Smoke-derived karrikin perception by the α/β-hydrolase KAI2 from Arabidopsis

Abstract

Genetic studies in Arabidopsis implicate an α/β-hydrolase, KARRIKIN-INSENSITIVE 2 (KAI2) as a receptor for karrikins, germination-promoting butenolide small molecules found in the smoke of burned plants. However, direct biochemical evidence for the interaction between KAI2 and karrikin and for the mechanism of downstream signaling by a KAI2–karrikin complex remain elusive. We report crystallographic analyses and ligand-binding experiments for KAI2 recognition of karrikins. The karrikin-1 (KAR₁) ligand sits in the opening to the active site abutting a helical domain insert but distal from the canonical catalytic triad (Ser95-His246-Asp217) of α/β-hydrolases, consistent with the lack of detectable hydrolytic activity by purified KAI2. The closest approach of KAR₁ to Ser95-His246-Asp217 is 3.8 Å from His246. Six aromatic side chains, including His246, encapsulate KAR₁ through geometrically defined aromatic–aromatic interactions. KAR₁ binding induces a conformational change in KAI2 at the active site entrance. A crevice of hydrophobic residues linking the polar edge of KAR₁ and the helical domain insert suggests that KAI2–KAR₁ creates a contiguous interface for binding signaling partners in a ligand-dependent manner.

Wildfires recycle plant environments by releasing nutrients stored in plant tissues and often stimulate the germination of quiescent seeds (1). Germination-stimulating compounds, generated in the smoke of burning plants, include a seed dormancy–breaking compound 3-methyl-2H-furo[2,3-c]pyran-2-one, a butenolide named karrikinolide-1 or karrikin-1 (KAR₁) (2). Previous reports reveal KAR₁ structure-activity relationships (SARs) and features of the physiological mode of action (3–7). Karrikins-insensitive (kai) mutants in the model plant Arabidopsis thaliana identify some of the genetic components underlying karrikins’ perception and signaling (8, 9). Seeds of kai2 mutants are insensitive to KAR₁-induced germination, suggesting that the KAI2 gene product is essential for KAR₁ perception and/or signal transduction (8, 9).

KAI2, also known as HYPOSENSITIVE TO LIGHT (HTL) (10), is a member of the α/β-hydrolase superfamily commonly containing a catalytic triad, Ser(Cys)-His-Asp (11). Notably, several other members of the α/β-hydrolase superfamily sense, bind, and/or catalytically modify phytohormones. For example, the α/β-hydrolase SALICYLIC ACID-BINDING PROTEIN 2 (SABP2) converts methyl salicylate to salicylic acid through its esterase activity. The product of the reaction, salicylic acid, retains relatively high binding affinity to SABP2, which inhibits SABP2 catalytic activity and fine tunes salicylic acid levels (12). Another α/β-hydrolase family member GIBBERELLIN-INSENSITIVE DWARF 1 (GID1) lacks the highly conserved catalytic triad, but serves as the receptor of gibberellins (13, 14). These previous studies have led to speculation that the KAI2 protein might be the receptor of karrikins (8, 9). This hypothesis is supported by the recent discovery that another α/β-hydrolase family member DECREASED APICAL DOMINANCE 2 (DAD2) from petunia (15) [Oryza sativa DWARF 14 (OsD14) from rice (16) and Arabidopsis thaliana DWARF 14 (AtD14) from Arabidopsis (9)], a close structural homolog of KAI2, is the putative receptor for another butenolide-containing class of chemicals, the strigolactones. This family of butenolides regulates several aspects of plant growth and 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-box protein MORE AXILLARY BRANCHES 2 (MAX2) is implicated as a downstream target for signaling by both karrikins and strigolactones (8). Although DAD2 possesses low but measurable hydrolase activity against the potent synthetic strigolactone analog GR24 (15), KAI2 lacks detectable hydrolase activity against karrikins, GR24, and synthetic α/β-hydrolase substrates including paranitrophenyl acetate and paranitrophenyl myristate (10). A recent crystal structure of apo-DAD2 confirmed the evolutionary linkage within the α/β-hydrolase superfamily but lacked a structural explanation for strigolactone perception (15).

Fig. 1.

Chemical structures of karrikins and synthetic strigolactone analog GR24. Karrikin activities were evaluated using Solanum orbiculatum and half maximal effective concentration (EC₅₀) values are shown in parentheses (18). The standard atom numbers for KAR₁ and GR24 are colored blue; the butenolide ring of KAR₁ is colored red. The butenolide rings of GR24, C and D, 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 annealing omit 2F_o-F_c electron-density map contoured at 0.7 σ and calculated at 1.35 Å resolution. (B) KAI2 chemical-shift perturbations induced by the addition of KAR₁. 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 cross peaks associated with KAR₁ binding. (C) KAR₁ fluorescence-based binding assay for KAR₁ ligand recognition by KAI2. A single binding site model was assumed for the KAI2–KAR₁ interaction and fit as described in Methods.

