The structure of the karrikin-insensitive protein (KAI2) in Arabidopsis thaliana.

KARRIKIN INSENSITIVE 2 (KAI2) is an α/β hydrolase involved in seed germination and seedling development. It is essential for plant responses to karrikins, a class of butenolide compounds derived from burnt plant material that are structurally similar to strigolactone plant hormones. The mechanistic basis for the function of KAI2 in plant development remains unclear. We have determined the crystal structure of Arabidopsis thaliana KAI2 in space groups P2(1) 2(1) 2(1) (a =63.57 Å, b =66.26 Å, c =78.25 Å) and P2(1) (a =50.20 Å, b =56.04 Å, c =52.43 Å, β =116.12°) to 1.55 and 2.11 Å respectively. The catalytic residues are positioned within a large hydrophobic pocket similar to that of DAD2, a protein required for strigolactone response in Petunia hybrida. KAI2 possesses a second solvent-accessible pocket, adjacent to the active site cavity, which offers the possibility of allosteric regulation. The structure of KAI2 is consistent with its designation as a serine hydrolase, as well as previous data implicating the protein in karrikin and strigolactone signalling

Structural modelling and transcriptional responses highlight a clade of PpKAI2-LIKE genes as candidate receptors for strigolactones in Physcomitrella patens

A set of PpKAI2 - LIKE paralogs that may encode strigolactone receptors in Physcomitrella patens were identified through evolutionary, structural, and transcriptional analyses, suggesting that strigolactone perception may have evolved independently in basal land plants in a similar manner as spermatophytes. Carotenoid-derived compounds known as strigolactones are a new class of plant hormones that modulate development and interactions with parasitic plants and arbuscular mycorrhizal fungi. The strigolactone receptor protein DWARF14 (D14) belongs to the alpha/beta hydrolase family. D14 is closely related to KARRIKIN INSENSITIVE2 (KAI2), a receptor of smoke-derived germination stimulants called karrikins. Strigolactone and karrikin structures share a butenolide ring that is necessary for bioactivity. Charophyte algae and basal land plants produce strigolactones that influence their development. However phylogenetic studies suggest that D14 is absent from algae, moss, and liverwort genomes, raising the question of how these basal plants perceive strigolactones. Strigolactone perception during seed germination putatively evolved in parasitic plants through gene duplication and neofunctionalization of KAI2 paralogs. The moss Physcomitrella patens shows an increase in KAI2 gene copy number, similar to parasitic plants. In this study we investigated whether P. patens KAI2-LIKE (PpKAI2L) genes may contribute to strigolactone perception. Based on phylogenetic analyses and homology modelling, we predict that a clade of PpKAI2L proteins have enlarged ligand-binding cavities, similar to D14. We observed that some PpKAI2L genes have transcriptional responses to the synthetic strigolactone GR24 racemate or its enantiomers. These responses were influenced by light and dark conditions. Moreover, (+)-GR24 seems to be the active enantiomer that induces the transcriptional responses of PpKAI2L genes. We hypothesize that members of specific PpKAI2L clades are candidate strigolactone receptors in moss

Evolution of strigolactone receptors by gradual neo-functionalization of KAI2 paralogues.

BackgroundStrigolactones (SLs) are a class of plant hormones that control many aspects of plant growth. The SL signalling mechanism is homologous to that of karrikins (KARs), smoke-derived compounds that stimulate seed germination. In angiosperms, the SL receptor is an α/β-hydrolase known as DWARF14 (D14); its close homologue, KARRIKIN INSENSITIVE2 (KAI2), functions as a KAR receptor and likely recognizes an uncharacterized, endogenous signal ('KL'). Previous phylogenetic analyses have suggested that the KAI2 lineage is ancestral in land plants, and that canonical D14-type SL receptors only arose in seed plants; this is paradoxical, however, as non-vascular plants synthesize and respond to SLs.ResultsWe have used a combination of phylogenetic and structural approaches to re-assess the evolution of the D14/KAI2 family in land plants. We analysed 339 members of the D14/KAI2 family from land plants and charophyte algae. Our phylogenetic analyses show that the divergence between the eu-KAI2 lineage and the DDK (D14/DLK2/KAI2) lineage that includes D14 occurred very early in land plant evolution. We show that eu-KAI2 proteins are highly conserved, and have unique features not found in DDK proteins. Conversely, we show that DDK proteins show considerable sequence and structural variation to each other, and lack clearly definable characteristics. We use homology modelling to show that the earliest members of the DDK lineage structurally resemble KAI2 and that SL receptors in non-seed plants likely do not have D14-like structure. We also show that certain groups of DDK proteins lack the otherwise conserved MORE AXILLARY GROWTH2 (MAX2) interface, and may thus function independently of MAX2, which we show is highly conserved throughout land plant evolution.ConclusionsOur results suggest that D14-like structure is not required for SL perception, and that SL perception has relatively relaxed structural requirements compared to KAI2-mediated signalling. We suggest that SL perception gradually evolved by neo-functionalization within the DDK lineage, and that the transition from KAI2-like to D14-like protein may have been driven by interactions with protein partners, rather than being required for SL perception per se

