Smoke-derived karrikin perception by theα/β-hydrolase KAI2 from ArabidopsisYongxia Guoa,b,1, Zuyu Zhenga,c,1, James J. La Claird,1, Joanne Chorya,c,2, and Joseph P. Noela,b,2

aHoward Hughes Medical Institute, bJack H. Skirball Center for Chemical Biology and Proteomics, and cPlant Biology Laboratory, Salk Institute for BiologicalStudies, La Jolla, CA 92037; and dDepartment of Chemistry and Biochemistry, University of California, San Diego, CA 92093

Contributed by Joanne Chory, April 2, 2013 (sent for review March 18, 2013)

Genetic studies inArabidopsis implicate an α/β-hydrolase, KARRIKIN-INSENSITIVE 2 (KAI2) as a receptor for karrikins, germination-pro-moting butenolide small molecules found in the smoke of burnedplants. However, direct biochemical evidence for the interaction be-tween KAI2 and karrikin and for the mechanism of downstream sig-naling by a KAI2–karrikin complex remain elusive. We reportcrystallographic analyses and ligand-binding experiments for KAI2 rec-ognition of karrikins. The karrikin-1 (KAR1) ligand sits in the opening tothe active site abutting a helical domain insert but distal from thecanonical catalytic triad (Ser95-His246-Asp217) of α/β-hydrolases, con-sistent with the lack of detectable hydrolytic activity by purified KAI2.The closest approach of KAR1 to Ser95-His246-Asp217 is 3.8 Å fromHis246. Six aromatic side chains, including His246, encapsulate KAR1through geometrically defined aromatic–aromatic interactions. KAR1binding induces a conformational change in KAI2 at the active siteentrance. A crevice of hydrophobic residues linking the polar edge ofKAR1 and the helical domain insert suggests that KAI2–KAR1 createsa contiguous interface for binding signaling partners in a ligand-dependent manner.

plant signaling | wildfires | seed dormancy

Wildfires recycle plant environments by releasing nutrientsstored in plant tissues and often stimulate the germination

of quiescent seeds (1). Germination-stimulating compounds, gen-erated in the smoke of burning plants, include a seed dormancy–breaking compound 3-methyl-2H-furo[2,3-c]pyran-2-one, a bute-nolide named karrikinolide-1 or karrikin-1 (KAR1) (2). Previousreports reveal KAR1 structure-activity relationships (SARs) andfeatures of the physiological mode of action (3–7). Karrikins-insensitive (kai) mutants in the model plant Arabidopsis thalianaidentify some of the genetic components underlying karrikins’perception and signaling (8, 9). Seeds of kai2 mutants are in-sensitive to KAR1-induced germination, suggesting that the KAI2gene product is essential for KAR1 perception and/or signaltransduction (8, 9).KAI2, also known as HYPOSENSITIVE TO LIGHT (HTL)

(10), is a member of the α/β-hydrolase superfamily commonlycontaining a catalytic triad, Ser(Cys)-His-Asp (11). Notably, severalother members of the α/β-hydrolase superfamily sense, bind, and/orcatalytically modify phytohormones. For example, the α/β-hydrolaseSALICYLIC ACID-BINDING PROTEIN 2 (SABP2) convertsmethyl salicylate to salicylic acid through its esterase activity. Theproduct of the reaction, salicylic acid, retains relatively high bindingaffinity to SABP2, which inhibits SABP2 catalytic activity and finetunes salicylic acid levels (12). Another α/β-hydrolase familymember GIBBERELLIN-INSENSITIVE DWARF 1 (GID1)lacks the highly conserved catalytic triad, but serves as the re-ceptor of gibberellins (13, 14). These previous studies have ledto speculation that the KAI2 protein might be the receptor ofkarrikins (8, 9). This hypothesis is supported by the recent dis-covery that another α/β-hydrolase family member DECREASEDAPICAL DOMINANCE 2 (DAD2) from petunia (15) [Oryzasativa DWARF 14 (OsD14) from rice (16) and Arabidopsisthaliana DWARF 14 (AtD14) from Arabidopsis (9)], a closestructural homolog of KAI2, is the putative receptor for another

butenolide-containing class of chemicals, the strigolactones. Thisfamily of butenolides regulates several aspects of plant growthand development, including leaf senescence, shoot branching,and root development (15, 17).The chemical structures of karrikins and strigolactones share

