA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:34:45Z dodecamer chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T09:21:18Z peptide structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:21:35Z EPIP protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:07Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T09:07:45Z HSL2 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T09:21:42Z transient assays taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:21:57Z tobacco structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:22:02Z This sequence motif protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:22:05Z highly conserved protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T10:19:03Z IDA family members protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T09:22:49Z IDA-LIKE PROTEINS protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T09:23:05Z IDLs residue_name SO: melaniev@ebi.ac.uk 2023-03-17T09:23:11Z Pro protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:23:35Z post-translationally modified residue_name SO: melaniev@ebi.ac.uk 2023-03-17T09:23:58Z hydroxyproline residue_name SO: melaniev@ebi.ac.uk 2023-03-17T09:24:15Z Hyp protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:43Z IDA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:07Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:43Z IDA protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T09:24:20Z receptor kinase protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:07Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-22T10:17:44Z IDA RESULTS title_1 8403 Results RESULTS title_2 8411 IDA directly binds to the LRR domain of HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:43Z IDA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:24:55Z LRR domain protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:07Z HAESA elife-15075-fig2.jpg fig2 FIG fig_title_caption 8457 Active IDA-family peptide hormones are hydroxyprolinated dodecamers. protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:12Z Active protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T09:25:46Z IDA-family peptide hormones protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:26:07Z hydroxyprolinated structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:26:18Z dodecamers elife-15075-fig2.jpg fig2 FIG fig_caption 8526 Close-up views of (A) IDA, (B) the N-terminally extended PKGV-IDA and (C) IDL1 bound to the HAESA hormone binding pocket (in bonds representation, in yellow) and including simulated annealing 2Fo–Fc omit electron density maps contoured at 1.0 σ. Note that Pro58IDA and Leu67IDA are the first residues defined by electron density when bound to the HAESA ectodomain. (D) Table summaries for equilibrium dissociation constants (Kd), binding enthalpies (ΔH), binding entropies (ΔS) and stoichoimetries (N) for different IDA peptides binding to the HAESA ectodomain ( ± fitting errors; n.d. no detectable binding). (E) Structural superposition of the active IDA (in bonds representation, in gray) and IDL1 peptide (in yellow) hormones bound to the HAESA ectodomain. Root mean square deviation (r.m.s.d.) is 1.0 Å comparing 100 corresponding atoms. protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:43Z IDA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:28:29Z N-terminally extended mutant MESH: melaniev@ebi.ac.uk 2023-03-17T09:30:14Z PKGV-IDA protein PR: melaniev@ebi.ac.uk 2023-03-17T09:29:55Z IDL1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:30:41Z bound to protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:07Z HAESA site SO: melaniev@ebi.ac.uk 2023-03-17T09:30:04Z hormone binding pocket experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T09:30:18Z simulated annealing evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:30:26Z 2Fo–Fc omit electron density maps residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:31:16Z Pro58 protein PR: melaniev@ebi.ac.uk 2023-03-17T09:30:56Z IDA residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:31:22Z Leu67 protein PR: melaniev@ebi.ac.uk 2023-03-17T09:31:07Z IDA evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:30:31Z electron density protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:30:41Z bound to protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:07Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:34:33Z ectodomain evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:31:30Z equilibrium dissociation constants evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:31:37Z Kd evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:31:42Z binding enthalpies evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:31:48Z ΔH evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:31:52Z binding entropies evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:31:59Z ΔS chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T16:10:22Z IDA peptides protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:08Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:34:33Z ectodomain experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T09:32:08Z Structural superposition protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:12Z active protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:43Z IDA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T16:10:54Z IDL1 peptide protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:30:41Z bound to protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:08Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:34:33Z ectodomain evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:33:22Z Root mean square deviation evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:33:38Z r.m.s.d. elife-15075-fig2.jpg fig2 FIG fig_caption 9382 DOI: http://dx.doi.org/10.7554/eLife.15075.007 elife-15075-fig3.jpg fig3 FIG fig_title_caption 9429 The receptor kinase SERK1 acts as a HAESA co-receptor and promotes high-affinity IDA sensing. protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T09:35:33Z receptor kinase protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:29Z SERK1 protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T09:36:35Z HAESA co-receptor protein PR: melaniev@ebi.ac.uk 2023-03-22T10:18:04Z IDA elife-15075-fig3.jpg fig3 FIG fig_caption 9523 (A) Petal break-strength assays measure the force (expressed in gram equivalents) required to remove the petals from the flower of serk mutant plants compared to haesa/hsl2 mutant and Col-0 wild-type flowers. Petal break-strength is measured from positions 1 to 8 along the primary inflorescence where positions 1 is defined as the flower at anthesis (n=15, bars=SD). This treatment-by-position balanced two-way layout was analyzed separately per position, because of the serious interaction, by means of a Dunnett-type comparison against the Col-0 control, allowing for heterogeneous variances. Petal break-strength was found significantly increased in almost all positions (indicated with a *) for haesa/hsl2 and serk1-1 mutant plants with respect to the Col-0 control. Calculations were performed in R (version 3.2.3). (B) Analytical size-exclusion chromatography. The HAESA LRR domain elutes as a monomer (black dotted line), as does the isolated SERK1 ectodomain (blue dotted line). A HAESA – IDA – SERK1 complex elutes as an apparent heterodimer (red line), while a mixture of HAESA and SERK1 yields two isolated peaks that correspond to monomeric HAESA and SERK1, respectively (black line). Void (V0) volume and total volume (Vt) are shown, together with elution volumes for molecular mass standards (A, Thyroglobulin, 669,000 Da; B, Ferritin, 440,00 Da, C, Aldolase, 158,000 Da; D, Conalbumin, 75,000 Da; E, Ovalbumin, 44,000 Da; F, Carbonic anhydrase, 29,000 Da). A SDS PAGE of the peak fractions is shown alongside. Purified HAESA and SERK1 are ~75 and ~28 kDa, respectively. (C) Isothermal titration calorimetry of wild-type and Hyp64→Pro IDA versus the HAESA and SERK1 ectodomains. The titration of IDA wild-type versus the isolated HAESA ectodomain from Figure 1B is shown for comparison (red line; n.d. no detectable binding) (D) Analytical size-exclusion chromatography in the presence of the IDA Hyp64→Pro mutant peptide reveals no complex formation between HAESA and SERK1 ectodomains. A SDS PAGE of the peak fractions is shown alongside. (E) In vitro kinase assays of the HAESA and SERK1 kinase domains. Wild-type HAESA and SERK1 kinase domains (KDs) exhibit auto-phosphorylation activities (lanes 1 + 3). Mutant (m) versions, which carry point mutations in their active sites (Asp837HAESA→Asn, Asp447SERK1→Asn) possess no autophosphorylation activity (lanes 2+4). Transphosphorylation activity from the active kinase to the mutated form can be observed in both directions (lanes 5+6). A coomassie-stained gel loading control is shown below. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T09:39:22Z Petal break-strength assays gene GENE: melaniev@ebi.ac.uk 2023-03-17T16:29:45Z serk protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:44:55Z mutant taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:33:49Z plants gene GENE: melaniev@ebi.ac.uk 2023-03-17T09:43:03Z haesa gene GENE: melaniev@ebi.ac.uk 2023-03-17T09:43:21Z hsl2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:44:44Z mutant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:44:50Z wild-type gene GENE: melaniev@ebi.ac.uk 2023-03-17T09:43:05Z haesa gene GENE: melaniev@ebi.ac.uk 2023-03-17T09:43:23Z hsl2 gene GENE: melaniev@ebi.ac.uk 2023-03-17T09:44:19Z serk1-1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:44:58Z mutant taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:33:49Z plants experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T09:39:31Z Analytical size-exclusion chromatography protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:08Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:44:40Z LRR domain oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:44:37Z monomer protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:29Z SERK1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:34:33Z ectodomain complex_assembly GO: melaniev@ebi.ac.uk 2023-03-17T09:45:13Z HAESA – IDA – SERK1 oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:45:27Z heterodimer protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:08Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:29Z SERK1 oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:45:22Z monomeric protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:08Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:29Z SERK1 protein PR: melaniev@ebi.ac.uk 2023-03-17T09:47:16Z Thyroglobulin protein PR: melaniev@ebi.ac.uk 2023-03-17T09:47:30Z Ferritin protein PR: melaniev@ebi.ac.uk 2023-03-17T09:47:46Z Aldolase protein PR: melaniev@ebi.ac.uk 2023-03-17T09:48:00Z Conalbumin protein PR: melaniev@ebi.ac.uk 2023-03-17T09:48:15Z Ovalbumin protein PR: melaniev@ebi.ac.uk 2023-03-17T09:48:30Z Carbonic anhydrase experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T09:45:35Z SDS PAGE protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:08Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:29Z SERK1 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T16:29:07Z Isothermal titration calorimetry protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:44:50Z wild-type mutant MESH: melaniev@ebi.ac.uk 2023-03-17T09:46:07Z Hyp64→Pro IDA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:08Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:29Z SERK1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:27:48Z ectodomains experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T09:46:53Z titration protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:44Z IDA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:44:50Z wild-type protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:08Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:34:33Z ectodomain experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T16:29:11Z Analytical size-exclusion chromatography protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:35:15Z presence of mutant MESH: melaniev@ebi.ac.uk 2023-03-17T09:46:15Z IDA Hyp64→Pro protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:46:39Z mutant chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T09:46:42Z peptide protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:08Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:29Z SERK1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:27:52Z ectodomains experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T09:45:35Z SDS PAGE experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T09:45:40Z In vitro kinase assays protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:08Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-22T10:18:32Z SERK1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:45:46Z kinase domains protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:44:50Z Wild-type protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:08Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:29Z SERK1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:27:56Z kinase domains structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:27:59Z KDs protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:28:35Z Mutant experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T09:45:42Z point mutations site SO: melaniev@ebi.ac.uk 2023-03-17T09:46:35Z active sites mutant MESH: melaniev@ebi.ac.uk 2023-03-17T09:46:22Z Asp837HAESA→Asn mutant MESH: melaniev@ebi.ac.uk 2023-03-17T09:46:29Z Asp447SERK1→Asn protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:12Z active protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:45:58Z mutated elife-15075-fig3.jpg fig3 FIG fig_caption 12096 DOI: http://dx.doi.org/10.7554/eLife.15075.008 tbl1.xml tbl1 TABLE table_caption 12143 Crystallographic data collection, phasing and refinement statistics for the isolated A. thaliana HAESA ectodomain. tbl1.xml tbl1 TABLE table_caption 12258 DOI: http://dx.doi.org/10.7554/eLife.15075.009 tbl1.xml tbl1 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="hsides" rules="groups"><thead><tr><th valign="top" rowspan="1" colspan="1"/><th valign="top" rowspan="1" colspan="1">HAESA NaI shortsoak</th><th valign="top" rowspan="1" colspan="1">HAESA apo</th></tr></thead><tbody><tr><td valign="top" rowspan="1" colspan="1"><bold>PDB-ID</bold></td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1">5IXO</td></tr><tr><td valign="top" rowspan="1" colspan="1"><bold>Data collection</bold></td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/></tr><tr><td valign="top" rowspan="1" colspan="1">Space group</td><td valign="top" rowspan="1" colspan="1"><italic>P</italic>3₁ 21</td><td valign="top" rowspan="1" colspan="1"><italic>P</italic>3₁ 21</td></tr><tr><td valign="top" rowspan="1" colspan="1">Cell dimensions</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/></tr><tr><td valign="top" rowspan="1" colspan="1"><italic>a</italic>, <italic>b, c</italic> (Å)</td><td valign="top" rowspan="1" colspan="1">148.55, 148.55, 58.30</td><td valign="top" rowspan="1" colspan="1">149.87, 149.87, 58.48</td></tr><tr><td valign="top" rowspan="1" colspan="1"><italic>α</italic>, β, γ (°)</td><td valign="top" rowspan="1" colspan="1">90, 90, 120</td><td valign="top" rowspan="1" colspan="1">90, 90, 120</td></tr><tr><td valign="top" rowspan="1" colspan="1">Resolution (Å)</td><td valign="top" rowspan="1" colspan="1">48.63–2.39 (2.45–2.39)</td><td valign="top" rowspan="1" colspan="1">45.75–1.74 (1.85–1.74)</td></tr><tr><td valign="top" rowspan="1" colspan="1">R_meas^#</td><td valign="top" rowspan="1" colspan="1">0.096 (0.866)</td><td valign="top" rowspan="1" colspan="1">0.038 (1.02)</td></tr><tr><td valign="top" rowspan="1" colspan="1">CC(1/2)<italic>^#</italic></td><td valign="top" rowspan="1" colspan="1">100/86.6</td><td valign="top" rowspan="1" colspan="1">100/75.6</td></tr><tr><td valign="top" rowspan="1" colspan="1"><italic>I/σ I^#</italic></td><td valign="top" rowspan="1" colspan="1">27.9 (4.9)</td><td valign="top" rowspan="1" colspan="1">18.7 (1.8)</td></tr><tr><td valign="top" rowspan="1" colspan="1">Completeness (%)<italic>^#</italic></td><td valign="top" rowspan="1" colspan="1">99.9 (98.6)</td><td valign="top" rowspan="1" colspan="1">99.6 (97.4)</td></tr><tr><td valign="top" rowspan="1" colspan="1">Redundancy<italic>^#</italic></td><td valign="top" rowspan="1" colspan="1">53.1 (29.9)</td><td valign="top" rowspan="1" colspan="1">14.4 (14.0)</td></tr><tr><td valign="top" rowspan="1" colspan="1">Wilson B-factor (Ų)<italic>^#</italic></td><td valign="top" rowspan="1" colspan="1">84.45</td><td valign="top" rowspan="1" colspan="1">81.10</td></tr><tr><td valign="top" rowspan="1" colspan="1"><bold>Refinement</bold></td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/></tr><tr><td valign="top" rowspan="1" colspan="1">Resolution (Å)</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1">45.75 – 1.74</td></tr><tr><td