To understand the architecture of karrikins’ perception by KAI2 and the evolution of the larger family of plant α/β-hydrolases, we determined the atomic resolution crystal structures of A. thaliana apo-KAI2 [Protein Data Bank (PDB) ID code 4JYP] and KAI2’s complex with KAR₁ (PDB ID code 4JYM). In tandem, we verified and quantified the energetics of KAR₁ binding to WT KAI2 using both ¹H-¹⁵N heteronuclear single-quantum coherence (¹H-¹⁵N HSQC) NMR spectroscopy and equilibrium microdialysis. Finally, we measured the energetic contribution of three of the observed KAR₁ binding elements by applying site-directed mutagenesis and determining the dissociation constants (K_d values) using equilibrium microdialysis.

Results and Discussion

Binding of KAR₁ to KAI2.

To verify KAR₁ binding by KAI2, we uniformly labeled KAI2 with ¹⁵N by expression and purification from Escherichia coli grown in ¹⁵N-enriched media. A ¹H-¹⁵N HSQC spectrum was first obtained in the absence of KAR₁ using 1.4 mM KAI2 and then after the addition of the KAR₁ ligand to a final concentration of 7 mM. Comparisons of the ¹H-¹⁵N HSQC spectra revealed clear shifts in select cross peaks of KAI2 when exposed to KAR₁, indicative of KAI2–KAR₁ complex formation (Fig. 2B). We next turned to equilibrium microdialysis to support this observation and quantify the energy of binding. Mixtures of KAI2 and KAR₁ were equilibrated overnight using conditions identical to that used in the NMR studies. Changes in the fluorescence emission intensities of KAR₁ at 379 nm were measured and fit to a single binding-site model resulting in a K_d value of 9.05 ± 2.03 µM (Fig. 2C).

Architecture of KAR₁ Recognition by KAI2.

KAI2 purified and crystallized as a monomer. The crystal structure of apo-KAI2 was solved and refined at 1.30 Å by molecular replacement (MR) using a 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 at 1.55 Å (20, 21) and 1.15 Å (22) resolution, respectively. We then solved and refined the structure of the KAI2–KAR₁ cocrystal structure at 1.35 Å resolution. Detailed methods and statistics for crystal growth and X-ray crystallographic analyses are provided in Methods and Table S1. The core domain consists of a seven-stranded mixed β-sheet and seven α-helices (Fig. 3A). The inserted helical lid (residues 136–194) resides between strands β6 and β7 (Fig. S1). This helical domain forms two parallel layers of V-shaped helices that bury an extensive surface area abutting the active site (Fig. S2). The kai2-1 Arabidopsis mutant that is fully insensitive to KAR₁-promoted germination contains a mutation of Gly133 to Glu (9). When modeled on the KAI2 structure reported here, a Glu133 change would likely induce conformational changes in the buried polypeptide backbone and/or steric clashes with Val219 assuming the mutant protein folds properly.

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 inserted lid domain is colored orange and the corresponding α-helix, β-sheet, and loop secondary structures are labeled. (B) KAI2 contains a canonical α/β-hydrolase catalytic 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 sticks and 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-binding site. Ribbon diagram of α-helices (gray) and β-sheet (cyan) are shown, together with part of the inserted helical lid (orange). KAR₁ contacting residues are shown 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.

KAI2’s catalytic triad, Ser95-His246-Asp217, occupies the end of a deep active site pocket (Fig. 3B). The putative α/β-hydrolase nucleophile, Ser95, projects from a sharp turn termed the nucleophilic elbow (11). Although most of the nucleophilic elbows of α/β-hydrolases span a conserved Gly-Xaa-Nuc(Ser/Cys)-Xaa-Gly sequence motif (23), the DAD2-KAI2 family stands out with a Gly-His-Ser-Val-Ser elbow. Moreover, the apo-KAI2 structure closely parallels the previously determined DAD2 structure with an rmsd of 0.8845 Å for alignment of backbone atoms (15).

Recognition of KAR₁ by KAI2.