a common butenolide moiety (18) (Figs. 1 and 2A), and the F-boxprotein MORE AXILLARY BRANCHES 2 (MAX2) is impli-cated as a downstream target for signaling by both karrikins andstrigolactones (8). Although DAD2 possesses low but measurablehydrolase activity against the potent synthetic strigolactone ana-log GR24 (15), KAI2 lacks detectable hydrolase activity againstkarrikins, GR24, and synthetic α/β-hydrolase substrates includingparanitrophenyl acetate and paranitrophenyl myristate (10). Arecent crystal structure of apo-DAD2 confirmed the evolutionarylinkage within the α/β-hydrolase superfamily but lacked a struc-tural explanation for strigolactone perception (15).To understand the architecture of karrikins’ perception byKAI2

and the evolution of the larger family of plant α/β-hydrolases, wedetermined the atomic resolution crystal structures of A. thalianaapo-KAI2 [Protein Data Bank (PDB) ID code 4JYP] and KAI2’scomplex with KAR1 (PDB ID code 4JYM). In tandem, we verifiedand quantified the energetics of KAR1 binding to WT KAI2 usingboth 1H-15N heteronuclear single-quantum coherence (1H-15NHSQC)NMR spectroscopy and equilibriummicrodialysis. Finally,we measured the energetic contribution of three of the observedKAR1 binding elements by applying site-directed mutagenesis anddetermining the dissociation constants (Kd values) using equilib-rium microdialysis.

Results and DiscussionBinding of KAR1 to KAI2. To verify KAR1 binding by KAI2, weuniformly labeled KAI2 with 15N by expression and purifica-tion from Escherichia coli grown in 15N-enriched media. A 1H-15NHSQC spectrum was first obtained in the absence of KAR1 using1.4 mM KAI2 and then after the addition of the KAR1 ligand toa final concentration of 7 mM. Comparisons of the 1H-15N HSQCspectra revealed clear shifts in select cross peaks of KAI2 whenexposed to KAR1, indicative of KAI2–KAR1 complex formation(Fig. 2B). We next turned to equilibrium microdialysis to supportthis observation and quantify the energy of binding. Mixtures ofKAI2 and KAR1 were equilibrated overnight using conditionsidentical to that used in the NMR studies. Changes in the fluo-rescence emission intensities of KAR1 at 379 nm were measured

Author contributions: Y.G., Z.Z., J.J.L.C., and J.P.N. designed research; Y.G., Z.Z., and J.J.L.C.performed research; Y.G., Z.Z., J.J.L.C., and J.P.N. analyzed data; and Y.G., Z.Z., J.J.L.C., J.C.,and J.P.N. wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.pdb.org (PDB ID codes 4JYM and 4JYP).1Y.G., Z.Z., and J.J.L.C. contributed equally to this work.2To whom correspondence may be addressed. E-mail: chory@salk.edu or noel@salk.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1306265110/-/DCSupplemental.

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and fit to a single binding-site model resulting in a Kd value of9.05 ± 2.03 μM (Fig. 2C).

Architecture of KAR1 Recognition by KAI2. KAI2 purified and crys-tallized as a monomer. The crystal structure of apo-KAI2 wassolved and refined at 1.30 Å by molecular replacement (MR) usinga search model based on the bacterial signaling protein RsbQ(PDB ID code 1WOM) (19). While preparing this manuscript,three independent groups reported apo-KAI2 crystal structures at1.55 Å (20, 21) and 1.15 Å (22) resolution, respectively. We then

solved and refined the structure of the KAI2–KAR1 cocrystalstructure at 1.35 Å resolution. Detailed methods and statistics forcrystal growth and X-ray crystallographic analyses are provided inMethods and Table S1. The core domain consists of a seven-stranded mixed β-sheet and seven α-helices (Fig. 3A). The insertedhelical lid (residues 136–194) resides between strands β6 and β7(Fig. S1). This helical domain forms two parallel layers of V-sha-ped helices that bury an extensive surface area abutting the activesite (Fig. S2). The kai2-1 Arabidopsismutant that is fully insensitiveto KAR1-promoted germination contains a mutation of Gly133 toGlu (9). When modeled on the KAI2 structure reported here,a Glu133 change would likely induce conformational changes inthe buried polypeptide backbone and/or steric clashes with Val219assuming the mutant protein folds properly.KAI2’s catalytic triad, Ser95-His246-Asp217, occupies the end