valign="top" rowspan="1" colspan="1">No. reflections</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1">71,213</td></tr><tr><td valign="top" rowspan="1" colspan="1"><italic>R</italic>_work/<italic>R</italic>_free^$</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1">0.188/0.218</td></tr><tr><td valign="top" rowspan="1" colspan="1"><bold>No. atoms</bold></td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/></tr><tr><td valign="top" rowspan="1" colspan="1">Protein/glycan</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1">4,533/126</td></tr><tr><td valign="top" rowspan="1" colspan="1">Water</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1">71</td></tr><tr><td valign="top" rowspan="1" colspan="1"><bold>Res. B-factors (Ų)</bold>^$</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/></tr><tr><td valign="top" rowspan="1" colspan="1">Protein</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1">77.54</td></tr><tr><td valign="top" rowspan="1" colspan="1">Glycan</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1">95.98</td></tr><tr><td valign="top" rowspan="1" colspan="1">Water</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1">73.20</td></tr><tr><td valign="top" rowspan="1" colspan="1"><bold>R.m.s deviations</bold>^$</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/></tr><tr><td valign="top" rowspan="1" colspan="1">Bond lengths (Å)</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1">0.0095</td></tr><tr><td valign="top" rowspan="1" colspan="1">Bond angles (°)</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1">1.51</td></tr></tbody></table> 12305 HAESA NaI shortsoak HAESA apo PDB-ID 5IXO Data collection Space group P31 21 P31 21 Cell dimensions a, b, c (Å) 148.55, 148.55, 58.30 149.87, 149.87, 58.48 α, β, γ (°) 90, 90, 120 90, 90, 120 Resolution (Å) 48.63–2.39 (2.45–2.39) 45.75–1.74 (1.85–1.74) Rmeas# 0.096 (0.866) 0.038 (1.02) CC(1/2)# 100/86.6 100/75.6 I/σ I# 27.9 (4.9) 18.7 (1.8) Completeness (%)# 99.9 (98.6) 99.6 (97.4) Redundancy# 53.1 (29.9) 14.4 (14.0) Wilson B-factor (Å2)# 84.45 81.10 Refinement Resolution (Å) 45.75 – 1.74 No. reflections 71,213 Rwork/Rfree$ 0.188/0.218 No. atoms Protein/glycan 4,533/126 Water 71 Res. B-factors (Å2)$ Protein 77.54 Glycan 95.98 Water 73.20 R.m.s deviations$ Bond lengths (Å) 0.0095 Bond angles (°) 1.51 tbl1.xml tbl1 TABLE table_footnote 13117 Highest resolution shell is shown in parenthesis. tbl1.xml tbl1 TABLE table_footnote 13167 #As defined in XDS. tbl1.xml tbl1 TABLE table_footnote 13187 $As defined in Refmac5. tbl2.xml tbl2 TABLE table_caption 13211 Crystallographic data collection and refinement statistics for the HAESA – IDA, – PKGV-IDA, – IDL1 and – IDA – SERK1 complexes. tbl2.xml tbl2 TABLE table_caption 13349 DOI: http://dx.doi.org/10.7554/eLife.15075.010 tbl2.xml tbl2 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="hsides" rules="groups"><thead><tr><th valign="top" rowspan="1" colspan="1"/><th valign="top" rowspan="1" colspan="1">HAESA – IDA</th><th valign="top" rowspan="1" colspan="1">HAESA – PKGV-IDA</th><th valign="top" rowspan="1" colspan="1">HAESA – IDL1</th><th valign="top" rowspan="1" colspan="1">HAESA – IDA – SERK1</th></tr></thead><tbody><tr><td valign="top" rowspan="1" colspan="1"><bold>PDB-ID</bold></td><td valign="top" rowspan="1" colspan="1">5IXQ</td><td valign="top" rowspan="1" colspan="1">5IXT</td><td valign="top" rowspan="1" colspan="1">5IYN</td><td valign="top" rowspan="1" colspan="1">5IYX</td></tr><tr><td valign="top" rowspan="1" colspan="1"><bold>Data collection</bold></td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/></tr><tr><td valign="top" rowspan="1" colspan="1">Space group</td><td valign="top" rowspan="1" colspan="1"><italic>P</italic>3₁ 21</td><td valign="top" rowspan="1" colspan="1"><italic>P</italic>3₁ 21</td><td valign="top" rowspan="1" colspan="1"><italic>P</italic>3₁ 21</td><td valign="top" rowspan="1" colspan="1"><italic>P</italic>2₁2₁2₁</td></tr><tr><td valign="top" rowspan="1" colspan="1">Cell dimensions</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/></tr><tr><td valign="top" rowspan="1" colspan="1"> <italic>a</italic>, <italic>b, c</italic> (Å)</td><td valign="top" rowspan="1" colspan="1">148.55, 148.55, 58.30</td><td valign="top" rowspan="1" colspan="1">148.92, 148.92, 58.02</td><td valign="top" rowspan="1" colspan="1">150.18, 150.18, 60.07</td><td valign="top" rowspan="1" colspan="1">74.51, 100.46, 142.76</td></tr><tr><td valign="top" rowspan="1" colspan="1"><italic>α</italic>, β, γ (°)</td><td valign="top" rowspan="1" colspan="1">90, 90, 120</td><td valign="top" rowspan="1" colspan="1">90, 90, 120</td><td valign="top" rowspan="1" colspan="1">90, 90, 120</td><td valign="top" rowspan="1" colspan="1">90, 90, 90</td></tr><tr><td valign="top" rowspan="1" colspan="1">Resolution (Å)</td><td valign="top" rowspan="1" colspan="1">48.54–1.86 <break/>(1.97–1.86)</td><td valign="top" rowspan="1" colspan="1">48.75–1.94 (2,06–1.94)</td><td valign="top" rowspan="1" colspan="1">49.16–2.56 (2.72–2.56)</td><td valign="top" rowspan="1" colspan="1">47.59–2.43 (2.57–2.43)</td></tr><tr><td valign="top" rowspan="1" colspan="1"><italic>R_meas^#</italic></td><td valign="top" rowspan="1" colspan="1">0.057 (1.35)</td><td valign="top" rowspan="1" colspan="1">0.037 (0.97)</td><td valign="top" rowspan="1" colspan="1">0.056 (1.27)</td><td valign="top" rowspan="1" colspan="1">0.113 (1.37)</td></tr><tr><td valign="top" rowspan="1" colspan="1">CC(1/2)<italic>^#</italic></td><td valign="top" rowspan="1" colspan="1">100/77.9</td><td valign="top" rowspan="1" colspan="1">100/80.3</td><td valign="top" rowspan="1" colspan="1">100/89.5</td><td valign="top" rowspan="1" colspan="1">100/77.6</td></tr><tr><td valign="top" rowspan="1" colspan="1"><italic>I</italic>/σ<italic>I^#</italic></td><td valign="top" rowspan="1" colspan="1">16.7 (2.0)</td><td valign="top" rowspan="1" colspan="1">20.9 (2.4)</td><td valign="top" rowspan="1" colspan="1">26.0 (1.9)</td><td valign="top" rowspan="1" colspan="1">16.12 (2.0)</td></tr><tr><td valign="top" rowspan="1" colspan="1">Completeness<italic>^#</italic> (%)</td><td valign="top" rowspan="1" colspan="1">99.8 (98.6)</td><td valign="top" rowspan="1" colspan="1">99.4 (97.9)</td><td valign="top" rowspan="1" colspan="1">99.5 (98.8)</td><td valign="top" rowspan="1" colspan="1">99.4 (96.4s</td></tr><tr><td valign="top" rowspan="1" colspan="1">Redundancy<italic>^#</italic></td><td valign="top" rowspan="1" colspan="1">20.3 (19.1)</td><td valign="top" rowspan="1" colspan="1">11.2 (11.1)</td><td valign="top" rowspan="1" colspan="1">14.7 (14.7)</td><td valign="top" rowspan="1" colspan="1">9.7 (9.3)</td></tr><tr><td valign="top" rowspan="1" colspan="1">Wilson B-factor (Ų)<italic>^#</italic></td><td valign="top" rowspan="1" colspan="1">80.0</td><td valign="top" rowspan="1" colspan="1">81.7</td><td valign="top" rowspan="1" colspan="1">89.5</td><td valign="top" rowspan="1" colspan="1">59.3</td></tr><tr><td valign="top" rowspan="1" colspan="1"><bold>Refinement</bold></td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/></tr><tr><td valign="top" rowspan="1" colspan="1">Resolution (Å)</td><td valign="top" rowspan="1" colspan="1">48.54–1.86</td><td valign="top" rowspan="1" colspan="1">48.75–1.94</td><td valign="top" rowspan="1" colspan="1">49.16–2.56</td><td valign="top" rowspan="1" colspan="1">47.59–2.43</td></tr><tr><td valign="top" rowspan="1" colspan="1">No. reflections</td><td valign="top" rowspan="1" colspan="1">58,551</td><td valign="top" rowspan="1" colspan="1">51,557</td><td valign="top" rowspan="1" colspan="1">23,835</td><td valign="top" rowspan="1" colspan="1">38,969</td></tr><tr><td valign="top" rowspan="1" colspan="1"><italic>R</italic>_work/<italic>R</italic>_free^$</td><td valign="top" rowspan="1" colspan="1">0.190/0.209</td><td valign="top" rowspan="1" colspan="1">0.183/0.208</td><td valign="top" rowspan="1" colspan="1">0.199/0.236</td><td valign="top" rowspan="1" colspan="1">0.199/0.235</td></tr><tr><td valign="top" rowspan="1" colspan="1"><bold>No. atoms</bold></td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/></tr><tr><td valign="top" rowspan="1" colspan="1">Protein/Glycan</td><td valign="top" rowspan="1" colspan="1">4,541/176</td><td valign="top" rowspan="1" colspan="1">4,545/176</td><td valign="top" rowspan="1" colspan="1">4,499/176</td><td valign="top" rowspan="1" colspan="1">5,965/168</td></tr><tr><td valign="top" rowspan="1" colspan="1">Peptide</td><td valign="top" rowspan="1" colspan="1">93</td><td valign="top" rowspan="1" colspan="1">93</td><td valign="top" rowspan="1" colspan="1">90</td><td valign="top" rowspan="1" colspan="1">112</td></tr><tr><td valign="top" rowspan="1" colspan="1">Water</td><td valign="top" rowspan="1" colspan="1">39</td><td valign="top" rowspan="1" colspan="1">40</td><td valign="top" rowspan="1" colspan="1">9</td><td valign="top" rowspan="1" colspan="1">136</td></tr><tr><td valign="top" rowspan="1" colspan="1"><bold>Res. B-factors (Ų)</bold>^$</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/></tr><tr><td valign="top" rowspan="1" colspan="1">Protein/Glycan</td><td valign="top" rowspan="1" colspan="1">79.48/109.02</td><td valign="top" rowspan="1" colspan="1">79.63/113.24</td><td valign="top" rowspan="1" colspan="1">102.12/132.49</td><td valign="top" rowspan="1" colspan="1">60.05/73.48</td></tr><tr><td valign="top" rowspan="1" colspan="1">Peptide</td><td valign="top" rowspan="1" colspan="1">87.19</td><td valign="top" rowspan="1" colspan="1">89.50</td><td valign="top" rowspan="1" colspan="1">125.74</td><td valign="top" rowspan="1" colspan="1">51.06</td></tr><tr><td valign="top" rowspan="1" colspan="1">Water</td><td valign="top" rowspan="1" colspan="1">75.32</td><td valign="top" rowspan="1" colspan="1">71.92</td><td valign="top" rowspan="1" colspan="1">74.65</td><td valign="top" rowspan="1" colspan="1">51.47</td></tr><tr><td valign="top" rowspan="1" colspan="1"><bold>R.m.s deviations</bold>^$</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/></tr><tr><td valign="top" rowspan="1" colspan="1">Bond lengths (Å)</td><td valign="top" rowspan="1" colspan="1">0.0087</td><td valign="top" rowspan="1" colspan="1">0.0091</td><td valign="top" rowspan="1" colspan="1">0.0081</td><td valign="top" rowspan="1" colspan="1">0.0074</td></tr><tr><td valign="top" rowspan="1" colspan="1">Bond angles (°)</td><td valign="top" rowspan="1" colspan="1">1.48</td><td valign="top" rowspan="1" colspan="1">1.47</td><td valign="top" rowspan="1" colspan="1">1.36</td><td valign="top" rowspan="1" colspan="1">1.34</td></tr></tbody></table> 13396 HAESA – IDA HAESA – PKGV-IDA HAESA – IDL1 HAESA – IDA – SERK1 PDB-ID 5IXQ 5IXT 5IYN 5IYX Data collection Space group P31 21 P31 21 P31 21 P212121 Cell dimensions a, b, c (Å) 148.55, 148.55, 58.30 148.92, 148.92, 58.02 150.18, 150.18, 60.07 74.51, 100.46, 142.76 α, β, γ (°) 90, 90, 120 90, 90, 120 90, 90, 120 90, 90, 90 Resolution (Å) 48.54–1.86 (1.97–1.86) 48.75–1.94 (2,06–1.94) 49.16–2.56 (2.72–2.56) 47.59–2.43 (2.57–2.43) Rmeas# 0.057 (1.35) 0.037 (0.97) 0.056 (1.27) 0.113 (1.37) CC(1/2)# 100/77.9 100/80.3 100/89.5 100/77.6 I/σI# 16.7 (2.0) 20.9 (2.4) 26.0 (1.9) 16.12 (2.0) Completeness# (%) 99.8 (98.6) 99.4 (97.9) 99.5 (98.8) 99.4 (96.4s Redundancy# 20.3 (19.1) 11.2 (11.1) 14.7 (14.7) 9.7 (9.3) Wilson B-factor (Å2)# 80.0 81.7 89.5 59.3 Refinement Resolution (Å) 48.54–1.86 48.75–1.94 49.16–2.56 47.59–2.43 No. reflections 58,551 51,557 23,835 38,969 Rwork/Rfree$ 0.190/0.209 0.183/0.208 0.199/0.236 0.199/0.235 No. atoms Protein/Glycan 4,541/176 4,545/176 4,499/176 5,965/168 Peptide 93 93 90 112 Water 39 40 9 136 Res. B-factors (Å2)$ Protein/Glycan 79.48/109.02 79.63/113.24 102.12/132.49 60.05/73.48 Peptide 87.19 89.50 125.74 51.06 Water 75.32 71.92 74.65 51.47 R.m.s deviations$ Bond lengths (Å) 0.0087 0.0091 0.0081 0.0074 Bond angles (°) 1.48 1.47 1.36 1.34 tbl2.xml tbl2 TABLE table_footnote 14804 Highest resolution shell is shown in parenthesis. tbl2.xml tbl2 TABLE table_footnote 14854 #As defined in XDS. tbl2.xml tbl2 TABLE table_footnote 14874 $As defined in Refmac5. RESULTS paragraph 14898 We purified the HAESA ectodomain (residues 20–620) from baculovirus-infected insect cells (Figure 1—figure supplement 1A, see Materials and methods) and quantified the interaction of the ~75 kDa glycoprotein with synthetic IDA peptides using isothermal titration calorimetry (ITC). A Hyp-modified dodecamer comprising the highly conserved PIP motif in IDA (Figure 1A) interacts with HAESA with 1:1 stoichiometry (N) and with a dissociation constant (Kd) of ~20 μM (Figure 1B). We next determined crystal structures of the apo HAESA ectodomain and of a HAESA-IDA complex, at 1.74 and 1.86 Å resolution, respectively (Figure 1C; Figure 1—figure supplement 1B–D; Tables 1,2). IDA binds in a completely extended conformation along the inner surface of the HAESA ectodomain, covering LRRs 2–14 (Figure 1C,D, Figure 1—figure supplement 2). The central Hyp64IDA is buried in a specific pocket formed by HAESA LRRs 8–10, with its hydroxyl group establishing hydrogen bonds with the strictly conserved Glu266HAESA and with a water molecule, which in turn is coordinated by the main chain oxygens of Phe289HAESA and Ser311HAESA (Figure 1E; Figure 1—figure supplement 3). The restricted size of the Hyp pocket suggests that IDA does not require arabinosylation of Hyp64IDA for activity in vivo, a modification that has been reported for Hyp residues in plant CLE peptide hormones. The C-terminal Arg-His-Asn motif in IDA maps to a cavity formed by HAESA LRRs 11–14 (Figure 1D,F). The COO- group of Asn69IDA is in direct contact with Arg407HAESA and Arg409HAESA and HAESA cannot bind a C-terminally extended IDA-SFVN peptide (Figures 1D,F, 2D). This suggests that the conserved Asn69IDA may constitute the very C-terminus of the mature IDA peptide in planta and that active IDA is generated by proteolytic processing from a longer pre-protein. Mutation of Arg417HSL2 (which corresponds to Arg409HAESA) causes a loss-of-function phenotype in HSL2, which indicates that the peptide binding pockets in different HAESA receptors have common structural and sequence features. Indeed, we find many of the residues contributing to the formation of the IDA binding surface in HAESA to be conserved in HSL2 and in other HAESA-type receptors in different plant species (Figure 1—figure supplement 3). A N-terminal Pro-rich motif in IDA makes contacts with LRRs 2–6 of the receptor (Figure 1D, Figure 1—figure supplement 2A–C). Other hydrophobic and polar interactions are mediated by Ser62IDA, Ser65IDA and by backbone atoms along the IDA peptide (Figure 1D, Figure 1—figure supplement 2A–C). experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T09:58:05Z purified protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:09Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:34:34Z ectodomain residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:58:08Z 20–620 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T09:58:11Z baculovirus-infected insect cells protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T09:58:14Z glycoprotein protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:58:36Z synthetic chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T16:11:53Z IDA peptides experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T09:58:41Z isothermal titration calorimetry experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T09:58:49Z ITC protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:59:09Z Hyp-modified structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:34:45Z dodecamer protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:59:16Z highly conserved structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:12:59Z PIP motif protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:44Z IDA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:09Z HAESA evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:59:53Z dissociation constant evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:31:37Z Kd evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:25Z crystal structures protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:00:06Z apo protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:09Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:34:34Z ectodomain complex_assembly GO: melaniev@ebi.ac.uk 2023-03-17T10:00:16Z HAESA-IDA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:44Z IDA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:00:36Z completely extended conformation protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:09Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:34:34Z ectodomain structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:04:34Z LRRs 2–14 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:01:07Z Hyp64 protein PR: melaniev@ebi.ac.uk 2023-03-17T10:01:33Z IDA site SO: melaniev@ebi.ac.uk 2023-03-17T10:01:41Z pocket protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:09Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T10:01:50Z LRRs 8–10 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:02:03Z