Evolution of strigolactone receptors by gradual neo-functionalization of KAI2 paralogues

Background: Strigolactones (SLs) are a class of plant hormones that control many aspects of plant growth. The SL signalling mechanism is homologous to that of karrikins (KARs), smoke-derived compounds that stimulate seed germination. In angiosperms, the SL receptor is an α/β-hydrolase known as DWARF14 (D14); its close homologue, KARRIKIN INSENSITIVE2 (KAI2), functions as a KAR receptor and likely recognizes an uncharacterized, endogenous signal (‘KL’). Previous phylogenetic analyses have suggested that the KAI2 lineage is ancestral in land plants, and that canonical D14-type SL receptors only arose in seed plants; this is paradoxical, however, as non-vascular plants synthesize and respond to SLs. Results: We have used a combination of phylogenetic and structural approaches to re-assess the evolution of the D14/KAI2 family in land plants. We analysed 339 members of the D14/KAI2 family from land plants and charophyte algae. Our phylogenetic analyses show that the divergence between the eu-KAI2 lineage and the DDK (D14/DLK2/KAI2) lineage that includes D14 occurred very early in land plant evolution. We show that eu-KAI2 proteins are highly conserved, and have unique features not found in DDK proteins. Conversely, we show that DDK proteins show considerable sequence and structural variation to each other, and lack clearly definable characteristics. We use homology modelling to show that the earliest members of the DDK lineage structurally resemble KAI2 and that SL receptors in non-seed plants likely do not have D14-like structure. We also show that certain groups of DDK proteins lack the otherwise conserved MORE AXILLARY GROWTH2 (MAX2) interface, and may thus function independently of MAX2, which we show is highly conserved throughout land plant evolution. Conclusions: Our results suggest that D14-like structure is not required for SL perception, and that SL perception has relatively relaxed structural requirements compared to KAI2-mediated signalling. We suggest that SL perception gradually evolved by neo-functionalization within the DDK lineage, and that the transition from KAI2-like to D14-like protein may have been driven by interactions with protein partners, rather than being required for SL perception per se.Science, Faculty ofNon UBCBotany, Department ofReviewedFacult

Crystal structure of KAI2.

<p>A. Stereoscopic ribbon diagram of KAI2 coloured from amino (blue) to carboxy (red) terminus. Every twentieth Cα is shown as a labelled sphere. B. Stereoscopic ribbon diagram of all three models of KAI2 (KAI2a blue, KAI2b orange, KAI2c black) and the models of DAD2 (purple) and RsbQ (brown) superposed. C. Stereoscopic cartoon diagram of KAI2. The α/β hydrolase domain is shown in blue and the cap domain shown in red.</p

Comparing KAI2 with DAD2.

<p>The active site cavities of KAI2 (A) and of DAD2 (B). Cavity-exposed phenylalanine and tyrosine residues are shown for both proteins. Tyr124 of KAI2 occludes a small adjacent pocket that is not occluded in DAD2. Other than this difference, the two pockets are similar in size and shape. C. Sequence alignments of KAI2 and DAD2. The <i>A. thaliana</i> KAI2 sequence is coloured by similarity within the KAI2 protein family, the <i>P. hybrida</i> DAD2 sequence is coloured by similarity within the D14 protein family and the consensus sequence was determined and coloured by similarity across both families. Darker green colouration indicates more conserved sequence. Capital letters in the consensus sequence denote a conserved residue across all KAI2 and D14 proteins. Amino acids of interest are those where aligned KAI2 and D14 residues are coloured darker than the corresponding consensus sequence residue.</p

KAI2 crystal.

<p>A multiple KAI2 crystal (32 μm×6 μm×6 μm) that was split for data collection (KAI2b).</p

Data collection and refinement statistics for <i>Arabidopsis thaliana</i> KAI2.

<p>Values in parentheses correspond to the highest resolution shell. ^a Values obtained using MOLPROBITY <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054758#pone.0054758-Chen1" target="_blank">[32]</a>.</p

Preparation and purification of mono-ubiquitinated proteins using Avi-tagged ubiquitin.

Site-specific conjugation of ubiquitin onto a range of DNA repair proteins regulates their critical functions in the DNA damage response. Biochemical and structural characterization of these functions are limited by an absence of tools for the purification of DNA repair proteins in purely the ubiquitinated form. To overcome this barrier, we designed a ubiquitin fusion protein that is N-terminally biotinylated and can be conjugated by E3 RING ligases onto various substrates. Biotin affinity purification of modified proteins, followed by cleavage of the affinity tag leads to release of natively-mono-ubiquitinated substrates. As proof-of-principle, we applied this method to several substrates of mono-ubiquitination in the Fanconi anemia (FA)-BRCA pathway of DNA interstrand crosslink repair. These include the FANCI:FANCD2 complex, the PCNA trimer and BRCA1 modified nucleosomes. This method provides a simple approach to study the role of mono-ubiquitination in DNA repair or any other mono-ubiquitination signaling pathways