Allosteric Regulation of Epoxide Opening Cascades by a Pair of

Dec 9, 2013 - Multistep catalysis of epoxide hydrolase/cyclase in the epoxide opening cascade is an intriguing issue in polyether biosynthesis. A pair...
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Allosteric Regulation of Epoxide Opening Cascades by a Pair of Epoxide Hydrolases in Monensin Biosynthesis Atsushi Minami,† Toyoyuki Ose,‡ Kyohei Sato,† Azusa Oikawa,‡ Kimiko Kuroki,‡ Katsumi Maenaka,‡ Hiroki Oguri,† and Hideaki Oikawa*,† †

Division of Chemistry, Graduate School of Science and ‡Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0810, Japan S Supporting Information *

ABSTRACT: Multistep catalysis of epoxide hydrolase/cyclase in the epoxide opening cascade is an intriguing issue in polyether biosynthesis. A pair of structurally homologous epoxide hydrolases was found in gene clusters of ionophore polyethers. In the epoxide opening reactions with MonBI and MonBII involved in monensin biosynthesis, we found that MonBII and catalytically inactive MonBI mutant catalyzed two-step reactions of bisepoxide substrate analogue to afford bicyclic product although MonBII alone catalyzed only the first cyclization. The X-ray crystal structure of MonBI dimers suggested the importance of the KSD motif in MonBI/MonBI interaction, which was further supported by gel filtration chromatography of wild-type MonBI and mutant MonBI. The involvement of the KSD motif in heterodimer formation was confirmed by in vitro assay. Direct evidence of MonBI/MonBII interaction was obtained by native mass spectrometry. Its dissociation constant was determined as 2.21 × 10−5 M by surface plasmon resonance. Our results suggested the involvement of an allosteric regulation mechanism by MonBI/MonBII interaction in monensin skeletal construction. These findings prompted us to identify the gene cluster of structurally simple ionophore polyether lasalocid.7,8 Utilizing several analogues installing key structural elements of lasalocid intermediate, we performed in vitro analysis of flavin-containing monooxygenase Lsd18 and epoxide hydrolase Lsd19.9−13 Lsd18 participates in two rounds of stereoselective epoxidation of diene precursor utilizing 4a-hydroperoxyflavin as an oxidizing agent.9 The epoxidations with Lsd18 proceed from terminal olefin to internal olefin. Lsd19 has a pseudodimeric architecture composed of an N-terminal domain (Lsd19A) and a C-terminal domain (Lsd19B), which contain a pair of acidic amino acids (Asp/Glu) as catalytic residues for cyclization in each active site. These domains sequentially catalyze 5-exo and 6-endo cyclizations in a stepwise manner with high regioselectivity.10−13 These results significantly advanced our understanding of epoxide opening cascade in polyether formation.14 The pair of EH domains, Lsd19A and Lsd19B, are functionally comparable to MonBI and MonBII (Supplementary Figure S1). However, lasalocid biosynthesis involves two epoxide opening reactions, while monensin biosynthesis involves three reactions affording the pentacyclic structure in 1 (Figure 1A). This suggests that one of the monensin EHs catalyzes two reactions. Multiple catalysis of EH is an intriguing

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olyethers possessing a wide range of biological activities comprise a unique family of secondary metabolites. Among them, ionophore polyethers have a characteristic skeleton composed of tetrahydrofuran (THF) and tetrahydropyran (THP).1 The structural diversity of the polyether moiety originates from the number of ether rings and the manner of ring closure. The structures and biological activities have continued to attract researchers, and extensive biosynthetic studies have been carried out over the past 40 years. On the basis of labeling patterns of the backbone structure along with stereochemical analysis, in 1983, the unified biosynthetic model for polyether construction was proposed by Cane, Celmer, and Westley.2 This model, designated the CCW model, postulates sequential epoxidation of polyene precursor followed by epoxide opening cascades of the resultant polyepoxide. Biomimetic synthesis utilizing a cascade reaction supports the CCW model.3 This simple yet versatile strategy for polyether construction can be extended to the biosynthetic machinery of all natural polyethers. In 2001, the landmark characterization of monensin (1) biosynthetic gene cluster (mon) was reported by Leadley and co-workers.4 The gene cluster consisted of characteristic open reading frames encoding a flavin-containing monooxygenase (EPX), monCI, and epoxide hydrolases/cyclases (EHs), monBI and monBII. A series of gene disruption experiments revealed that these EPX and EHs catalyze epoxidation and epoxide opening reactions predicted in the CCW model (Figure 1A).5,6 © 2013 American Chemical Society