strictly conserved residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:02:08Z Glu266 protein PR: melaniev@ebi.ac.uk 2023-03-17T10:02:15Z HAESA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T09:15:02Z water residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:02:29Z Phe289 protein PR: melaniev@ebi.ac.uk 2023-03-17T10:02:37Z HAESA residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:02:42Z Ser311 protein PR: melaniev@ebi.ac.uk 2023-03-17T10:02:56Z HAESA site SO: melaniev@ebi.ac.uk 2023-03-17T10:03:01Z Hyp pocket protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:44Z IDA ptm MESH: melaniev@ebi.ac.uk 2023-03-17T10:03:05Z arabinosylation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:03:11Z Hyp64 protein PR: melaniev@ebi.ac.uk 2023-03-17T10:03:28Z IDA residue_name SO: melaniev@ebi.ac.uk 2023-03-17T10:03:54Z Hyp taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:01Z plant protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T10:04:09Z CLE peptide hormones structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:01:51Z Arg-His-Asn motif protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:44Z IDA site SO: melaniev@ebi.ac.uk 2023-03-17T10:06:21Z cavity protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:09Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T10:06:28Z LRRs 11–14 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:06:35Z Asn69 protein PR: melaniev@ebi.ac.uk 2023-03-17T10:06:46Z IDA residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:06:54Z Arg407 protein PR: melaniev@ebi.ac.uk 2023-03-17T10:06:56Z HAESA residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:06:59Z Arg409 protein PR: melaniev@ebi.ac.uk 2023-03-17T10:07:02Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:09Z HAESA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:07:10Z C-terminally extended mutant MESH: melaniev@ebi.ac.uk 2023-03-17T10:07:16Z IDA-SFVN protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:07:20Z conserved residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:07:23Z Asn69 protein PR: melaniev@ebi.ac.uk 2023-03-17T10:07:32Z IDA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:07:37Z mature chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T16:09:00Z IDA peptide taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:08:00Z planta protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:12Z active protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:44Z IDA experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T10:08:09Z Mutation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:08:11Z Arg417 protein PR: melaniev@ebi.ac.uk 2023-03-17T10:08:14Z HSL2 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:08:17Z Arg409 protein PR: melaniev@ebi.ac.uk 2023-03-17T10:08:19Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T09:07:45Z HSL2 site SO: melaniev@ebi.ac.uk 2023-03-17T10:08:23Z peptide binding pockets protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T10:08:25Z HAESA receptors site SO: melaniev@ebi.ac.uk 2023-03-17T10:08:29Z IDA binding surface protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:09Z HAESA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:08:32Z conserved protein PR: melaniev@ebi.ac.uk 2023-03-17T09:07:45Z HSL2 protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T10:08:36Z HAESA-type receptors taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:01Z plant structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:02:59Z Pro-rich motif protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:44Z IDA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T10:08:48Z LRRs 2–6 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:09:02Z Ser62 protein PR: melaniev@ebi.ac.uk 2023-03-17T10:09:13Z IDA residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:09:16Z Ser65 protein PR: melaniev@ebi.ac.uk 2023-03-17T10:09:25Z IDA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T16:09:00Z IDA peptide RESULTS title_2 17511 HAESA specifically senses IDA-family dodecamer peptides protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:09Z HAESA protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:12:54Z IDA-family structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:06:17Z dodecamer chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T16:13:06Z peptides RESULTS paragraph 17567 We next investigated whether HAESA binds N-terminally extended versions of IDA. We obtained a structure of HAESA in complex with a PKGV-IDA peptide at 1.94 Å resolution (Table 2). In this structure, no additional electron density accounts for the PKGV motif at the IDA N-terminus (Figure 2A,B). Consistently, PKGV-IDA and IDA have similar binding affinities in our ITC assays, further indicating that HAESA senses a dodecamer peptide comprising residues 58-69IDA (Figure 2D). protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:09Z HAESA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:12:37Z N-terminally extended protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:44Z IDA evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:13:01Z structure protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:09Z HAESA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:35:29Z in complex with mutant MESH: melaniev@ebi.ac.uk 2023-03-17T09:30:14Z PKGV-IDA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T10:13:12Z peptide evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:13:16Z structure evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:13:19Z electron density structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:13:30Z PKGV motif protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:44Z IDA mutant MESH: melaniev@ebi.ac.uk 2023-03-17T09:30:14Z PKGV-IDA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:44Z IDA evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:13:35Z binding affinities experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T10:13:38Z ITC assays protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:10Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:34:45Z dodecamer chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T10:14:02Z peptide residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:14:06Z 58-69 protein PR: melaniev@ebi.ac.uk 2023-03-17T10:14:20Z IDA RESULTS paragraph 18046 We next tested if HAESA binds other IDA peptide family members. IDL1, which can rescue IDA loss-of-function mutants when introduced in abscission zone cells, can also be sensed by HAESA, albeit with lower affinity (Figure 2D). A 2.56 Å co-crystal structure with IDL1 reveals that different IDA family members use a common binding mode to interact with HAESA-type receptors (Figure 2A–C,E, Table 2). We do not detect interaction between HAESA and a synthetic peptide missing the C-terminal Asn69IDA (ΔN69), highlighting the importance of the polar interactions between the IDA carboxy-terminus and Arg407HAESA/Arg409HAESA (Figures 1F, 2D). Replacing Hyp64IDA, which is common to all IDLs, with proline impairs the interaction with the receptor, as does the Lys66IDA/Arg67IDA → Ala double-mutant discussed below (Figure 1A, 2D). Notably, HAESA can discriminate between IDLs and functionally unrelated dodecamer peptides with Hyp modifications, such as CLV3 (Figures 2D, 7). protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:10Z HAESA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T16:14:03Z IDA peptide family members protein PR: melaniev@ebi.ac.uk 2023-03-17T09:29:55Z IDL1 protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:44Z IDA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:10Z HAESA evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:18:36Z affinity evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:18:39Z co-crystal structure protein PR: melaniev@ebi.ac.uk 2023-03-17T09:29:55Z IDL1 protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T10:19:03Z IDA family members protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T10:19:14Z HAESA-type receptors protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:10Z HAESA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:19:21Z synthetic chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T10:19:25Z peptide protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:19:28Z missing the C-terminal residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:19:34Z Asn69 protein PR: melaniev@ebi.ac.uk 2023-03-17T10:19:46Z IDA mutant MESH: melaniev@ebi.ac.uk 2023-03-17T10:19:53Z ΔN69 protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:45Z IDA residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:20:03Z Arg407 protein PR: melaniev@ebi.ac.uk 2023-03-17T10:20:06Z HAESA residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:20:09Z Arg409 protein PR: melaniev@ebi.ac.uk 2023-03-17T10:20:12Z HAESA experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T10:20:15Z Replacing residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:20:19Z Hyp64 protein PR: melaniev@ebi.ac.uk 2023-03-17T10:20:32Z IDA protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T09:23:05Z IDLs residue_name SO: melaniev@ebi.ac.uk 2023-03-17T10:20:44Z proline mutant MESH: melaniev@ebi.ac.uk 2023-03-17T10:20:55Z Lys66IDA/Arg67IDA → Ala protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:20:58Z double-mutant protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:10Z HAESA protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T09:23:05Z IDLs protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:21:07Z functionally unrelated structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:34:45Z dodecamer chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T10:21:28Z peptides ptm MESH: melaniev@ebi.ac.uk 2023-03-17T10:21:33Z Hyp modifications protein PR: melaniev@ebi.ac.uk 2023-03-17T10:21:54Z CLV3 RESULTS title_2 19026 The co-receptor kinase SERK1 allows for high-affinity IDA sensing protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T10:22:26Z co-receptor kinase protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:29Z SERK1 RESULTS paragraph 19092 Our binding assays reveal that IDA family peptides are sensed by the isolated HAESA ectodomain with relatively weak binding affinities (Figures 1B, 2A–D). It has been recently reported that SOMATIC EMBRYOGENESIS RECEPTOR KINASES (SERKs) are positive regulators of floral abscission and can interact with HAESA and HSL2 in an IDA-dependent manner. As all five SERK family members appear to be expressed in the Arabidopsis abscission zone, we quantified their relative contribution to floral abscission in Arabidopsis using a petal break-strength assay. Our experiments suggest that among the SERK family members, SERK1 is a positive regulator of floral abscission. We found that the force required to remove the petals of serk1-1 mutants is significantly higher than that needed for wild-type plants, as previously observed for haesa/hsl2 mutants, and that floral abscission is delayed in serk1-1 (Figure 3A). The serk2-2, serk3-1, serk4-1 and serk5-1 mutant lines showed a petal break-strength profile not significantly different from wild-type plants. Possibly because SERKs have additional roles in plant development such as in pollen formation and brassinosteroid signaling, we found that higher-order SERK mutants exhibit pleiotropic phenotypes in the flower, rendering their analysis and comparison by quantitative petal break-strength assays difficult. We thus focused on analyzing the contribution of SERK1 to HAESA ligand sensing and receptor activation. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T10:27:24Z binding assays chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T10:27:28Z IDA family peptides protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:27:31Z isolated protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:10Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:34:34Z ectodomain evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:27:34Z binding affinities protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T10:27:38Z SOMATIC EMBRYOGENESIS RECEPTOR KINASES protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T10:27:47Z SERKs protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:10Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T09:07:45Z HSL2 protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T10:28:50Z SERK family members taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:28:16Z Arabidopsis taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:28:38Z Arabidopsis experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T10:28:43Z petal break-strength assay protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T10:28:50Z SERK family members protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:29Z SERK1 gene GENE: melaniev@ebi.ac.uk 2023-03-17T09:44:19Z serk1-1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:29:05Z mutants protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:44:51Z wild-type taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:33:49Z plants gene GENE: melaniev@ebi.ac.uk 2023-03-17T09:43:05Z haesa gene GENE: melaniev@ebi.ac.uk 2023-03-17T09:43:23Z hsl2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:29:10Z mutants gene GENE: melaniev@ebi.ac.uk 2023-03-17T09:44:19Z serk1-1 gene GENE: melaniev@ebi.ac.uk 2023-03-17T10:29:18Z serk2-2 gene GENE: melaniev@ebi.ac.uk 2023-03-17T10:29:27Z serk3-1 gene GENE: melaniev@ebi.ac.uk 2023-03-17T10:29:36Z serk4-1 gene GENE: melaniev@ebi.ac.uk 2023-03-17T10:29:45Z serk5-1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:29:49Z mutant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:44:51Z wild-type taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:33:49Z plants protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T10:27:47Z SERKs experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T10:30:01Z quantitative petal break-strength assays protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:29Z SERK1 protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:10Z HAESA RESULTS paragraph 20558 In vitro, the LRR ectodomain of SERK1 (residues 24–213) forms stable, IDA-dependent heterodimeric complexes with HAESA in size exclusion chromatography experiments (Figure 3B). We next quantified the contribution of SERK1 to IDA recognition by HAESA. We found that HAESA senses IDA with a ~60 fold higher binding affinity in the presence of SERK1, suggesting that SERK1 is involved in the specific recognition of the peptide hormone (Figure 3C). We next titrated SERK1 into a solution containing only the HAESA ectodomain. In this case, there was no detectable interaction between receptor and co-receptor, while in the presence of IDA, SERK1 strongly binds HAESA with a dissociation constant in the mid-nanomolar range (Figure 3C). This suggests that IDA itself promotes receptor – co-receptor association, as previously described for the steroid hormone brassinolide and for other LRR-RK complexes. Importantly, hydroxyprolination of IDA is critical for HAESA-IDA-SERK1 complex formation (Figure 3C,D). Our calorimetry experiments now reveal that SERKs may render HAESA, and potentially other receptor kinases, competent for high-affinity sensing of their cognate ligands. structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:10:10Z LRR ectodomain protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:29Z SERK1 residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:34:12Z 24–213 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:34:19Z stable protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:34:23Z IDA-dependent oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:34:39Z heterodimeric protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:34:43Z complexes with protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:10Z HAESA experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T10:34:47Z size exclusion chromatography protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:29Z SERK1 protein PR: melaniev@ebi.ac.uk 2023-03-22T10:19:27Z IDA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:10Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:10Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:45Z IDA evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:34:51Z binding affinity protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:35:13Z presence of protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:30Z SERK1 protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:30Z SERK1 protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T08:29:24Z peptide hormone experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T10:35:24Z titrated protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:30Z SERK1 protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:10Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:34:34Z ectodomain protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:35:15Z presence of protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:45Z