of a deep active site pocket (Fig. 3B). The putative α/β-hydrolasenucleophile, Ser95, projects from a sharp turn termed the nu-cleophilic elbow (11). Although most of the nucleophilic elbowsof α/β-hydrolases span a conserved Gly-Xaa-Nuc(Ser/Cys)-Xaa-Gly sequence motif (23), the DAD2-KAI2 family stands out witha Gly-His-Ser-Val-Ser elbow. Moreover, the apo-KAI2 structureclosely parallels the previously determined DAD2 structure withan rmsd of 0.8845 Å for alignment of backbone atoms (15).

Recognition of KAR1 by KAI2. The KAR1 ligand sits at the outeredge of the active site distal from the catalytic triad, consistentwith the lack of detectable hydrolase activity by purified KAI2.The closest approach of KAR1 to the catalytic triad is 3.8 Å fromHis246. Six aromatic side chains including the imidazole moietyof His246 and the side chains of Phe26, Tyr124, Phe134, Phe157,and Phe194 encapsulate KAR1 through geometrically definedaromatic–aromatic interactions including edge-to-face, face-to-face, and parallel displaced orientations (24). This hydrophobicpocket includes residues from loops 3, 10, and 18 and from thelid domain helices LαA, LαB, and LαD (Fig. 3 C and D and Fig.S3). KAR1 is enveloped by the side chains of aforementionedaromatic residues and buttressed by the alkyl side chains ofLeu139 and Leu142 (Fig. 3 C and D). The methyl group at the 3position of KAR1 (Fig. 1 and Fig. 2A) points into the active sitein the general direction of the putative nucleophile, Ser95,leaving the oxygen-bearing edge of KAR1 exposed to solvent(Fig. 3B). Finally, the bicyclic butenolide ring system of KAR1

Fig. 1. Chemical structures of karrikins and synthetic strigolactone analogGR24. Karrikin activities were evaluated using Solanum orbiculatum and halfmaximal effective concentration (EC50) values are shown in parentheses (18).The standard atom numbers for KAR1 and GR24 are colored blue; thebutenolide ring of KAR1 is colored red. The butenolide rings of GR24, C andD, are colored green and pink, respectively.

Fig. 2. Binding of KAR1 to KAI2. (A) The chemical structure of KAR1 and the KAI2-bound KAR1 ligand as color-coded sticks, including a simulated annealingomit 2Fo-Fc electron-density map contoured at 0.7 σ and calculated at 1.35 Å resolution. (B) KAI2 chemical-shift perturbations induced by the addition ofKAR1. The

1H-15N HSQC spectra of 1.4 mM KAI2 were recorded in the absence (blue) and presence (orange) of 7 mM KAR1. Arrows denote shifted 1H-15N crosspeaks associated with KAR1 binding. (C) KAR1 fluorescence-based binding assay for KAR1 ligand recognition by KAI2. A single binding site model was assumedfor the KAI2–KAR1 interaction and fit as described in Methods.

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forms parallel stacking interactions with the aromatic rings ofPhe134 and Phe194 (Fig. 3 C and D and Fig. 4B).

KAR1 Binding Induces a KAI2 Conformational Change. Comparisonof apo-KAI2 to the KAI2–KAR1 complex shows that Gln141,Arg147, Ser148, Lys151, Met166, Leu169, Glu173, Arg176,Phe194, Lys216, and Leu218 undergo conformational shifts oftheir side chains upon KAR1 binding (Fig. 4A). The most pro-nounced conformational change occurs for Phe194, which abutsthe helical lid domain and forms part of the aromatic pairsandwiching KAR1 at the active site entrance (Fig. 4B). KAR1

and Phe194 form part of an extended crevice spanning the activesite entrance and the KAI2 helical domain. The exposed polarrim of KAR1 is devoid of hydrogen-bonding interactions, sug-gesting it forms part of a larger interaction surface for a pro-tein partner.