The KAR₁ ligand sits at the outer edge of the active site distal from the catalytic triad, consistent with the lack of detectable hydrolase activity by purified KAI2. The closest approach of KAR₁ to the catalytic triad is 3.8 Å from His246. Six aromatic side chains including the imidazole moiety of His246 and the side chains of Phe26, Tyr124, Phe134, Phe157, and Phe194 encapsulate KAR₁ through geometrically defined aromatic–aromatic interactions including edge-to-face, face-to-face, and parallel displaced orientations (24). This hydrophobic pocket includes residues from loops 3, 10, and 18 and from the lid domain helices LαA, LαB, and LαD (Fig. 3 C and D and Fig. S3). KAR₁ is enveloped by the side chains of aforementioned aromatic residues and buttressed by the alkyl side chains of Leu139 and Leu142 (Fig. 3 C and D). The methyl group at the 3 position of KAR₁ (Fig. 1 and Fig. 2A) points into the active site in 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 forms parallel stacking interactions with the aromatic rings of Phe134 and Phe194 (Fig. 3 C and D and Fig. 4B).

Fig. 4.

Conformational changes in KAI2 induced by KAR₁. (A) Transparent accessible surface area of the KAI2–KAR₁ complex highlighting a hydrophobic concave surface linking the helical lid domain and the KAI2 active site entrance. Side chain rearrangements of helical lid domain residues are shown as color-coded sticks with nitrogen as blue, oxygen as red, sulfur as yellow, and carbon as salmon (apo-KAI2) or purple (KAI2–KAR₁ complex). The transparent lid domain–accessible surface is colored pink. KAR₁ is shown as color-coded sticks with carbon yellow and oxygen red. (B) Close-up ribbon diagrams of the KAR₁-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.

KAR₁ Binding Induces a KAI2 Conformational Change.

Comparison of apo-KAI2 to the KAI2–KAR₁ complex shows that Gln141, Arg147, Ser148, Lys151, Met166, Leu169, Glu173, Arg176, Phe194, Lys216, and Leu218 undergo conformational shifts of their side chains upon KAR₁ binding (Fig. 4A). The most pronounced conformational change occurs for Phe194, which abuts the helical lid domain and forms part of the aromatic pair sandwiching KAR₁ at the active site entrance (Fig. 4B). KAR₁ and Phe194 form part of an extended crevice spanning the active site entrance and the KAI2 helical domain. The exposed polar rim of KAR₁ is devoid of hydrogen-bonding interactions, suggesting it forms part of a larger interaction surface for a protein partner.

Energetic Contribution of KAI2 Residues to KAR₁ Recognition.

We next replaced three of the most direct KAR₁-binding residues, Phe134, Phe194, and His246, with Ala residues by site-directed mutagenesis. Again using equilibrium microdialysis, we measured changes in the energy of protein-ligand binding upon partial disruption of the KAR₁-binding site of KAI2 by single-site mutants. All three mutant proteins were expressed and purified to homogeneity. Although indistinguishable from WT KAI2 in terms of solubility and overall stability, all three mutants displayed measurable 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 network of aromatic–aromatic interactions with KAR₁ and the KAI2-binding site (Fig. 3 B–D). Binding studies demonstrated a threefold loss in His246Ala KAI2 affinity for KAR₁ with a K_d value of 35.5 ± 9.68 µM (Fig. S4). The aromatic rings of Phe134 and Phe194 form parallel stacking interactions with the bicyclic butenolide ring system of KAR₁. Equilibrium microdialysis demonstrated fivefold losses in KAI2 affinity for KAR₁ for each mutant, with K_d values of 45.5 ± 11.2 µM and 50.9 ± 11.1 µM for the Phe134Ala and Phe194Ala mutants, respectively (Figs. S5 and S6).

Adaptation of the α/β-Hydrolase Fold for Butenolide Perception in Plants.

Based on the crystallographic and binding studies presented here, KAI2’s active site does not appear to be used catalytically for the karrikins. Instead, His246, which is one component of the canonical catalytic triad of α/β-hydrolases, recognizes the planar face of KAR₁ using an aromatic–aromatic edge-to-face arrangement (24). Moreover, other identified karrikins including KAR_2–6 (Fig. 1) are readily superimposed on KAR₁ without any concomitant steric clashes with KAI2.