Received: August 27, 2013 Accepted: December 9, 2013 Published: December 9, 2013 562

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Figure 1. (A) Enzymatic epoxide opening cascade catalyzed by MonBI/MonBII in monensin biosynthesis. (B) Comparison of putative substrate 3 and substrate analogues. To simplify the discussion, we used the same numbering system of substrate analogues as that of 3.

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RESULTS AND DISCUSSION Enzymatic Reaction of Hydroxybisepoxide with MonBI and MonBII. To study enzymatic epoxide opening reaction, especially in energetically favored 5-exo cyclization,16 it is necessary to consider the reactivity of the epoxyalcohol against non-enzymatic cyclization under standard assay conditions. In our previous study, we found that hydroxyepoxide 4 carrying a tertiary alcohol was chemically stable and also served as a substrate of enzymatic 5-exo cyclization.15 This enabled us to detect MonBI and MonBII activities. However, extremely low reactivity of 4 mimicking the C12−C19 region of 3 was problematic for further biochemical study. To increase the binding affinity against the EHs, we designed a new hydroxybisepoxide analogue 5 mimicking the C8−C19 region of 3 (Figure 1B). Previously, we optimized the chain length of the acyl group on the substrate analogue mimicking the putative substrate 3.15 Synthesis of 5 was essentially the same as the established procedure15 (Supplementary Scheme S1). Shi’s

issue in epoxide opening cascades. To elucidate the mechanism of enzymatic cyclization catalyzed by the MonBI/MonBII pair, we carried out biochemical analysis with the simple monoepoxide analogue 4.15 This led to interesting result that desired 5-exo cyclization was mediated only in the presence of both enzymes (Figure 1B). For a better understanding of this unexpected cooperative effect between MonBI and MonBII in cyclization, we carried out extensive biochemical, structural, and physicochemical analyses of MonBI and MonBII. Here, we describe enzymatic reactions of elaborated substrate analogue 5 harboring bisepoxide moieties with wild-type and mutant EHs, native ESI-MS spectrometry, surface plasmon resonance analyses of MonBI and MonBII, and crystal structural analysis of MonBI dimer. Taken together, our results suggested an unexpected allosteric regulation mechanism by MonBI/MonBII interaction for epoxide opening cascade in monensin biosynthesis. 563

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Figure 2. LC−MS profiles of in vitro experiments of 5: (1) with MonBI and MonBII, (2) with MonBII, (3) with MonBI, (4) with MonBI-D38A and MonBII, and (5) with MonBI-K7A and MonBII.

Figure 3. Overall structure and active site of MonBI: (A) A view of MonBI dimer: one monomer is colored blue, and the second monomer is colored green. (B) Close-up of the possible active site. (C) Close-up of the interaction site.

asymmetric epoxidation17 of key diene intermediate S7 gave the desired (R,R)-epoxide. As in the case of 4, 5 was not converted to THF-intermediate 6 and THF-THF product 7 under standard conditions (37 °C, 2 h). Enzyme assays were performed with 5 (100 μM) in the presence of overexpressed MonBI and MonBII (0.5 μM each) at 37 °C with a 20-min incubation. The results of LC−MS analysis of the reaction products under various conditions are shown in Figure 2. Unlike the enzymatic cyclization of 4, nearly complete consumption of 5 and production of 6 (24%) and 7 (68%) were observed in the standard reaction with two EHs, indicating that two rounds of 5-exo cyclization occurred in a stepwise manner. More importantly, these results also showed that MonBI and MonBII had sufficient reactivity against substrate 5. While removal of MonBI from the reaction mixture resulted in accumulation of 6 (86%) and gave no 7, removal of MonBII resulted in no product. For detailed understanding of this unusual enzymatic cyclization, we performed the reaction using catalytically inactive mutants, MonBI-E64A and MonBIID38A.15 In the reaction with MonBI-E64A and MonBII, bicyclic product 7 (57%) was observed. In contrast, no product was detected in the reaction with MonBI and MonBII-D38A (data not shown). These results established that MonBII catalyzes the first cyclization and MonBII complexed with either wild-type or catalytically inactive MonBI catalyzes the