Energetic Contribution of KAI2 Residues to KAR1 Recognition. Wenext replaced three of the most direct KAR1-binding residues,Phe134, Phe194, and His246, with Ala residues by site-directedmutagenesis. Again using equilibrium microdialysis, we measuredchanges in the energy of protein-ligand binding upon partial dis-ruption of the KAR1-binding site of KAI2 by single-site mutants.All three mutant proteins were expressed and purified to homo-geneity. Although indistinguishable from WT KAI2 in terms of

solubility and overall stability, all three mutants displayed mea-surable losses in affinity for KAR1 (Figs. S4–S6).His246 is well outside the expected hydrogen-bonding distance

to KAR1, but, as mentioned, forms part of an extensive networkof aromatic–aromatic interactions with KAR1 and the KAI2-binding site (Fig. 3 B–D). Binding studies demonstrated athreefold loss in His246Ala KAI2 affinity for KAR1 with a Kdvalue of 35.5 ± 9.68 μM (Fig. S4). The aromatic rings of Phe134and Phe194 form parallel stacking interactions with the bicyclicbutenolide ring system of KAR1. Equilibrium microdialysis dem-onstrated fivefold losses in KAI2 affinity for KAR1 for each mu-tant, with Kd values of 45.5 ± 11.2 μM and 50.9 ± 11.1 μM for thePhe134Ala and Phe194Ala mutants, respectively (Figs. S5 and S6).

Adaptation of the α/β-Hydrolase Fold for Butenolide Perception inPlants. Based on the crystallographic and binding studies pre-sented here, KAI2’s active site does not appear to be used cata-lytically for the karrikins. Instead, His246, which is one componentof the canonical catalytic triad of α/β-hydrolases, recognizes theplanar face of KAR1 using an aromatic–aromatic edge-to-facearrangement (24). Moreover, other identified karrikins includingKAR2–6 (Fig. 1) are readily superimposed on KAR1 without anyconcomitant steric clashes with KAI2.Curiously, although DAD2, implicated in strigolactone signaling,

andKAI2, associated with karrikin signaling, share homologous 3Dstructures and bind molecules possessing a common butenolide

Fig. 3. Three-dimensional X-ray crystal structures of apo-KAI2 and the KAI2–KAR1 complex. (A) The ribbon representation of the overall structure of KAI2.KAI2 contains a typical α/β-hydrolase fold. The core domain of KAI2 is a six-stranded parallel β-sheet (cyan) surrounded by seven α-helices (gray). The insertedlid domain is colored orange and the corresponding α-helix, β-sheet, and loop secondary structures are labeled. (B) KAI2 contains a canonical α/β-hydrolasecatalytic triad. Ser95, Asp217, and His246 are depicted as sticks in which carbon is colored salmon, nitrogen is blue, and oxygen is red. KAR1 is shown as sticksand carbon is yellow. The distances from Asp217 to His246, His246 to Ser95, and His246 to KAR1 are shown. (C) Close-up view highlighting the KAR1-bindingsite. Ribbon diagram of α-helices (gray) and β-sheet (cyan) are shown, together with part of the inserted helical lid (orange). KAR1 contacting residues areshown as color-coded sticks in which carbon is cyan, nitrogen is blue, and oxygen is red. KAR1 carbon atoms are colored yellow. (D) 180° rotation of the KAI2–KAR1 complex around a vertical axis relative to C.

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moiety, DAD2 recognizes amuch larger ligand.When theGR24C-ring is superimposed on the equivalent moiety of KAR1 withmaximal overlap of common atoms, the chemically reactive stri-golactone projects into the active site with minimal steric clashesand is juxtaposed next to the conserved catalytic triad (Fig. 1 andFig. 5A). However, when the GR24 C-ring is superimposed on the

butenolide moiety of KAR1 but flipped 180° around an axis passingthrough the carbonyl moieties, the D-ring of GR24 clashes with thelid domain helix LαB and side chains of Trp153 and Phe157 (Fig. 1and Fig. 5B). Superimposing GR24’s D-ring on KAR1’s butenolidering with maximal overlap of common atoms results in the C-ring ofGR24 in close steric contact to Leu142 in the ligand-binding pocketof KAI2 (Fig. 1 and Fig. 5C). Finally, superimposingGR24’s D-ringon the butenolide moiety of KAR1 but flipped 180° around anaxis passing through the carbonyl moieties of each results in the A,B, and C rings of GR24 in close steric contact to Tyr124 (Fig. 1 andFig. 5D).Fig. 5 A and D are most consistent with a cleavage reaction