IDA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:30Z SERK1 protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:10Z HAESA evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:35:53Z dissociation constant protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:45Z IDA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T10:35:59Z steroid hormone chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T10:36:07Z brassinolide complex_assembly GO: melaniev@ebi.ac.uk 2023-03-21T18:20:42Z LRR-RK ptm MESH: melaniev@ebi.ac.uk 2023-03-17T10:36:41Z hydroxyprolination protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:45Z IDA complex_assembly GO: melaniev@ebi.ac.uk 2023-03-21T18:20:42Z HAESA-IDA-SERK1 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T10:37:07Z calorimetry protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T10:27:47Z SERKs protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:10Z HAESA protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T10:37:12Z receptor kinases RESULTS paragraph 21738 Upon IDA binding at the cell surface, the kinase domains of HAESA and SERK1, which have been shown to be active protein kinases, may interact in the cytoplasm to activate each other. Consistently, the HAESA kinase domain can transphosphorylate SERK1 and vice versa in in vitro transphosphorylation assays (Figure 3E). Together, our genetic and biochemical experiments implicate SERK1 as a HAESA co-receptor in the Arabidopsis abscission zone. protein PR: melaniev@ebi.ac.uk 2023-03-22T10:19:41Z IDA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T10:39:05Z kinase domains protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:10Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:30Z SERK1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:12Z active protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T10:39:17Z protein kinases protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:10Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T10:39:23Z kinase domain protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:30Z SERK1 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T10:39:26Z transphosphorylation assays experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T10:39:45Z genetic and biochemical experiments protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:30Z SERK1 protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T10:39:59Z HAESA co-receptor taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:40:29Z Arabidopsis RESULTS title_2 22181 SERK1 senses a conserved motif in IDA family peptides protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:30Z SERK1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:41:04Z conserved structure_element SO: melaniev@ebi.ac.uk 2023-03-17T10:41:07Z motif chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T10:41:10Z IDA family peptides elife-15075-fig4.jpg fig4 FIG fig_title_caption 22235 Crystal structure of a HAESA – IDA – SERK1 signaling complex. evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:41:33Z Crystal structure complex_assembly GO: melaniev@ebi.ac.uk 2023-03-17T09:45:14Z HAESA – IDA – SERK1 elife-15075-fig4.jpg fig4 FIG fig_caption 22301 (A) Overview of the ternary complex with HAESA in blue (surface representation), IDA in yellow (bonds representation) and SERK1 in orange (surface view). (B) The HAESA ectodomain undergoes a conformational change upon SERK1 co-receptor binding. Shown are Cα traces of a structural superposition of the unbound (yellow) and SERK1-bound (blue) HAESA ectodomains (r.m.s.d. is 1.5 Å between 572 corresponding Cα atoms). SERK1 (in orange) and IDA (in red) are shown alongside. The conformational change in the C-terminal LRRs and capping domain is indicated by an arrow. (C) SERK1 forms an integral part of the receptor's peptide binding pocket. The N-terminal capping domain of SERK1 (in orange) directly contacts the C-terminal part of IDA (in yellow, in bonds representation) and the receptor HAESA (in blue). Polar contacts of SERK1 with IDA are shown in magenta, with the HAESA LRR domain in gray. (D) Details of the zipper-like SERK1-HAESA interface. Ribbon diagrams of HAESA (in blue) and SERK1 (in orange) are shown with selected interface residues (in bonds representation). Polar interactions are highlighted as dotted lines (in magenta). protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:10Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:45Z IDA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:30Z SERK1 protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:10Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:34:34Z ectodomain protein PR: melaniev@ebi.ac.uk 2023-03-22T10:20:00Z SERK1 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T09:32:08Z structural superposition protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:44:20Z unbound protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:44:23Z SERK1-bound protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:11Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:28:03Z ectodomains evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:44:48Z r.m.s.d. protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:30Z SERK1 protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:45Z IDA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T10:45:02Z LRRs structure_element SO: melaniev@ebi.ac.uk 2023-03-17T10:51:15Z capping domain protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:30Z SERK1 site SO: melaniev@ebi.ac.uk 2023-03-17T09:14:20Z peptide binding pocket structure_element SO: melaniev@ebi.ac.uk 2023-03-17T10:51:15Z capping domain protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:30Z SERK1 protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:45Z IDA protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T10:45:34Z receptor protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:11Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:30Z SERK1 protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:45Z IDA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:11Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T10:45:41Z LRR domain structure_element SO: melaniev@ebi.ac.uk 2023-03-17T10:46:04Z zipper-like site SO: melaniev@ebi.ac.uk 2023-03-17T10:46:09Z SERK1-HAESA interface protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:11Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:30Z SERK1 site SO: melaniev@ebi.ac.uk 2023-03-17T10:46:15Z interface residues elife-15075-fig4.jpg fig4 FIG fig_caption 23453 DOI: http://dx.doi.org/10.7554/eLife.15075.011 RESULTS paragraph 23500 To understand in molecular terms how SERK1 contributes to high-affinity IDA recognition, we solved a 2.43 Å crystal structure of the ternary HAESA – IDA – SERK1 complex (Figure 4A, Table 2). HAESA LRRs 16–21 and its C-terminal capping domain undergo a conformational change upon SERK1 binding (Figure 4B). The SERK1 ectodomain interacts with the IDA peptide binding site using a loop region (residues 51-59SERK1) from its N-terminal cap (Figure 4A,C). SERK1 loop residues establish multiple hydrophobic and polar contacts with Lys66IDA and the C-terminal Arg-His-Asn motif in IDA (Figure 4C). SERK1 LRRs 1–5 and its C-terminal capping domain form an additional zipper-like interface with residues originating from HAESA LRRs 15–21 and from the HAESA C-terminal cap (Figure 4D). SERK1 binds HAESA using these two distinct interaction surfaces (Figure 1—figure supplement 3), with the N-cap of the SERK1 LRR domain partially covering the IDA peptide binding cleft. protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:30Z SERK1 protein PR: melaniev@ebi.ac.uk 2023-03-22T10:20:17Z IDA evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:50:47Z crystal structure complex_assembly GO: melaniev@ebi.ac.uk 2023-03-17T09:45:14Z HAESA – IDA – SERK1 protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:11Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T10:51:05Z LRRs 16–21 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T10:51:15Z capping domain protein PR: melaniev@ebi.ac.uk 2023-03-22T10:20:28Z SERK1 protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:30Z SERK1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:34:34Z ectodomain site SO: melaniev@ebi.ac.uk 2023-03-17T10:51:25Z IDA peptide binding site structure_element SO: melaniev@ebi.ac.uk 2023-03-17T10:51:29Z loop region residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:51:32Z 51-59 protein PR: melaniev@ebi.ac.uk 2023-03-17T10:51:35Z SERK1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T10:51:43Z cap protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:30Z SERK1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T10:51:46Z loop residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:51:55Z Lys66 protein PR: melaniev@ebi.ac.uk 2023-03-17T10:52:10Z IDA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:01:51Z Arg-His-Asn motif protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:45Z IDA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:30Z SERK1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T10:52:15Z LRRs 1–5 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T10:51:15Z capping domain structure_element SO: melaniev@ebi.ac.uk 2023-03-17T10:52:29Z zipper-like site SO: melaniev@ebi.ac.uk 2023-03-17T10:52:33Z interface protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:11Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T10:52:36Z LRRs 15–21 protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:11Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T10:52:39Z cap protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:30Z SERK1 protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:11Z HAESA site SO: melaniev@ebi.ac.uk 2023-03-17T10:52:44Z interaction surfaces structure_element SO: melaniev@ebi.ac.uk 2023-03-17T10:52:47Z N-cap protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:30Z SERK1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T10:52:51Z LRR domain site SO: melaniev@ebi.ac.uk 2023-03-17T10:52:56Z IDA peptide binding cleft elife-15075-fig5.jpg fig5 FIG fig_title_caption 24478 The IDA C-terminal motif is required for HAESA-SERK1 complex formation and for IDA bioactivity. protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:45Z IDA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T10:56:03Z C-terminal motif complex_assembly GO: melaniev@ebi.ac.uk 2023-03-21T18:20:42Z HAESA-SERK1 elife-15075-fig5.jpg fig5 FIG fig_caption 24574 (A) Size exclusion chromatography experiments similar to Figure 3B,D reveal that IDA mutant peptides targeting the C-terminal motif do not form biochemically stable HAESA-IDA-SERK1 complexes. Deletion of the C-terminal Asn69IDA completely inhibits complex formation. Void (V0) volume and total volume (Vt) are shown, together with elution volumes for molecular mass standards (A, Thyroglobulin, 669,000 Da; B, Ferritin, 440,00 Da, C, Aldolase, 158,000 Da; D, Conalbumin, 75,000 Da; E, Ovalbumin, 44,000 Da; F, Carbonic anhydrase, 29,000 Da). Purified HAESA and SERK1 are ~75 and ~28 kDa, respectively. Left panel: IDA K66A/R67A; center: IDA ΔN69, right panel: SDS-PAGE of peak fractions. Note that the HAESA and SERK1 input lanes have already been shown in Figure 3D. (B) Isothermal titration thermographs of wild-type and mutant IDA peptides titrated into a HAESA - SERK1 mixture in the cell. Table summaries for calorimetric binding constants and stoichoimetries for different IDA peptides binding to the HAESA – SERK1 ectodomain mixture ( ± fitting errors; n.d. no detectable binding) are shown alongside. (C) Quantitative petal break-strength assay for Col-0 wild-type flowers and 35S::IDA wild-type and 35S::IDA K66A/R67A mutant flowers. Petal break is measured from positions 1 to 8 along the primary inflorescence where positions 1 is defined as the flower at anthesis (n=15, bars=SD). The three treatment groups in this unbalanced one-way layout were compared by Tukey’s all-pairs comparison procedure using the package multcomp in R (version 3.2.3). 35S::IDA plants showed significantly increased abscission compared to Col-0 controls in inflorescence positions 2 and 3 (a). Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. 15 out of 15 35S::IDA plants, 0 out of 15 Col-0 plants and 0 out of 15 35S::IDA K66A/R67A double-mutant plants, showed an enlarged abscission zone, respectively (3 independent lines were analyzed). experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T14:45:52Z Size exclusion chromatography protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:45Z IDA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T14:45:44Z mutant chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T14:45:47Z peptides structure_element SO: melaniev@ebi.ac.uk 2023-03-17T14:45:50Z C-terminal motif protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T14:45:56Z biochemically stable complex_assembly GO: melaniev@ebi.ac.uk 2023-03-17T14:45:41Z HAESA-IDA-SERK1 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T14:45:59Z Deletion residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T14:46:02Z Asn69 protein PR: melaniev@ebi.ac.uk 2023-03-17T14:44:34Z IDA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T14:45:11Z inhibits protein PR: melaniev@ebi.ac.uk 2023-03-17T09:47:16Z Thyroglobulin protein PR: melaniev@ebi.ac.uk 2023-03-17T09:47:30Z Ferritin protein PR: melaniev@ebi.ac.uk 2023-03-17T09:47:46Z Aldolase protein PR: melaniev@ebi.ac.uk 2023-03-17T09:48:00Z Conalbumin protein PR: melaniev@ebi.ac.uk 2023-03-17T09:48:15Z Ovalbumin protein PR: melaniev@ebi.ac.uk 2023-03-17T09:48:30Z Carbonic anhydrase experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T16:29:17Z Purified protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:11Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:30Z SERK1 mutant MESH: melaniev@ebi.ac.uk 2023-03-17T14:46:11Z IDA K66A/R67A mutant MESH: melaniev@ebi.ac.uk 2023-03-17T14:46:14Z IDA ΔN69 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T14:46:23Z SDS-PAGE protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:11Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:30Z SERK1 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T14:46:31Z Isothermal titration thermographs protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:44:51Z wild-type protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T14:46:34Z mutant chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T16:11:55Z IDA peptides experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T14:47:02Z titrated protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:11Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:30Z SERK1 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T14:47:09Z calorimetric binding constants chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T16:11:55Z IDA peptides protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:11Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:30Z SERK1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:34:34Z ectodomain experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T14:47:42Z petal break-strength assay protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:44:51Z wild-type gene GENE: melaniev@ebi.ac.uk 2023-03-17T14:52:18Z 35S protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:46Z IDA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:44:51Z wild-type gene GENE: melaniev@ebi.ac.uk 2023-03-17T14:52:19Z 35S mutant MESH: melaniev@ebi.ac.uk 2023-03-17T14:51:06Z IDA K66A/R67A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T14:51:10Z mutant gene GENE: melaniev@ebi.ac.uk 2023-03-17T14:52:20Z 35S protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:46Z IDA taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:33:49Z plants gene GENE: melaniev@ebi.ac.uk 2023-03-17T14:52:20Z 35S mutant MESH: melaniev@ebi.ac.uk 2023-03-17T14:54:27Z IDA K66A/R67A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T14:54:31Z mutant taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:33:49Z plants taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:33:49Z plants gene GENE: melaniev@ebi.ac.uk 2023-03-17T14:52:20Z 35S protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:46Z IDA taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:33:49Z plants gene GENE: melaniev@ebi.ac.uk 2023-03-17T14:52:20Z 35S protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:46Z IDA gene GENE: melaniev@ebi.ac.uk 2023-03-17T14:52:20Z 35S mutant MESH: melaniev@ebi.ac.uk 2023-03-17T14:55:03Z IDA K66A/R67A protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:46Z IDA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:44:51Z wild-type protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T14:55:07Z mutant experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T14:55:28Z 35S promoter over-expression lines gene GENE: melaniev@ebi.ac.uk 2023-03-17T14:52:20Z 35S protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:46Z IDA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:44:51Z