Curiously, although DAD2, implicated in strigolactone signaling, and KAI2, associated with karrikin signaling, share homologous 3D structures and bind molecules possessing a common butenolide moiety, DAD2 recognizes a much larger ligand. When the GR24 C-ring is superimposed on the equivalent moiety of KAR₁ with maximal overlap of common atoms, the chemically reactive strigolactone projects into the active site with minimal steric clashes and is juxtaposed next to the conserved catalytic triad (Fig. 1 and Fig. 5A). However, when the GR24 C-ring is superimposed on the butenolide moiety of KAR₁ but flipped 180° around an axis passing through the carbonyl moieties, the D-ring of GR24 clashes with the lid domain helix LαB and side chains of Trp153 and Phe157 (Fig. 1 and Fig. 5B). Superimposing GR24’s D-ring on KAR₁’s butenolide ring with maximal overlap of common atoms results in the C-ring of GR24 in close steric contact to Leu142 in the ligand-binding pocket of KAI2 (Fig. 1 and Fig. 5C). Finally, superimposing GR24’s D-ring on the butenolide moiety of KAR₁ but flipped 180° around an axis 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 and Fig. 5D).

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 matched to 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 GR24 atoms 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 red and 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.

Fig. 5 A and D are most consistent with a cleavage reaction catalyzed by DAD2 on strigolactones given the proximity of the larger strigolactone with the homologous catalytic machinery of KAI2 and DAD2. Nevertheless, DAD2 (PDB ID code 4DNP) maintains slightly larger access to the catalytic machinery. For DAD2, the backbone alpha carbons of Val143 and His218, located on opposite sides of the active site periphery, are separated by 8.02 Å, whereas the equivalent residues in KAI2, Leu142, and Leu218, are separated by 6.56 Å. Overall, DAD2 and KAI2 maintain highly similar 3D structures with a rmsd of 0.8845 Å for alignment of equivalent backbone atoms. These observations suggest that DAD2 cleaves the larger strigolactone to induce a signaling event, whereas KAI2 relies on fire to spark formation of the core butenolide trigger for awakening seeds for germination. Notably, the conserved architecture of KAI2 and DAD2, together with the binding of small molecules containing a common butenolide moiety, suggests that karrikin perception is a more recent selective event rooted in more ancient recognition of strigolactones by ancestors of the contemporary DAD2-KAI2 clade.

Conclusion

The combined use of protein X-ray crystallography, site-directed mutagenesis, and energetic binding measurements provides a structural basis for placing KAI2 within the signal transduction pathway associated with the perception of smoke-derived chemical signals such as KAR₁. Notably, KAR₁ and Phe194 form an exposed and central structural element that brings together a largely hydrophobic crevice spanning the active site entrance and the KAI2 helical domain. Unexpectedly, the exposed edge of KAR₁ lacks obvious hydrogen-bonding interactions in the static crystal structures. First, this observation implies that the exposed periphery of KAR₁ serves as a binding interface for KAI2-signaling partners. Second, KAI2’s solvent-accessible, aromatic-rich, hydrophobic patch surrounding KAR₁ and harboring KAR₁-induced conformational switches extends this ligand-dependent interface. Finally, the helical domain insert into the core α/β-hydrolase fold and abutting these two features is a third likely binding unit. These three components of the KAI2–KAR₁ complex appear poised to modulate interactions of KAI2 with downstream components of the karrikin-signaling pathway in a ligand-dependent manner.

Methods

Protein Expression and Purification.

The KAI2 gene was amplified from an A. thaliana cDNA library and inserted between the NcoI and BamHI sites of the expression vector pHIS8 encoding an N-terminal His₈-tag and thrombin cleavage site for tag removal. E. coli BL21 (DE3) cells harboring the KAI2 expression vector were grown at 37 °C in Terrific broth (Invitrogen) until the D_600nm reached ∼1.0. Isopropyl-β-d-thiogalactoside (0.5 mM) was added to induce protein expression under control of T7 RNA polymerase regulated by a modified lac promoter. Cells were grown overnight at 18 °C and harvested by centrifugation. Cell pellets were resuspended in lysis buffer [50 mM Tris⋅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 sonication. The soluble lysates were passed through a Ni²⁺-nitrilotriacetic acid (NTA) agarose column and eluted with lysis buffer supplemented with 250 mM imidazole (pH 8.0). The N-terminal His₈ tag was removed by treatment with thrombin during overnight dialysis in 50 mM Tris⋅HCl (pH 8.0), 500 mM NaCl, and 20 mM 2-mercaptoethanol. Thrombin and uncleaved His₈-KAI2 were removed by passing over benzamidine Sepharose then Ni²⁺-NTA agarose. Full-length KAI2 was further purified by size exclusion chromatography on a Superdex 75 HR26/60 column (Pharmacia Biosystems), dialyzed into crystallization 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 grown by 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) polyethylene glycol 8000, 0.1 M Na⁺-Pipes (pH 6.5), 0.3 M NaBr, and 2 mM DTT. Crystal growth occurred over 2–3 d and was improved by streak seeding. For cocrystallization with the KAR₁ ligand, the protein solutions included 5 mM KAR₁ (Toronto Research Chemical) diluted from a 100 mM stock solution in 100% methanol. Crystals were flash frozen by immersion in liquid nitrogen following 10 s incubations in a cryoprotectant solution consisting of reservoir solution supplemented with 17% (vol/vol) ethylene glycol. Single-wavelength data (λ = 1.0 Å) were collected at beamline 8.2.1 of the Advanced Light Source (Lawrence Berkeley National Laboratory) using an ADSC Quantum 315 CCD detector.