catalyzed by DAD2 on strigolactones given the proximity of thelarger strigolactone with the homologous catalytic machinery ofKAI2 and DAD2. Nevertheless, DAD2 (PDB ID code 4DNP)maintains slightly larger access to the catalytic machinery. ForDAD2, the backbone alpha carbons of Val143 and His218,located on opposite sides of the active site periphery, are sepa-rated by 8.02 Å, whereas the equivalent residues in KAI2, Leu142,and Leu218, are separated by 6.56 Å. Overall, DAD2 and KAI2maintain highly similar 3D structures with a rmsd of 0.8845 Å foralignment of equivalent backbone atoms. These observationssuggest that DAD2 cleaves the larger strigolactone to inducea signaling event, whereas KAI2 relies on fire to spark formationof the core butenolide trigger for awakening seeds for germina-tion. Notably, the conserved architecture of KAI2 and DAD2,together with the binding of small molecules containing a com-mon butenolidemoiety, suggests that karrikin perception is a morerecent selective event rooted in more ancient recognition of stri-golactones by ancestors of the contemporary DAD2-KAI2 clade.

ConclusionThe combined use of protein X-ray crystallography, site-directedmutagenesis, and energetic binding measurements provides astructural basis for placing KAI2 within the signal transductionpathway associated with the perception of smoke-derived chem-ical signals such as KAR1. Notably, KAR1 and Phe194 form anexposed and central structural element that brings togethera largely hydrophobic crevice spanning the active site entranceand the KAI2 helical domain. Unexpectedly, the exposed edge ofKAR1 lacks obvious hydrogen-bonding interactions in the staticcrystal structures. First, this observation implies that the exposedperiphery of KAR1 serves as a binding interface for KAI2-sig-naling partners. Second, KAI2’s solvent-accessible, aromatic-rich,hydrophobic patch surrounding KAR1 and harboring KAR1-in-duced conformational switches extends this ligand-dependentinterface. Finally, the helical domain insert into the core α/β-hydrolase fold and abutting these two features is a third likelybinding unit. These three components of the KAI2–KAR1 com-plex appear poised to modulate interactions of KAI2 with down-stream components of the karrikin-signaling pathway in a ligand-dependent manner.

MethodsProtein Expression and Purification. The KAI2 gene was amplified from anA. thaliana cDNA library and inserted between the NcoI and BamHI sites ofthe expression vector pHIS8 encoding an N-terminal His8-tag and thrombincleavage site for tag removal. E. coli BL21 (DE3) cells harboring the KAI2expression vector were grown at 37 °C in Terrific broth (Invitrogen) until theD600nm reached ∼1.0. Isopropyl-β-D-thiogalactoside (0.5 mM) was added toinduce protein expression under control of T7 RNA polymerase regulated bya modified lac promoter. Cells were grown overnight at 18 °C and harvestedby centrifugation. Cell pellets were resuspended in lysis buffer [50 mMTris·HCl (pH 8.0), 500 mM NaCl, 20 mM imidazole, 1% (vol/vol) Tween 20,10% (vol/vol) glycerol, and 20 mM 2-mercaptoethanol] and lysed by soni-cation. The soluble lysates were passed through a Ni2+-nitrilotriacetic acid(NTA) agarose column and eluted with lysis buffer supplemented with 250mM imidazole (pH 8.0). The N-terminal His8 tag was removed by treatmentwith thrombin during overnight dialysis in 50 mM Tris·HCl (pH 8.0), 500 mM

Fig. 4. Conformational changes in KAI2 induced by KAR1. (A) Transparentaccessible surface area of the KAI2–KAR1 complex highlighting a hydrophobicconcave surface linking the helical lid domain and the KAI2 active site en-trance. Side chain rearrangements of helical lid domain residues are shown ascolor-coded sticks with nitrogen as blue, oxygen as red, sulfur as yellow, andcarbon as salmon (apo-KAI2) or purple (KAI2–KAR1 complex). The transparentlid domain–accessible surface is colored pink. KAR1 is shown as color-codedsticks with carbon yellow and oxygen red. (B) Close-up ribbon diagrams of theKAR1-binding site depicting KAI2 in the absence (teal) and presence (purple)of KAR1 superimposed. Phe134 and Phe194 are shown as teal (apo) or purple(complex) sticks. KAR1 carbons are yellow and oxygen is red.