wild-type gene GENE: melaniev@ebi.ac.uk 2023-03-17T14:52:20Z 35S mutant MESH: melaniev@ebi.ac.uk 2023-03-17T14:56:01Z IDA K66A/R67A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T14:56:05Z double-mutant experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T14:56:07Z T3 transgenic lines gene GENE: melaniev@ebi.ac.uk 2023-03-17T14:52:20Z 35S protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:46Z IDA taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:33:49Z plants taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:33:49Z plants gene GENE: melaniev@ebi.ac.uk 2023-03-17T14:52:20Z 35S mutant MESH: melaniev@ebi.ac.uk 2023-03-17T14:56:22Z IDA K66A/R67A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T14:56:25Z double-mutant taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:33:49Z plants elife-15075-fig5.jpg fig5 FIG fig_caption 27046 DOI: http://dx.doi.org/10.7554/eLife.15075.012 RESULTS paragraph 27093 The four C-terminal residues in IDA (Lys66IDA-Asn69IDA) are conserved among IDA family members and are in direct contact with SERK1 (Figures 1A, 4C). We thus assessed their contribution to HAESA – SERK1 complex formation. Deletion of the buried Asn69IDA completely inhibits receptor – co-receptor complex formation and HSL2 activation (Figure 5A,B). A synthetic Lys66IDA/Arg67IDA → Ala mutant peptide (IDA K66A/R66A) showed a 10 fold reduced binding affinity when titrated in a HAESA/SERK1 protein solution (Figures 5A,B, 2D). We over-expressed full-length wild-type IDA or this Lys66IDA/Arg67IDA → Ala double-mutant to similar levels in Col-0 Arabidopsis plants (Figure 5D). We found that over-expression of wild-type IDA leads to early floral abscission and an enlargement of the abscission zone (Figure 5C–E). In contrast, over-expression of the IDA Lys66IDA/Arg67IDA → Ala double mutant significantly delays floral abscission when compared to wild-type control plants, suggesting that the mutant IDA peptide has reduced activity in planta (Figure 5C–E). Comparison of 35S::IDA wild-type and mutant plants further indicates that mutation of Lys66IDA/Arg67IDA → Ala may cause a weak dominant negative effect (Figure 5C–E). In agreement with our structures and biochemical assays, this experiment suggests a role of the conserved IDA C-terminus in the control of floral abscission. protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:46Z IDA residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:01:23Z Lys66IDA-Asn69IDA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:01:26Z conserved protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T10:19:03Z IDA family members protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:30Z SERK1 complex_assembly GO: melaniev@ebi.ac.uk 2023-03-21T18:20:42Z HAESA – SERK1 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T15:02:08Z Deletion residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:02:11Z Asn69 protein PR: melaniev@ebi.ac.uk 2023-03-17T15:02:22Z IDA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:02:41Z completely inhibits protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:03:03Z synthetic mutant MESH: melaniev@ebi.ac.uk 2023-03-17T15:03:05Z Lys66IDA/Arg67IDA → Ala protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:03:08Z mutant chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T15:03:10Z peptide mutant MESH: melaniev@ebi.ac.uk 2023-03-17T15:03:20Z IDA K66A/R66A evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:03:24Z binding affinity experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T15:03:27Z titrated protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:12Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:31Z SERK1 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T15:03:29Z over-expressed protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:19:56Z full-length protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:44:51Z wild-type protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:46Z IDA mutant MESH: melaniev@ebi.ac.uk 2023-03-17T15:03:32Z Lys66IDA/Arg67IDA → Ala protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:03:35Z double-mutant taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:03:48Z Arabidopsis taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:33:49Z plants experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T15:03:57Z over-expression protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:44:51Z wild-type protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:46Z IDA experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T15:04:00Z over-expression mutant MESH: melaniev@ebi.ac.uk 2023-03-17T15:04:04Z IDA Lys66IDA/Arg67IDA → Ala protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:04:06Z double mutant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:44:51Z wild-type taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:33:49Z plants protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:04:14Z mutant chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T16:09:00Z IDA peptide taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:08:00Z planta gene GENE: melaniev@ebi.ac.uk 2023-03-17T14:52:20Z 35S protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:46Z IDA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:44:51Z wild-type protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:04:49Z mutant taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:33:50Z plants experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T15:04:52Z mutation mutant MESH: melaniev@ebi.ac.uk 2023-03-17T15:04:54Z Lys66IDA/Arg67IDA → Ala evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:04:56Z structures experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T15:05:00Z biochemical assays protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:05:02Z conserved protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:46Z IDA DISCUSS title_1 28494 Discussion DISCUSS paragraph 28505 In contrast to animal LRR receptors, plant LRR-RKs harbor spiral-shaped ectodomains and thus they require shape-complementary co-receptor proteins for receptor activation. For a rapidly growing number of plant signaling pathways, SERK proteins act as these essential co-receptors (; ). SERK1 has been previously reported as a positive regulator in plant embryogenesis, male sporogenesis, brassinosteroid signaling and in phytosulfokine perception. Recent findings by and our mechanistic studies now also support a positive role for SERK1 in floral abscission. As serk1-1 mutant plants show intermediate abscission phenotypes when compared to haesa/hsl2 mutants, SERK1 likely acts redundantly with other SERKs in the abscission zone (Figure 3A). It has been previously suggested that SERK1 can inhibit cell separation. However our results show that SERK1 also can activate this process upon IDA sensing, indicating that SERKs may fulfill several different functions in the course of the abscission process. taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:08:22Z animal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T15:08:27Z LRR receptors taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:01Z plant structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:19:47Z LRR-RKs protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:08:34Z spiral-shaped structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:28:09Z ectodomains protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:08:38Z shape-complementary protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T15:08:42Z co-receptor proteins taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:01Z plant protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T15:08:47Z SERK proteins protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T15:08:50Z co-receptors protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:31Z SERK1 taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:02Z plant protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:31Z SERK1 gene GENE: melaniev@ebi.ac.uk 2023-03-17T09:44:19Z serk1-1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:28:40Z mutant taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:33:50Z plants gene GENE: melaniev@ebi.ac.uk 2023-03-17T09:43:05Z haesa gene GENE: melaniev@ebi.ac.uk 2023-03-17T09:43:23Z hsl2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:28:44Z mutants protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:31Z SERK1 protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T10:27:47Z SERKs protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:31Z SERK1 protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:31Z SERK1 protein PR: melaniev@ebi.ac.uk 2023-03-22T10:21:16Z IDA protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T10:27:47Z SERKs DISCUSS paragraph 29513 While the sequence of the mature IDA peptide has not been experimentally determined in planta, our HAESA-IDA complex structures and calorimetry assays suggest that active IDLs are hydroxyprolinated dodecamers. It will be thus interesting to see if proteolytic processing of full-length IDA in vivo is regulated in a cell-type or tissue-specific manner. The central Hyp residue in IDA is found buried in the HAESA peptide binding surface and thus this post-translational modification may regulate IDA bioactivity. Our comparative structural and biochemical analysis further suggests that IDLs share a common receptor binding mode, but may preferably bind to HAESA, HSL1 or HSL2 in different plant tissues and organs. protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:11:07Z mature chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T16:09:00Z IDA peptide taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:08:00Z planta complex_assembly GO: melaniev@ebi.ac.uk 2023-03-17T10:00:16Z HAESA-IDA evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:11:39Z structures evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:11:42Z calorimetry assays protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:12Z active protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T09:23:05Z IDLs protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:11:57Z hydroxyprolinated structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:16:45Z dodecamers protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:19:56Z full-length protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:46Z IDA residue_name SO: melaniev@ebi.ac.uk 2023-03-17T15:12:16Z Hyp protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:46Z IDA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:12Z HAESA site SO: melaniev@ebi.ac.uk 2023-03-17T15:12:28Z peptide binding surface protein PR: melaniev@ebi.ac.uk 2023-03-22T10:21:31Z IDA experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T15:12:43Z comparative structural and biochemical analysis protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T09:23:05Z IDLs protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:12Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T16:27:35Z HSL1 protein PR: melaniev@ebi.ac.uk 2023-03-17T09:07:46Z HSL2 taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:02Z plant DISCUSS paragraph 30229 In our quantitative biochemical assays, the presence of SERK1 dramatically increases the HAESA binding specificity and affinity for IDA. This observation is consistent with our complex structure in which receptor and co-receptor together form the IDA binding pocket. The fact that SERK1 specifically interacts with the very C-terminus of IDLs may allow for the rational design of peptide hormone antagonists, as previously demonstrated for the brassinosteroid pathway. Importantly, our calorimetry assays reveal that the SERK1 ectodomain binds HAESA with nanomolar affinity, but only in the presence of IDA (Figure 3C). This ligand-induced formation of a receptor – co-receptor complex may allow the HAESA and SERK1 kinase domains to efficiently trans-phosphorylate and activate each other in the cytoplasm. It is of note that our reported binding affinities for IDA and SERK1 have been measured using synthetic peptides and the isolated HAESA and SERK1 ectodomains, and thus might differ in the context of the full-length, membrane-embedded signaling complex. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T16:29:22Z quantitative biochemical assays protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:35:15Z presence of protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:31Z SERK1 protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:12Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:46Z IDA evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:31:21Z structure site SO: melaniev@ebi.ac.uk 2023-03-17T09:01:38Z IDA binding pocket protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:31Z SERK1 protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T09:23:05Z IDLs chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T15:31:45Z peptide hormone antagonists experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T15:31:48Z calorimetry assays protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:31Z SERK1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T08:34:34Z ectodomain protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:32:04Z binds protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:12Z HAESA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T10:35:15Z presence of protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:46Z IDA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:12Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:31Z SERK1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T15:32:32Z kinase domains evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:28:18Z binding affinities protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:46Z IDA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:31Z SERK1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:32:35Z synthetic chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T15:32:38Z peptides experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T15:32:41Z isolated protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:12Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:31Z SERK1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T15:32:49Z ectodomains protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:19:56Z full-length protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:32:45Z membrane-embedded elife-15075-fig6.jpg fig6 FIG fig_title_caption 31293 SERK1 uses partially overlapping surface areas to activate different plant signaling receptors. protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:31Z SERK1 taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:02Z plant protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T15:33:24Z signaling receptors elife-15075-fig6.jpg fig6 FIG fig_caption 31389 (A) Structural comparison of plant steroid and peptide hormone membrane signaling complexes. Left panel: Ribbon diagram of HAESA (in blue), SERK1 (in orange) and IDA (in bonds and surface represention). Right panel: Ribbon diagram of the plant steroid receptor BRI1 (in blue) bound to brassinolide (in gray, in bonds representation) and to SERK1, shown in the same orientation (PDB-ID. 4lsx). (B) View of the inner surface of the SERK1 LRR domain (PDB-ID 4lsc, surface representation, in gray). A ribbon diagram of SERK1 in the same orientation is shown alongside. Residues interacting with the HAESA or BRI1 LRR domains are shown in orange or magenta, respectively. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T16:29:27Z Structural comparison taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:02Z plant chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T15:44:58Z steroid protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T15:51:22Z peptide hormone protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T15:45:38Z membrane signaling complexes protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:12Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:31Z SERK1 protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:46Z IDA taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:02Z plant protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T15:45:44Z steroid receptor protein PR: melaniev@ebi.ac.uk 2023-03-17T15:45:52Z BRI1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T09:30:41Z bound to chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T15:46:03Z brassinolide protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:31Z SERK1 protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:31Z SERK1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T15:46:33Z LRR domain protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:31Z SERK1 protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:12Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T15:45:52Z BRI1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T15:46:36Z LRR domains elife-15075-fig6.jpg fig6 FIG fig_caption 32056 DOI: http://dx.doi.org/10.7554/eLife.15075.013 DISCUSS paragraph 32103 Comparison of our HAESA – IDA – SERK1 structure with the brassinosteroid receptor signaling complex, where SERK1 also acts as co-receptor, reveals an overall conserved mode of SERK1 binding, while the ligand binding pockets map to very different areas in the corresponding receptors (LRRs 2 – 14; HAESA; LRRs 21 – 25, BRI1) and may involve an island domain (BRI1) or not (HAESA) (Figure 6A). Several residues in the SERK1 N-terminal capping domain (Thr59SERK1, Phe61SERK1) and the LRR inner surface (Asp75SERK1, Tyr101SERK1, SER121SERK1, Phe145SERK1) contribute to the formation of both complexes (Figures 4C,D, 6B). In addition, residues 53-55SERK1 from the SERK1 N-terminal cap mediate specific interactions with the IDA peptide (Figures 4C, 6B). These residues are not involved in the sensing of the steroid hormone brassinolide. In both cases however, the co-receptor completes the hormone binding pocket. This fact together with the largely overlapping SERK1 binding surfaces in HAESA and BRI1 allows us to speculate that SERK1 may promote high-affinity peptide hormone and brassinosteroid sensing by simply slowing down dissociation of the ligand from its cognate receptor. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T15:52:04Z Comparison complex_assembly GO: melaniev@ebi.ac.uk 2023-03-17T09:45:14Z HAESA – IDA – SERK1 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:52:17Z structure protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:31Z SERK1 protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T15:52:55Z co-receptor protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:52:32Z conserved protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:31Z SERK1 site SO: melaniev@ebi.ac.uk 2023-03-17T15:52:39Z ligand binding pockets structure_element SO: melaniev@ebi.ac.uk 2023-03-17T15:53:01Z LRRs 2 – 14 protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:12Z HAESA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T15:53:05Z LRRs 21 – 25 protein PR: melaniev@ebi.ac.uk 2023-03-17T15:45:52Z BRI1 protein PR: melaniev@ebi.ac.uk 2023-03-17T15:45:52Z BRI1 protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:12Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:31Z SERK1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T10:51:15Z capping domain residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:53:40Z Thr59 protein PR: melaniev@ebi.ac.uk 2023-03-17T15:53:42Z SERK1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:53:46Z Phe61 protein PR: melaniev@ebi.ac.uk 2023-03-17T15:53:49Z SERK1 site SO: melaniev@ebi.ac.uk 2023-03-17T15:53:58Z LRR inner surface residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:54:01Z Asp75 protein PR: melaniev@ebi.ac.uk 2023-03-17T15:54:03Z SERK1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:54:06Z Tyr101 protein PR: melaniev@ebi.ac.uk 2023-03-17T15:54:09Z SERK1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:54:12Z SER121 protein PR: melaniev@ebi.ac.uk 2023-03-17T15:54:15Z SERK1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:54:18Z Phe145 protein PR: melaniev@ebi.ac.uk 2023-03-17T15:54:21Z SERK1 residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-17T15:54:27Z 53-55 protein PR: melaniev@ebi.ac.uk 2023-03-17T15:54:31Z SERK1 protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:31Z SERK1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T15:54:42Z cap chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T16:09:00Z IDA peptide chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T15:55:13Z steroid hormone chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T15:55:15Z brassinolide site SO: melaniev@ebi.ac.uk 2023-03-17T09:30:04Z hormone binding pocket site SO: melaniev@ebi.ac.uk 2023-03-17T15:55:24Z SERK1 binding surfaces protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:12Z HAESA protein PR: melaniev@ebi.ac.uk 2023-03-17T15:45:52Z BRI1 protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:31Z SERK1 protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T08:29:25Z peptide hormone elife-15075-fig7.jpg fig7 FIG fig_title_caption 33296 Different plant peptide hormone families contain a C-terminal (Arg)-His-Asn motif, which in IDA represents the co-receptor recognition site. taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:02Z plant protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T15:56:16Z peptide hormone families structure_element SO: melaniev@ebi.ac.uk 2023-03-17T15:56:20Z (Arg)-His-Asn motif protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:47Z IDA site SO: melaniev@ebi.ac.uk 2023-03-17T15:56:23Z co-receptor recognition site elife-15075-fig7.jpg fig7 FIG fig_caption 33437 Structure-guided multiple sequence alignment of IDA and IDA-like peptides with other plant peptide hormone families, including CLAVATA3 – EMBRYO SURROUNDING REGION-RELATED (CLV3/CLE), ROOT GROWTH FACTOR – GOLVEN (RGF/GLV), PRECURSOR GENE PROPEP1 (PEP1) from Arabidopsis thaliana. The conserved (Arg)-His-Asn motif is highlighted in red, the central Hyp residue in IDLs and CLEs is marked in blue. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T15:57:40Z Structure-guided multiple sequence alignment protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:47Z IDA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T15:58:54Z IDA-like peptides taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:02Z plant protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T15:59:07Z peptide hormone families protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T15:59:18Z CLAVATA3 – EMBRYO SURROUNDING REGION-RELATED protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T15:59:28Z CLV3/CLE protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T15:59:39Z ROOT GROWTH FACTOR – GOLVEN protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T15:59:49Z RGF/GLV protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T15:59:59Z PRECURSOR GENE PROPEP1 protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:00:10Z PEP1 species MESH: melaniev@ebi.ac.uk 2023-03-17T08:47:13Z Arabidopsis thaliana protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:00:15Z conserved structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:00:17Z (Arg)-His-Asn motif residue_name SO: melaniev@ebi.ac.uk 2023-03-17T16:00:30Z Hyp protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T09:23:05Z IDLs protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:00:44Z CLEs elife-15075-fig7.jpg fig7 FIG fig_caption 33838 DOI: http://dx.doi.org/10.7554/eLife.15075.014 DISCUSS paragraph 33885 Our experiments reveal that SERK1 recognizes a C-terminal Arg-His-Asn motif in IDA. Importantly, this motif can also be found in other peptide hormone families (Figure 7). Among these are the CLE peptides regulating stem cell maintenance in the shoot and the root. It is interesting to note, that CLEs in their mature form are also hydroxyprolinated dodecamers, which bind to a surface area in the BARELY ANY MERISTEM 1 receptor that would correspond to part of the IDA binding cleft in HAESA. Diverse plant peptide hormones may thus also bind their LRR-RK receptors in an extended conformation along the inner surface of the LRR domain and may also use small, shape-complementary co-receptors for high-affinity ligand binding and receptor activation. protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:31Z SERK1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T09:01:51Z Arg-His-Asn motif protein PR: melaniev@ebi.ac.uk 2023-03-17T08:32:47Z IDA structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:03:02Z this motif protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:03:13Z peptide hormone families chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T16:03:28Z CLE peptides protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:03:38Z CLEs protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:03:41Z mature form protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:04:05Z hydroxyprolinated structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:16:25Z dodecamers site SO: melaniev@ebi.ac.uk 2023-03-17T16:04:41Z surface area protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:04:44Z BARELY ANY MERISTEM 1 receptor site SO: melaniev@ebi.ac.uk 2023-03-17T16:04:48Z IDA binding cleft protein PR: melaniev@ebi.ac.uk 2023-03-17T08:33:12Z HAESA taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T08:34:02Z plant protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:05:02Z peptide hormones protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:05:07Z LRR-RK receptors protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:05:10Z extended conformation structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:28:14Z LRR domain protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:28:49Z small protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:28:53Z shape-complementary protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:05:29Z co-receptors METHODS title_1 34637 Materials and methods METHODS title_2 34659 Protein expression and purification METHODS paragraph 34695 Synthetic genes coding for the Arabidopsis thaliana HAESA (residues 20–620) and SERK1 ectodomains (residues 24–213, carrying Asn115→Asp and Asn163→Gln mutations), codon optimized for expression in Trichoplusia ni (Geneart, Germany), were cloned into a modified pBAC-6 transfer vector (Novagen, Billerica, MA), providing an azurocidin signal peptide and a C-terminal TEV (tobacco etch virus protease) cleavable Strep-9xHis tandem affinity tag. Recombinant baculoviruses were generated by co-transfecting transfer vectors with linearised baculovirus DNA (ProFold-ER1, AB vector, San Diego, CA) followed by viral amplification in Spodoptera frugiperda Sf9 cells. The HAESA and SERK1 ectodomains were individually expressed in Trichoplusia ni Tnao38 cells using a multiplicity of infection of 3, and harvested from the medium 2 days post infection by tangential flow filtration using 30 kDa MWCO and 10 kDa MWCO (molecular weight cut-off) filter membranes (GE Healthcare Life Sciences, Pittsburgh, PA), respectively. Proteins were purified separately by sequential Ni2+ (HisTrap HP, GE Healthcare) and Strep (Strep-Tactin Superflow high-capacity, IBA, Germany) affinity chromatography. Next, affinity tags were removed by incubating the purified proteins with recombinant Strep-tagged TEV protease in 1:100 molar ratio. The cleaved tag and the protease were separated from HAESA and SERK1 by a second Strep affinity step. The purified HAESA ectodomain was incubated with a synthetic IDA peptide (YVPIPPSA-Hyp-SKRHN, the N-terminal Tyr residue was added to allow for peptide quantification by UV absorbance) and the SERK1 ectodomain in 1:1:1.5 molar ratio. The HAESA-IDA-SERK1 complex was purified by size exclusion chromatography on a Superdex 200 HR10/30 column (GE Healthcare) equilibrated in 20 mM citric acid pH 5.0, 100 mM NaCl). Peak fractions containing the complex were concentrated to ~10 mg/mL and immediately used for crystallization. About 0.2 mg of purified HAESA and 0.1 mg of purified SERK1 protein were obtained from 1 L of insect cell culture, respectively. METHODS title_2 36785 Crystallization and data collection METHODS paragraph 36821 Hexagonal crystals of the isolated HAESA ectodomain developed at room-temperature in hanging drops composed of 1.0 μL of protein solution (5.5 mg/mL) and 1.0 μL of crystallization buffer (21% PEG 3,350, 0.2 M MgCl2 · 6 H2O, 0.1 M citric acid pH 4.0), suspended above 1.0 mL of crystallization buffer. For structure solution crystals were derivatized and cryo-protected by serial transfer into crystallization buffer supplemented with 0.5 M NaI and 15% ethylene glycol and cryo-cooled in liquid nitrogen. Redundant single-wavelength anomalous diffraction (SAD) data to 2.39 Å resolution were collected at beam-line PXII at the Swiss Light Source (SLS), Villigen, CH with λ=1.7 Å. A native data set to 1.74 Å resolution was collected on a crystal from the same drop cryo-protected by serial transfer into crystallization buffer supplemented with 15% (v/v) ethylene glycol only (λ=1.0 Å; Table 1). METHODS paragraph 37745 HAESA complexes with IDA (PIPPSA-Hyp-SKRHN), PKGV-IDA (YPKGVPIPPSA-Hyp-SKRHN) and IDL1 (LVPPSG-Hyp-SMRHN) peptide hormones were obtained by soaking apo crystals in crystallization buffer containing the respective synthetic peptide at a final concentration of 15 mM. Soaked crystals diffracted to 1.86 Å (HAESA – IDA), 1.94 Å (HAESA-PKGV-IDA) and 2.56 Å resolution (HAESA – IDL1), respectively (Table 2). Orthorhombic crystals of the HAESA-IDA-SERK1 complex developed in 18% PEG 8000, MgCl2 · 6 H2O, 0.1 M citric acid and diffracted to 2.43 Å resolution (Table 2). Data processing and scaling was done in XDS (version: Nov 2014). METHODS title_2 38395 Structure solution and refinement METHODS paragraph 38429 The SAD method was used to determine the structure of the isolated HAESA ectodomain. SHELXD located 32 iodine sites (CC All/Weak 37.7/14.9). 20 consistent sites were input into the program SHARP for phasing and identification of 8 additional sites at 2.39 Å resolution. Refined heavy atom sites and phases were provided to PHENIX.AUTOBUILD for density modification and automated model building. The structure was completed in alternating cycles of model building in COOT and restrained TLS refinement in REFMAC5 (version 5.8.0107) against an isomorphous high resolution native data set. Crystals contain one HAESA monomer per asymmetric unit with a solvent content of ~55%, the final model comprises residues 20 – 615. The refined structure has excellent stereochemistry, with 93.8% of all residues in the favored region of the Ramachandran plot, no outliers and a PHENIX.MOLPROBITY score of 1.34 (Table 1). METHODS paragraph 39342 The HAESA – IDA – SERK1 complex structure was determined by molecular replacement with the program PHASER, using the isolated HAESA and SERK1 (PDB-ID: 4LSC) LRR domain structures as search models. The solution comprises one HASEA-IDA-SERK1 complex in the asymmetric unit. The structure was completed in iterative cycles of manual model-building in COOT and restrained TLS refinement in REFMAC5. Amino acids whose side-chain position could not be modeled with confidence were truncated to alanine (0.6 – 1% of total residues), the stereochemistry of N-linked glycan structures was assessed with the CCP4 program PRIVATEER-VALIDATE. The refined model has 94.44% of all residues in the favored region of the Ramachandran plot, no outliers and a PHENIX.MOLPROBITY score of 1.17 (Table 2). Structural visualization was done with POVScript+ and POV-Ray (http://www.povray.org). METHODS title_2 40220 Size-exclusion chromatography METHODS paragraph 40250 Gel filtration experiments were performed using a Superdex 200 HR 10/30 column (GE Healthcare) pre-equilibrated in 20 mM citric acid (pH 5) and 100 mM NaCl. 100 μL of the isolated HAESA ectodomain (5.5 mg/mL), of the purified SERK1 LRR domain (3 mg/mL) or of mixtures of HAESA and SERK1 (either in the presence or absence of synthetic wild-type IDA, wild-type IDL1 or mutant IDA peptides at a concentration of 25 μM; 10 mg/mL; samples contained HAESA and SERK1 in 1:1 molar ratio) were loaded sequentially onto the column and elution at 0.5 mL/min was monitored by ultraviolet absorbance at 280 nm. METHODS title_2 40861 Isothermal titration calorimetry METHODS paragraph 40894 ITC experiments were performed using a Nano ITC (TA Instruments, New Castle, DE) with a 1.0 mL standard cell and a 250 μL titration syringe. Proteins were dialyzed extensively against ITC buffer (20 mM citric acid pH 5.0, 100 mM NaCl) and synthetic wild-type or point-mutant peptides (with wild-type IDA sequence YVPIPPSA-Hyp-SKRHN, PKGV-IDA YPKGVPIPPSA-Hyp-SKRHN, IDA-SFVN YPIPPSA-Hyp-SKRHNSFVN, IDL1 YLVPPSG-Hyp-SMRHN and CLV3 sequence YRTV-Hyp-SG-Hyp-DPLHH) were dissolved in ITC buffer prior to all titrations. Molar protein concentrations for SERK1 and HAESA were calculated using their molar extinction coefficient and a molecular weight of 27,551 and 74,896 Da, respectively (determined by MALDI-TOF mass spectrometry). Experiments were performed at 25°C. A typical experiment consisted of injecting 10 μL aliquots of peptide solution (250 μM) into 20 μM HAESA. The concentrations for the complex titrations were 150 μM of ligand (either wild-type or point-mutant IDA peptides) in the syringe and 10 μM of a 1:1 HAESA – SERK1 protein mixture in the cell at time intervals of 150 s to ensure that the titration peak returned to the baseline. Binding of SERK1 to HAESA was assessed by titrating SERK1 (100 μM) into a solution containing HAESA (10 μM) in the pre- or absence of 150 μM wild-type IDA peptide. ITC data were corrected for the heat of dilution by subtracting the mixing enthalpies for titrant solution injections into protein free ITC buffer. Data were analyzed using the NanoAnalyze program (version 2.3.6) as provided by the manufacturer. METHODS title_2 42490 In vitro kinase trans-phosphorylation assay METHODS paragraph 42534 Coding sequences of SERK1 