Data Processing, Structure Determination, and Refinement.

The observed reflections 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 search model 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 electron density maps and manual rebuilding of atomic models. Positional and isotropic B-factor refinement was performed with Phenix (28). A refined model of the KAI2 monomer served as a search model for MR for the KAI2–KAR₁ complex. The final structures were evaluated with PROCHECK (31). The apo-KAI2 structure had 93.2%, 6.4%, and 0.4% of residues in the most favored, additional allowed, and generously allowed regions of the Ramachandran plot, respectively, with no residues in the disallowed region. The KAI2–KAR₁ structure had 92.9%, 6.6%, and 0.4% of residues in the most favored, additional allowed, and generously allowed regions of the Ramachandran plot, respectively, with no residues in the disallowed region. Structural superpositions were calculated with SSM (32). All structure figures were prepared and rendered using PyMOL (DeLano Scientific; The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC.).

¹H-¹⁵N HSQC NMR Binding Study.

For NMR measurements, ¹⁵N-labeled KAI2 was produced in M9 minimal media containing 0.1% (wt/vol) ¹⁵NH₄Cl. The stable isotope–labeled protein was purified by the method described. Before NMR analysis, KAI2 at 1.4 mM was buffer-exchanged into 5 mM Tris⋅HCl (pH 8.0), 20 mM NaCl, and 0.5 mM DTT containing 10% (vol/vol) D₂O. ¹H-¹⁵N HSQC spectra were collected on a Varian VS800 NMR machine with water suppression. Spectra were collected at 25 °C on a sample of apo-KAI2 over a period of 3–4 h. Data collection was stopped and KAR₁ ligand dissolved in 100% DMSO was added to 7 mM and 3.5% (vol/vol) DMSO final concentrations. KAI2–KAR₁ mixtures were incubated at 25 °C for 1.5 h or 4 °C for 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 KAI2 remained stable.

Equilibrium Dialysis.

Equilibrium microdialysis coupled to KAR₁ fluorescence intensity detection was used to measure the K_d values for binding of KAR₁ to WT and mutant KAI2 proteins. In a standard equilibrium dialysis assay, two chambers (ligand chamber and protein chamber) are separated by a dialysis membrane preventing protein equilibration. At equilibrium, the ligand concentration in the ligand chamber is reduced by the total amount of ligand, bound and free, in the protein chamber. For this study, we used disposable DispoEquilibrium Dialyzers with a 5 kDa MWCO membrane (Harvard Apparatus). Briefly, a 50-µL aliquot of 10 µM KAI2 (or KAI2 mutant) was placed into the protein chambers of the dialyzer and 50 µL of a given concentration of KAR₁ placed into the ligand chambers. Total concentrations of KAR₁ were varied as follows: 0.1–60 µM (WT KAI2); 1–200 µM (Phe194Ala KAI2 mutant and Phe134Ala mutant); and 0.25–100 µM (His246Ala KAI2 mutant). The DispoEquilibrium Dialyzers were placed on a shaker at 23 °C and moderately agitated overnight to reach equilibrium. Once at equilibrium, the samples from substrate chambers, which contained unbound KAR₁, were collected by centrifugation and fluorescence intensities were measured using a Sapphire fluorescence plate reader (Tecan). The concentration of free KAR₁ was determined by fluorescence spectroscopy using an excitation wavelength of 260 nm and monitoring emission intensities at 379 nm. Three replicates were collected for each concentration. The mean change in fluorescence intensity was determined by subtracting the observed fluorescence intensity from the equilibrated ligand chamber from half the initial KAR₁ fluorescence for each chamber and plotted against the total KAR₁ concentrations. The points were then fit by nonlinear regression using Graphpad Prism 5 (GraphPad Software, Inc.) to Y = (B_max × X) × (K_d + X)⁻¹, where Y is the mean change in fluorescent intensity, B_max is maximum specific binding in fluorescent intensity, X is the concentration of KAR₁, and K_d value is measured in micromolar (μM). Graphpad Prism uses the method of linear descent in early iterations and then gradually switches to the Gauss-Newton approach to find the values of B_max and K_d that fit the data best (Levenberg-Marquardt method) (33).