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NaCl, and 20 mM 2-mercaptoethanol. Thrombin and uncleaved His8-KAI2were removed by passing over benzamidine Sepharose then Ni2+-NTA aga-rose. Full-length KAI2 was further purified by size exclusion chromatographyon a Superdex 75 HR26/60 column (Pharmacia Biosystems), dialyzed intocrystallization buffer [12.5 mM Tris·HCl (pH 8.0), 50 mM NaCl, and 2 mM DTT]and concentrated to 12 mg·mL−1.

Protein Crystallization and X-Ray Data Collection. Crystals of KAI2 were grownby vapor diffusion at 4 °C from 1:1 mixtures of protein solution (12 mg·mL−1)and reservoir solution. The reservoir contained 22% (wt/vol) polyethyleneglycol 8000, 0.1 M Na+-Pipes (pH 6.5), 0.3 M NaBr, and 2 mM DTT. Crystalgrowth occurred over 2–3 d and was improved by streak seeding. For coc-rystallization with the KAR1 ligand, the protein solutions included 5 mMKAR1 (Toronto Research Chemical) diluted from a 100 mM stock solution in100% methanol. Crystals were flash frozen by immersion in liquid nitrogenfollowing 10 s incubations in a cryoprotectant solution consisting of reservoirsolution supplemented with 17% (vol/vol) ethylene glycol. Single-wavelengthdata (λ = 1.0 Å) were collected at beamline 8.2.1 of the Advanced Light Source(Lawrence Berkeley National Laboratory) using an ADSC Quantum 315CCD detector.

Data Processing, Structure Determination, and Refinement. The observed re-flections were indexed, integrated, and scaled using iMosflm (25) and SCALA(26) in the CCP4 suite (27). Initial crystallographic phases for the apo-KAI2 structure were determined using MR in Phenix (28). The starting searchmodel for Phenix MR was a homology model built for a monomer of KAI2,based on a sequence alignment and structure of RsbQ (PDB ID code 1WOM)rendered using Modeler (29). Coot (30) was used for visualization of electrondensity maps and manual rebuilding of atomic models. Positional and

isotropic B-factor refinement was performed with Phenix (28). A refinedmodel of the KAI2 monomer served as a search model for MR for the KAI2–KAR1 complex. The final structures were evaluated with PROCHECK (31). Theapo-KAI2 structure had 93.2%, 6.4%, and 0.4% of residues in the most fa-vored, additional allowed, and generously allowed regions of the Ram-achandran plot, respectively, with no residues in the disallowed region. TheKAI2–KAR1 structure had 92.9%, 6.6%, and 0.4% of residues in the mostfavored, additional allowed, and generously allowed regions of the Ram-achandran plot, respectively, with no residues in the disallowed region.Structural superpositions were calculated with SSM (32). All structure figureswere prepared and rendered using PyMOL (DeLano Scientific; The PyMOLMolecular Graphics System, Version 1.5.0.4 Schrödinger, LLC.).

1H-15N HSQC NMR Binding Study. For NMR measurements, 15N-labeled KAI2was produced in M9 minimal media containing 0.1% (wt/vol) 15NH4Cl. Thestable isotope–labeled protein was purified by the method described. BeforeNMR analysis, KAI2 at 1.4 mM was buffer-exchanged into 5 mM Tris·HCl (pH8.0), 20 mM NaCl, and 0.5 mM DTT containing 10% (vol/vol) D2O. 1H-15NHSQC spectra were collected on a Varian VS800 NMR machine with watersuppression. Spectra were collected at 25 °C on a sample of apo-KAI2 overa period of 3–4 h. Data collection was stopped and KAR1 ligand dissolved in100% DMSO was added to 7 mM and 3.5% (vol/vol) DMSO final concen-trations. KAI2–KAR1 mixtures were incubated at 25 °C for 1.5 h or 4 °Cfor 24 h before recollection of the 1H-15N HSQC spectra over a period of 3–4 h. Spectra were obtained for multiple samples, during which time KAI2remained stable.