kinase domain (SERK1-KD) (residues 264–625) and HAESA-KD (residues 671–969) were cloned into a modified pET (Novagen) vector providing an TEV-cleavable N-terminal 8xHis-StrepII-Thioredoxin tag. Point mutations were introduced into the SERK1 (Asp447→Asn; mSERK1) and HAESA (Asp837→Asn; mHAESA) coding sequences by site directed mutagenesis, thereby rendering the kinases inactive. The plasmids were transformed into E.coli Rosetta 2 (DE3) (Novagen). Protein expression was induced by adding IPTG to final concentration of 0.5 mM to cell cultures grown to an OD600 = 0.6. Cells were then incubated at 16°C for 18 hr, pelleted by centrifugation at 5000 x g and 4°C for 15 min, and resuspended in buffer A (20 mM Tris-HCl pH 8, 500 mM NaCl, 4 mM MgCl2 and 2 mM β-Mercaptoethanol) supplemented with 15 mM Imidazole and 0.1% (v/v) Igepal. After cell lysis by sonication, cell debris was removed by centrifugation at 35,000 x g and 4°C for 30 min. The recombinant proteins were isolated by Co2+ metal affinity purification using a combination of batch and gravity flow approaches (HIS-Select Cobalt Affinity Gel, Sigma, St. Louis, MO). After washing the resin with the wash buffer (buffer A + 15 mM Imidazole) proteins were eluted in buffer A supplemented with 250 mM Imidazole. All elutions were then dialyzed against 20 mM Tris-HCl pH 8, 250 mM NaCl, 4 mM MgCl2 and 0.5 mM TCEP. For SERK1-KD and mSERK1-KD the 8xHis-StrepII-Thioredoxin tag was removed with 6xHis tagged TEV protease. TEV and the cleaved tag were removed by a second metal affinity purification step. Subsequently, all proteins were purified by gel filtration on a Superdex 200 10/300 GL column equilibrated in 20 mM Tris pH 8, 250 mM NaCl, 4 mM MgCl2 and 0.5 mM TCEP. Peak fractions were collected and concentrated using Amicon Ultra centrifugation devices (10,000 MWCO). For in vitro kinase assays, 1 μg of HAESA-KD, 0.25 μg of SERK1-KD and 2 μg of mSERK1 and mHAESA were used in a final reaction volume of 20 μl. The reaction buffer consisted of 20 mM Tris pH 8, 250 mM NaCl, 4 mM MgCl2 and 0.5 mM TCEP. The reactions were started by the addition of 4 μCi [γ-32P]-ATP (Perkin-Elmer, Waltham, MA), incubated at room temperature for 45 min and stopped by the addition of 6x SDS-loading dye immediately followed by incubating the samples at 95°C. Proteins of the whole reaction were subsequently separated via SDS-PAGE in 4–15% gradient gels (TGX, Biorad, Hercules, CA) and stained with Instant Blue (Expedeon, San Diego, CA). After pictures were taken of the stained gel, 32P-derived signals were visualized by exposing the gel to an X-ray film (Fuji, SuperRX, Valhalla, NY). METHODS title_2 45274 Plant material and generation of transgenic lines METHODS paragraph 45324 35S::IDA wild-type and 35S::IDA (R66 → Ala/K67 → Ala) over-expressing transgenic lines in Col-0 background were generated as follows: The constructs were introduced in the destination vector pB7m34GW2 and transferred to A. tumefaciens strain pGV2260. Plants were transformed using the floral dip method. Transformants were selected in medium supplemented with BASTA up to the T3 generation. For phenotyping, plants were grown at 21°C with 50% humidity and a 16h light: 8 hr dark cycle. METHODS title_2 45816 RNA analyses METHODS paragraph 45829 Plants were grown on ½ Murashige and Skoog (MS) plates supplemented with 1% sucrose. After 7 d, ∼30 to 40 seedlings were collected and frozen in liquid nitrogen. Total RNA was extracted using a RNeasy plant mini kit (Qiagen, Valencia, CA), and 1 μg of the RNA solution obtained was reverse-transcribed using the SuperScritpVILO cDNA synthesis kit (Invitrogen, Grand Island, NY). RT-qPCR amplifications and measurements were performed using a 7900HT Fast Real Time PCR-System by Applied Biosystems (Carlsbad, CA). RT-qPCR amplifications were monitored using SYBR-Green fluorescent stain (Applied Biosystems). Relative quantification of gene expression data was performed using the 2−ΔΔCT (or comparative CT) method. Expression levels were normalized using the CT values obtained for the actin2 gene (forward: TGCCAATCTACGAGGGTTTC; reverse: TTCTCGATGGAAGAGCTGGT). For detection and amplification of IDA sequence we used specific primers (forward: TCGTACGATGATGGTTCTGC; reverse: GAATGGGAACGCCTTTAGGT). The presence of a single PCR product was further verified by dissociation analysis in all amplifications. All quantifications were made in quadruplicates on RNA samples obtained from three independent experiments. METHODS title_2 47053 Petal break measurements METHODS paragraph 47078 serk1-1, serk2-2, serk3-1, serk4-1 and serk5-1 and Col-0 wild-type plants were grown in growth chambers at 22°C under long days (16 hr day/8 hr dark) at a light intensity of 100 µE·m-2·sec-1. Petal break-strength was quantified as the force in gram equivalents required for removal of a petal from a flower when the plants had a minimum of twenty flowers and siliques. Measurements were performed using a load transducer as described in. Break-strength was measured for 15 plants and a minimum of 15 measurements at each position. ACK_FUND title_1 47617 Funding Information ACK_FUND paragraph 47637 This paper was supported by the following grants: ACK_FUND paragraph 47687 to Michael Hothorn. ACK_FUND paragraph 47709 to Michael Hothorn. ACK_FUND paragraph 47731 to Michael Hothorn. ACK_FUND paragraph 47753 to Melinka A Butenko. ACK_FUND paragraph 47777 to Benjamin Brandt. ACK_FUND paragraph 47799 to Julia Santiago. ACK_FUND title_1 47820 Additional information COMP_INT title_1 47843 Competing interests COMP_INT footnote 47863 The authors declare that no competing interests exist. AUTH_CONT title_1 47918 Author contributions AUTH_CONT footnote 47939 JS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. AUTH_CONT footnote 48058 BB, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. AUTH_CONT footnote 48154 MW, Acquisition of data, Analysis and interpretation of data. AUTH_CONT footnote 48216 UH, Acquisition of data, Analysis and interpretation of data. AUTH_CONT footnote 48278 LAH, Analysis and interpretation of data, Drafting or revising the article. AUTH_CONT footnote 48354 MAB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. AUTH_CONT footnote 48474 MH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. 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A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included. REVIEW_INFO paragraph 53746 Thank you for submitting your article "Mechanistic insight into a peptide hormone signaling complex mediating floral organ abscission" to eLife for consideration by eLife. Your article has been favorably evaluated by John Kuyiyan (Senior editor) and three reviewers, one of whom, (Mingjie Zhang) is a member of our Board of Reviewing Editors. REVIEW_INFO paragraph 54089 The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. As you will see that the required revisions are essentially clarifications and some additional analysis of existing data in nature. REVIEW_INFO paragraph 54372 Summary: REVIEW_INFO paragraph 54381 In this work, Hothorn and colleagues investigated the structural basis governing the recognition of peptide hormone IDA during plant floral abscission process. Through an array of complex structures, supplemented with biochemical and genetic experiments, the authors uncovered the IDA recognition mechanism by a co-receptor (HAESA and SERK1) detection mechanism. The structures also reveal the specific recognition mechanism of the 12-residue IDA core peptide sequence by the co-receptors, and suggest that this 12-residue IDA sequence is likely to be the mature peptide hormone functioning in plants. The comparison of the structures of the HAESA/SERK1/IDA complex and the previously determined BRA1/SERK1/brassinolide complex by the same group also suggests a co-receptor pairing mechanism for various plant hormones. The story gives detailed and novel mechanistic insights in the perception of IDA during floral abscission, and is convincing and worthy to be considered for publication in eLife with the following revisions. REVIEW_INFO paragraph 55409 Key issues which need to be addressed: REVIEW_INFO paragraph 55448 1) As the results reported here rest largely on the interpretation of the structural data, the following points need to be addressed by the authors. i) Temperature factors (Wilson B and residual) are unusually high for the reported resolution. Are there portions of the structure that exhibit more disorder or are these high temperature factors throughout the structure? Are there potential problems with radiation damage due to the high multiplicity? ii) A simulated annealing omit map figure should be provided as the peptides all exhibit very high temperature factors. iii) How many side chains were trimmed due to poor electron density? If this is a significant percentage, it should be noted. REVIEW_INFO paragraph 56146 2) A further discussion comparing the mechanisms of BRI1-BL-SERK1 and HAESA-IDA-SERK1 would be helpful as a structural comparison is presented. As BR1 binds its ligand with high affinity, the low affinity of HAESA for IDA could be further discussed. REVIEW_INFO paragraph 56396 3) Are the results here applicable to the related receptor HSL2? Are the residues that interact with SERK1 and IDA conserved in HSL2? REVIEW_INFO paragraph 56530 4) The authors show that N-terminal extension of the peptide does not impact on binding efficiency, but what would happen if the peptide was extended at the C-terminal end, at the suggested cleavage site? Would cleavage be required for recognition? A brief discussion on this point may help. REVIEW_INFO paragraph 56822 5) Figure 3C: Could the authors comment on the difference between the blue (SERK1 vs. HAESA-IDA) and the black (IDA vs. HAESA-SERK1) line? SERK1 vs. HAESA-IDA gives a Kd of 75 nM, yet IDA vs. SERK1-HAESA gives a Kd of 350 nM. In the text the authors keep referring to the 75 nM Kd, but not the 350 nM Kd. Is it really fair to say the Kd is 75 nM? REVIEW_INFO paragraph 57169 6) In the Discussion, can the authors comment further on the discrepancy between their study and the study of Lewis et al. (Plant J, 2010) concerning the role of SERK1 in floral abscission? Similarly, could the authors comment on the fact that the LRR-RLP EVD/SOBIR seems to be a negative regulator of the HAESA/HSL2 pathway (Leslie et al., Development, 2010), which seems puzzling given that EVD/SOBIR function is normally restricted to LRR-RLPs (Gust & Felix, Curr Op Plant Biol, 2014). REVIEW_INFO paragraph 57658 7) Given that the central hydroxyproline in IDA is of such crucial importance for binding, isn't it surprising that IDAΔ69N does not bind to HAESA at all? Wouldn't it be expected that the remaining part of the peptide still binds to HAESA? REVIEW_INFO paragraph 57902 8) Figure 1—figure supplement 2: How is it possible that charged amino acids are involved in hydrophobic interactions? REVIEW_INFO paragraph 58023 9) Are the distances shown in the graphical representation of the structures proportional? It would seem that some of the aromatic rings could cause steric hindrance. REVIEW_INFO paragraph 58190 10) Why did the authors decide to express HAESA and SERK1 without signal peptide? Would it make a difference for binding of IDA, if they leave the SP on? REVIEW_INFO paragraph 58344 11) Figure 7: Are the homologous regions also the active parts of these peptides? And could the authors display amino acids numbers on either side of the fragments? REVIEW_INFO paragraph 58509 12) Have the authors ever measured dissociation of the peptide from the complex? And in this regard, to what does "highly stable receptor – co-receptor complex" refer/compare to? REVIEW_INFO paragraph 58690 13) Figures 3A and 5C require statistical analyses. REVIEW_INFO paragraph 58742 10.7554/eLife.15075.018 REVIEW_INFO title 58766 Author response REVIEW_INFO paragraph 58782 1) As the results reported here rest largely on the interpretation of the structural data, the following points need to be addressed by the authors. i) Temperature factors (Wilson B and residual) are unusually high for the reported resolution. REVIEW_INFO paragraph 59028 We thank the reviewer(s) for pointing out this issue: Indeed our mean B-values deviate substantially from the expected mean B-values (resolution 1.75 – 2.00 A, B(Wilson) ~ 27.0 over 5,510 structures in the PDB; Pavel Afonine, personal communication). We would like to note that due to the many N-glycosylation sites dispersed over the HAESA LRR domain (shown in Figure 1—figure supplement 1D), we find relatively few crystal contacts in our P3121 crystal form, which may rationalize our high B-values. We have reanalyzed our space group assignment (using the CCP4 program ZANUDA) and checked for any signs of problems during data collection (ice rings, multiple crystal lattices, splitting, using the programs XDS and XDSSTAT), as well as for twinning and pseudosymmetry (using phenix.xtriage). No such problems appear to exist, our structures refine very well and our refined B-values are in good agreement with our Wilson B-factors (see Table 2). Thus, the high B-values appear to represent an intrinsic property of our crystals and are not the result of a poor data collection strategy or inappropriate crystallographic analysis. REVIEW_INFO paragraph 60165 Are there portions of the structure that exhibit more disorder or are these high temperature factors throughout the structure? elife-15075-resp-fig1.jpg fig8 FIG fig_title_caption 60293 Cα trace of the HAESA LRR domain and IDA peptide colored according to B-factor from low (60.9, in blue) to high (134.7, in red). elife-15075-resp-fig1.jpg fig8 FIG fig_caption 60426 Mean B-value is 79.5. elife-15075-resp-fig1.jpg fig8 FIG fig_caption 60448 DOI: http://dx.doi.org/10.7554/eLife.15075.015 REVIEW_INFO paragraph 60495 Yes. As shown in revised Figure 4B, the Cterminal LRRs of HAESA in contact with SERK1 in our complex structure appear to be somewhat flexible. Author response image 1. illustrates that the B-values are significantly higher in the C-terminal part of the HAESA LRR domain (with the Cterminal capping domain being the most flexible), while both the N-terminal LRRs of HAESA (with exception of the LRR N-terminal capping domain) and the IDA peptide appear better ordered in our P3121 crystals form. REVIEW_INFO paragraph 60990 Are there potential problems with radiation damage due to the high multiplicity? REVIEW_INFO paragraph 61072 No. Data were collected at SLS beamline PXIII equipped with a Dectris Pilatus 2M-F detector. We perform all our data collections at very low dose and high multiplicity of measurement, which at this beam-line produces similar results compared to exposing the crystal at higher dose for a smaller angular range. We collected 360 deg with 0.1 deg slices and obtained a Wilson B-value of 80, with no sign of radiation damage in our data processing (subroutine COLSPOT in XDS over all frames). To test the reviewer's hypothesis we cut the data after 90 deg (when completeness approaches 100%) and we obtained a Wilson B-value of 78 and a refined mean B-value of around 75. These value do not significantly differ from our presented 360 deg data set and thus it is unlikely that radiation damage produces these high B-values. Again, they rather appear to be an intrinsic property of our crystals. REVIEW_INFO paragraph 61963 ii) A simulated annealing omit map figure should be provided as the peptides all exhibit very high temperature factors. REVIEW_INFO paragraph 62083 Thank you for this suggestion. In our first submission, we presented 2Fo-Fc omit electron density maps for our HAESA-IDA/IDL complex structures. As suggested, we now present simulated annealing omit maps in revised Figure 2A, B, C. The maps were generated like this: phenix.composite_omit_map *.pdb *.mtz *.cif nproc=8 anneal=True We would like to note that our peptide are well ordered in our structures, and their B-values match the B-values of their interacting LRR surface (compare Author response image 1). REVIEW_INFO paragraph 62595 iii) How many side chains were trimmed due to poor electron density? If this is a significant percentage, it should be noted. REVIEW_INFO paragraph 62722 Here are the requested numbers (trimmed residues out of total residues in asymmetric unit, percentage): REVIEW_INFO paragraph 62826 HAESA apo: 7 out of 595 (1%) REVIEW_INFO paragraph 62855 HAESA IDA: 6 out of 597 + 12 (1%) REVIEW_INFO paragraph 62889 HAESA IDL1: 6 out of 597 + 12 (1%) REVIEW_INFO paragraph 62924 HAESA – IDA – SERK1: 5 out of 594 + 12 + 185 (0.6%) REVIEW_INFO paragraph 62980 We have included a statement in the Methods section that reads: “Amino acids whose side-chain position could not be modeled with confidence were truncated to alanine (0.6 – 1% of total residues)[…]” REVIEW_INFO paragraph 63187 2) A further discussion comparing the mechanisms of BRI1-BL-SERK1 and HAESA-IDA-SERK1 would be helpful as a structural comparison is presented. REVIEW_INFO paragraph 63333 We have expanded our discussion of the HAESA – SERK1 and BRI1 – SERK1 interfaces. We now specify the SERK1 residues in contact with both receptors and the SERK1 residues unique to HAESA/IDA sensing. We also comment on the very different ligand binding modes in HAESA and BRI1 and specify that different LRR segments contribute to the formation of the respective steroid and peptide hormone binding pockets. We feel however that an in-depth comparison of the interacting surfaces is beyond the scope of this report and partially redundant with our earlier work (Santiago et al., Science, 2013). In our opinion, such an analysis seems more appropriate for a review on the subject, which we are currently preparing. Our revised Discussion now reads: “Comparison of our HAESA – IDA – SERK1 structure with the brassinosteroid receptor signaling complex, where SERK1 also acts as co-receptor (Santiago, Henzler, and Hothorn 2013), reveals an overall conserved mode of SERK1 binding, while the ligand binding pockets map to very different areas in the corresponding receptors (LRRs 2 – 14; HAESA; LRRs 21 – 25, BRI1) and may involve an island domain (BRI1) or not (HAESA) (Figure 6A).