Equilibrium Dialysis. Equilibrium microdialysis coupled to KAR1 fluorescenceintensity detection was used tomeasure the Kd values for binding of KAR1 to

Fig. 5. Docking of strigolactone GR24 on KAI2-bound KAR1. (A) GR24’s C-ring superimposed on KAR1’s butenolide ring (KAR1 atoms 1, 2, 3, 3a, and 7a matchedto GR24 atoms 1, 2, 3, 3a, and 8b, respectively). (B) GR24’s C-ring superimposed on KAR1’s butenolide ring (KAR1 atoms 1, 2, 3, 3a, and 7a matched to GR24atoms 3, 2, 1, 8b, and 3a, respectively). (C) GR24’s D-ring superimposed on KAR1’s butenolide ring (KAR1 atoms 1, 2, 3, 3a, and 7a matched to GR24 atoms 1′, 5′,4′, 3′, and 2′, respectively). (D) GR24’s D-ring superimposed on KAR1’s butenolide ring (KAR1 atoms 1, 2, 3, 3a, and 7a matched to GR24 atoms 4′, 5′, 1′, 2′, and 3′,respectively). Close-up ribbon diagrams are shown with the inserted helical domain colored orange. GR24 is shown as van der Waals spheres with oxygen redand carbons colored green and pink for the C and D rings, respectively. Ser95, Asp217, and His246 are depicted as sticks in which carbon is cyan, nitrogen is blue,and oxygen is red. Tyr124, Leu142, Trp153, and Phe157 are depicted as sticks in which carbon is orange, oxygen is red, and nitrogen is blue.

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WT and mutant KAI2 proteins. In a standard equilibrium dialysis assay, twochambers (ligand chamber and protein chamber) are separated by a dialysismembrane preventing protein equilibration. At equilibrium, the ligandconcentration in the ligand chamber is reduced by the total amount of li-gand, bound and free, in the protein chamber. For this study, we used dis-posable DispoEquilibriumDialyzers with a 5 kDaMWCOmembrane (HarvardApparatus). Briefly, a 50-μL aliquot of 10 μM KAI2 (or KAI2 mutant) wasplaced into the protein chambers of the dialyzer and 50 μL of a given con-centration of KAR1 placed into the ligand chambers. Total concentrations ofKAR1 were varied as follows: 0.1–60 μM (WT KAI2); 1–200 μM (Phe194AlaKAI2 mutant and Phe134Ala mutant); and 0.25–100 μM (His246Ala KAI2mutant). The DispoEquilibrium Dialyzers were placed on a shaker at 23 °Cand moderately agitated overnight to reach equilibrium. Once at equilib-rium, the samples from substrate chambers, which contained unbound KAR1,were collected by centrifugation and fluorescence intensities were mea-sured using a Sapphire fluorescence plate reader (Tecan). The concentrationof free KAR1 was determined by fluorescence spectroscopy using an excita-tion wavelength of 260 nm and monitoring emission intensities at 379 nm.Three replicates were collected for each concentration. The mean change influorescence intensity was determined by subtracting the observed fluo-rescence intensity from the equilibrated ligand chamber from half the initialKAR1 fluorescence for each chamber and plotted against the total KAR1

concentrations. The points were then fit by nonlinear regression usingGraphpad Prism 5 (GraphPad Software, Inc.) to Y = (Bmax × X) × (Kd + X)−1,where Y is the mean change in fluorescent intensity, Bmax is maximum spe-cific binding in fluorescent intensity, X is the concentration of KAR1, and Kd

value is measured in micromolar (μM). Graphpad Prism uses the method oflinear descent in early iterations and then gradually switches to the Gauss-Newton approach to find the values of Bmax and Kd that fit the data best(Levenberg-Marquardt method) (33).

ACKNOWLEDGMENTS. We thank Dr. Xuemei Huang for assisting with theNMR studies, Dr. Gordon Louie for critical reading and comments on themanuscript, Dr. Zheng Xiang and members of the J.P.N. laboratory fordiscussion, and the staff at Advanced Light Source for assistance with X-raydata collection. This work was supported by National Institutes of Health(NIH) Grants 5R01GM52413 and GM094428 (to J.C.), the National ScienceFoundation under Awards EEC-0813570 and MCB-0645794 (to J.P.N.), andthe Howard Hughes Medical Institute (Z.Z., J.C., and J.P.N.). Portions of thisresearch were conducted at Advanced Light Source, a national user facilityoperated by Lawrence Berkeley National Laboratory, on behalf of the USDepartment of Energy, Office of Basic Energy Sciences. The Berkeley Centerfor Structural Biology is supported in part by the Department of Energy,Office of Biological and Environmental Research and by NIH, NationalInstitute of General Medical Sciences.

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