[…] These residues are not involved in the sensing of the steroid hormone brassinolide (Santiago, Henzler, and Hothorn 2013). In both cases however, the co-receptor completes the hormone binding pocket.” REVIEW_INFO paragraph 64732 As BR1 binds its ligand with high affinity, the low affinity of HAESA for IDA could be further discussed. REVIEW_INFO paragraph 64839 High affinity brassinosteroid binding to BRI1 was previously shown using BRI1-enriched plant extracts and radiolabeled brassinolide (Wang et al., Nature410:380-383, 2001). We now know that co-immunoprecipitations of BRI1 from Arabidopsis contain SERK proteins (compare for example Jaillais et al., PNAS, 2011) and thus the reported binding constants likely correspond to steroid binding to BRI1-SERK complexes, not to BRI1 alone. We would thus prefer not to compare the binding affinities for brassinosteroid and peptide hormone ligands at this point. REVIEW_INFO paragraph 65391 3) Are the results here applicable to the related receptor HSL2? Are the residues that interact with SERK1 and IDA conserved in HSL2? REVIEW_INFO paragraph 65526 Yes. We present a structure-based sequence alignment of AtHAESA and AtHSL2, as well as other HAESA-type receptors from different plant species in Figure 1—figure supplement 3. In the peptide binding surface, 17 out of 26 contributing amino-acids are conserved among AtHAESA and AtHSL2. 13 out of 19 interacting residues in the HAESA – SERK1 complex are also present in AtHSL2. We feel that this is strong conservation given that the AtHAESA and AtHSL2 ectodomains share 45% overall sequence identity. We have included a statement in our manuscript that reads: “Indeed, we find many of the residues contributing to the formation of the IDA binding surface in HAESA to be conserved in HSL2 and other HAESA-type receptors in different plant species (Figure 1—figure supplement 3).” REVIEW_INFO paragraph 66315 4) The authors show that N-terminal extension of the peptide does not impact on binding efficiency, but what would happen if the peptide was extended at the C-terminal end, at the suggested cleavage site? elife-15075-resp-fig2.jpg fig9 FIG fig_title_caption 66521 Isothermal titration calorimetry thermograph of the C-terminally extended IDA-SFVN peptide (200 μM) titrated into a solution containing 20 μM of the purified HAESA ectodomain. elife-15075-resp-fig2.jpg fig9 FIG fig_caption 66699 No detectable binding is observed. elife-15075-resp-fig2.jpg fig9 FIG fig_caption 66734 DOI: http://dx.doi.org/10.7554/eLife.15075.016 REVIEW_INFO paragraph 66781 Thank you for suggesting this experiment. We synthesized a C-terminally extended version of the IDA peptide (IDA-SFVN with sequence YPIPPSA-Hyp- SKRHN SFVN) and performed quantitative binding assays by ITC. As shown in Author response image 2, we cannot observe any detectable binding of this C-terminally extended peptide to the HAESA ectodomain, consistent with our crystallographic models that suggest that HAESA specifically senses an active IDA 12mer. We have incorporated this new result in Figure 2D. We have included a new statement in the manuscript that reads: “The COO- group of Asn69IDA is in direct contact with Arg407HAESA and Arg409HAESA and HAESA cannot bind a C-terminally extended IDA-SFVN peptide (Figures 1D, F, 2D).” REVIEW_INFO paragraph 67523 Would cleavage be required for recognition? A brief discussion on this point may help. REVIEW_INFO paragraph 67611 Yes. We have modified our manuscript accordingly: “This suggests that the conserved Asn69 may constitute the very C-terminus of the mature IDA peptide in planta and that active IDA is generated by proteolytic processing from a longer pre-protein (Stenvik et al. 2008).“ REVIEW_INFO paragraph 67885 5) Figure 3C: Could the authors comment on the difference between the blue (SERK1 vs. HAESA-IDA) and the black (IDA vs. HAESA-SERK1) line? SERK1 vs. HAESA-IDA gives a Kd of 75 nM, yet IDA vs. SERK1-HAESA gives a Kd of 350 nM. In the text the authors keep referring to the 75 nM Kd, but not the 350 nM Kd. Is it really fair to say the Kd is 75 nM? REVIEW_INFO paragraph 68232 Yes and No. These are two different experiments. We first measured (by ITC) the binding affinity of IDA (in the syringe) binding to a protein solution containing equimolar ratios of HAESA and SERK1 (Kdis 350 nM in this case). Next, we titrated a concentrated SERK1 solution (in the syringe) into a solution of HAESA containing IDA in 10fold molar excess (Kdin this case is 75 nM). Given that the experimental conditions (protein and peptide concentrations and molar ratios between the components) are very different, we feel that the Kd 's obtained by these experiments are in good agreement (4.5 fold difference vs. a 60-260 fold difference when compared to the isolated HAESA ectodomain). Nevertheless, we addressed the reviewer's concern by modifying our manuscript which now states: “In this case, there was no detectable interaction between receptor and co-receptor, while in the presence of IDA, SERK1 strongly binds HAESA with a dissociation constant in the mid-nanomolar range. (Figure 3C).” REVIEW_INFO paragraph 69236 6) In the Discussion, can the authors comment further on the discrepancy between their study and the study of Lewis et al. (Plant J, 2010) concerning the role of SERK1 in floral abscission? REVIEW_INFO paragraph 69427 The process of floral organ abscission in Arabidopsis is divided into distinct steps where a gradual loosening of the cell wall between abscising cells can be measured as a reduction in petal breakstrength (Bleecker and Patterson, 1997). During floral abscission in wild-type plants a significant drop in breakstrength occurs shortly before the petals drop (shown in Figure 3A in our manuscript). Previously reported negative regulators of abscission, such as the transcription factor KNAT1, have an earlier reduction in breakstrength, indicative of early cell wall remodeling (Shi et al. 2011). Our results show that serk1 mutant plants, contrary to knat1 mutants and wild type, have a delay in cell wall loosening and organ separation (Figure 3A) thus positively regulating organ separation during abscission. The weaker phenotype when compared to haesa/hsl2 mutants is likely due to the redundant nature of other SERKs inthe abscission zone (recent work of Meng et al. 2016, cited in the Results and Discussion sections of our manuscript). REVIEW_INFO paragraph 70470 It has previously been reported that mutations in SERK1 can rescue the block in abscission in plants without the functional ADP-ribosylation factor GTPase-activating protein NEVERSHED (NEV) (Lewis et al. 2010). However, as a mutation in SERK1 is not capable of rescuing the ida mutant phenotype (Lewis et al. 2010) and revertant mutants capable of rescuing the abscission defect of ida do not complement nev, it has been suggested that NEV and IDA function in parallel pathways to promote cell separation (Liu 2013). Our work does not rule out a function for SERK1 in such a parallel pathway, we merely report SERK1 can ALSO act as a positive regulator of abscission by interacting with HAESA in an IDA-dependent manner. We do not observe negative regulation of floral abscission using our SERK1 mutant alleles. Based on the available evidence there is thus little to discuss and speculate about the different functions of SERK1 in abscission, as no molecular mechanism for the negative role of SERK1 in this pathway has been reported thus far. We feel that it is beyond the scope of our manuscript to clarify the different roles of SERK1 in the Arabidopsis abscission zone. REVIEW_INFO paragraph 71645 Similarly, could the authors comment on the fact that the LRR-RLP EVD/SOBIR seems to be a negative regulator of the HAESA/HSL2 pathway (Leslie et al., Development, 2010), which seems puzzling given that EVD/SOBIR function is normally restricted to LRR-RLPs (Gust & Felix, Curr Op Plant Biol, 2014). REVIEW_INFO paragraph 71944 We did attempt to express and purify the EVR/SOBIR extracellular domain, but in our hands the protein is not properly secreted and hence unfolded. We thus could not further investigate the potential mechanism of EVR/SOBIR in the HAESA pathway. REVIEW_INFO paragraph 72188 7) Given that the central hydroxyproline in IDA is of such crucial importance for binding, isn't it surprising that IDAΔ69N does not bind to HAESA at all? Wouldn't it be expected that the remaining part of the peptide still binds to HAESA? REVIEW_INFO paragraph 72433 We thank the reviewers for pointing this out to us. Indeed, we find several structural and sequence features in IDA peptide to be important determinants for HAESA binding, namely the correct size of the peptide, the presence of a central Hyp residue and an intact C-terminal Arg-His-Asn motif that is buried in the structure. In the revised manuscript we now provide new experiments (binding of a C-terminal extended IDA peptide to HAESA) that clarifies this point (summarized in revised Figure 2D). We have revised our statement in the Discussion accordingly: “The central Hyp residue in IDA is found buried in the HAESA peptide binding surfaceand thus this post-translational modification may regulateIDA bioactivity.” REVIEW_INFO paragraph 73158 8) Figure 1—figure supplement 2: How is it possible that charged amino acids are involved in hydrophobic interactions? REVIEW_INFO paragraph 73280 We apologize for this confusing statement. It now reads: “A N-terminal Pro-rich motif in IDA makes contacts LRRs 2-6 of the receptor(Figure 1D, Figure 1—figure supplement 2A-C).” REVIEW_INFO paragraph 73465 9) Are the distances shown in the graphical representation of the structures proportional? It would seem that some of the aromatic rings could cause steric hindrance. REVIEW_INFO paragraph 73633 No. The graphical representation are proportional and e.g. Trp218 in the back of the binding pocket is not producing steric clashes with the peptide with the closest distance being 4.5 A. REVIEW_INFO paragraph 73821 10) Why did the authors decide to express HAESA and SERK1 without signal peptide? Would it make a difference for binding of IDA, if they leave the SP on? REVIEW_INFO paragraph 73976 No. We did express both the HAESA and SERK1 ectodomains fused to the signal peptide of human azurocidin, which provides very efficient secretion of LRR proteins in insect cells (Olczak & Olczak, Anal. Biochem., 2006) (see Methods, subsection “Protein Expression and Purification“). Both the native signal peptides for SERK1 and HAESA as well as the azurocidin signal peptide are being recognized and cleaved by the Trichoplusia ni signal peptidase. This results, just like in planta, in a mature receptor/coreceptor ectodomain starting with the first α-helix of the N-terminal capping domain (residues 20 and 24, respectively). Thus, there is no reason to believe that the signal peptide would play a role in IDA sensing. Using our system, we cannot produce HAESA and/or SERK1 ectodomains with an intact signal peptide, as this would impair folding and proper secretion of the recombinant proteins. REVIEW_INFO paragraph 74880 11) Figure 7: Are the homologous regions also the active parts of these peptides? REVIEW_INFO paragraph 74963 Yes. We have included three additional references in the Discussion section of our manuscript, which report the bioactive regions of CLV3/CLE, RGF and PEP peptides shown in Figure 7. The revised section now reads: “Importantly, this motif can also be found in other peptide hormone families (Kondo et al. 2006; Matsuzaki et al. 2010; Tang et al. 2015)(Figure 7). Among these are the CLE peptides regulating stem cell maintenance in the shoot and the root (Clark, Running, and Meyerowitz 1995). It is interesting to note, that CLEs in their mature form are also hydroxyprolinated dodecamers, which bind to a surface area in the BARELY ANY MERISTEM 1 receptor that would correspond to part of the IDA binding cleft in HAESA (Kondo et al. 2006;Ogawa et al. 2008; Shinohara et al. 2012).” REVIEW_INFO paragraph 75752 And could the authors display amino acids numbers on either side of the fragments? REVIEW_INFO paragraph 75836 Yes. We have now included the residues number of each peptide in Figure 7. REVIEW_INFO paragraph 75911 12) Have the authors ever measured dissociation of the peptide from the complex? REVIEW_INFO paragraph 75993 No. We have not performed any biochemical experiment that would allow us to quantify the dissociation of the peptide from the ternary complex. In qualitative terms it is however of note that HAESA-IDA-SERK1 complexes do not dissociate in size exclusion chromatography experiments, even when the peptide is not provided in excess or supplied in the running buffer. REVIEW_INFO paragraph 76357 And in this regard, to what does "highly stable receptor – co-receptor complex" refer/compare to? REVIEW_INFO paragraph 76458 The reviewers are correct, we should not claim that the complex is 'highly stable' if we have not quantified the dissociation rate. The revised sentence reads: “This ligand-induced formation of a receptor – co-receptor complex may allow the HAESA and SERK1 kinase domains to efficiently trans-phosphorylate and activate each other in the cytoplasm.” REVIEW_INFO paragraph 76814 13) Figures 3A and 5C require statistical analyses. REVIEW_INFO paragraph 76866 Thank you for pointing this out to us. The statistical analysis of the petal break-strength assays shown in Figures 3A and 5C has been carried out by Prof. Ludwig A. Hothorn, Institute for Biostatistics, University of Hannover, Germany, whom we have added as an author on our manuscript: REVIEW_INFO paragraph 77154 Statistical analysis for Figure 3A: The statistical analysis is described in the figure legend of Figure 3A; statistical significant changes are indicated by a * in the Figure itself. The revised figure legend reads: “Petal break-strength assays measure the force (expressed in gram equivalents) required to remove the petals from the flower of serk mutant plants compared to haesa/hsl2 mutant and Col-0 wild-type flowers. […] Petal break was found significantly increased in almost all positions (indicated with a *) for haesa/hsl2 and serk1-1 mutant plants with respect to the Col-0 control. Calculations were performed in R (R Core Team 2014) (version 3.2.3).” The two new references have been added to the Reference section of the manuscript. REVIEW_INFO paragraph 77907 We have changed our Results section accordingly: “Our experiments suggest that among the SERK family members, SERK1 is a positive regulator offloral abscission. We found that the force required to remove the petals of serk1-1 mutants is significantlyhigher than that needed for wild-type plants, as previously observed for haesa/hsl2 mutants (Stenvik et al. 2008), and that floral abscission is delayed in serk1-1 (Figure 3A). The serk2-2, serk3-1, serk4-1 and serk5-1 mutant lines (Albrecht et al. 2008) showed a petal break-strength profile not significantly differentfrom wild-type plants.” REVIEW_INFO paragraph 78505 Statistical analysis for Figure 5C: The statistical analysis is described in the figure legend of Figure 5C; statistical significant changes are indicated by * and # symbols in the Figure itself. The revised figure legend reads:”Quantitative petal break assay for Col-0 wild-type flowers and 35S::IDA wild-type and 35S::IDA K66A/R67A mutant flowers. […] Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c).” REVIEW_INFO paragraph 79041 We have changed our Results section accordingly: “We overexpressed full-length wild-type IDA or this Lys66IDA/Arg67IDA → Ala double-mutant to similar levels in Col-0 Arabidopsis plants (Figure 5D). […] Comparison of 35S::IDA wild-type and mutant plants further indicates that mutation of Lys66IDA/Arg67IDA→ Ala may cause a weak dominant negative effect (Figure 5C-E).” REVIEW_INFO paragraph 79420 Following the suggestions from for example Nuzzo (Nature506:150-152, 2014) and Trafirmow and Marks (Basic and Applied Social Psychology37:1-2, 2015), we decided not to report p-values.