second cyclization. This unique cooperative effect between MonBI and MonBII in the second cyclization was further examined with varying MonBI concentration. As shown in Supplementary Figure S2, production of 7 was increased in parallel with MonBI concentration (up to a molar ratio of ∼1) and reached a plateau. This strongly suggested that MonBI is essential for the second cyclization. Structural and Mutational Analysis of MonBI. Structural analysis was performed to investigate the cooperative catalytic mechanism by MonBI and MonBII. Although we tested a significant number of crystallization conditions, only MonBI gave suitable crystals. It is worth noting that the homodimeric form of MonBI showed essentially the same overall crystal structure as pseudo 2-fold dimeric (heterodimeric) Lsd19 architecture. The final model of MonBI consisted of residues 1−145 and formed a dimer with a pseudo 2-fold axis in an asymmetric unit; there is one dimer in an asymmetric unit (Figure 3A). Each MonBI monomer consists of four α-helices and six-stranded β-sheets resembling Lsd19A (122 Cα atoms can be superimposed with RMSD of 0.809) and Lsd19B (127 Cα atoms can be superimposed with RMSD of 1.208).13 The region between strands 3 and 4, consisting of residues 89−112, includes one helix and has a relatively high b-factor. This region corresponds to a loop-helixloop region, which provides a cap for the active site to increase 564

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Figure 4. Native ESI-MS spectra (deconvoluted) of MonBI and MonBII. (A) MonBI/MonBII complex. (B) MonBI. (C) MonBII. Overexpressed MonBI mainly consists of MonBI lacking L-Met at the N-terminal. On the other hand, overexpressed MonBII consists of two species (MonBII and MonBII lacking L-Met at the N-terminal).

Tyr134, and Thr137. Further specific electrostatic and hydrogen bonding interactions by three amino acid residues, Lys7, Ser138, and Asp139, were found at “hinge” sites in the 2fold symmetric dimer (Figure 3C). Lys7 at one monomer specifically interacts with Ser138 (2.94 Å) and Asp139 (2.65 Å) from the other monomer. The corresponding interactions at the opposite side were determined as Lys−Ser (2.80 Å) and Lys−Asp (3.00 Å). Importance of these residues for homodimer formation was examined by mutational analysis. Mutants (MonBI-K7A and MonBI-S138A) were successfully obtained in an electrophoretically homogeneous state (Supplementary Figure S3). The purified MonBI, MonBI-K7A, and

binding affinity of the substrate. The active site is formed by His13, Asp37, Val44, His53, Glu64, Tyr95, Arg98, Arg114, Ala116, Leu118, Leu131, and Trp135 (Figure 3B). The location of the acidic amino acid pair (Asp37 and Glu64) seems to be catalytically relevant in comparison with Lsd19A and Lsd19B. In our previous study, we actually found that MonBI in the presence of catalytically inactive MonBII-D38A mutant showed weak cyclization activity using monoepoxide 4 as a substrate.15 The interface of MonBI homodimeric structure is mainly composed of hydrophobic interactions involving Glu3, Val70, Leu79, Gln81, Trp100, Thr115, Met117, Val119, Lys132, 565

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MonBI-S138A were independently subjected to gel filtration chromatography. The elution volume of these proteins suggested that MonBI and MonBI-S138A exist as a dimeric structure while MonBI-K7A exists as a monomeric form under the same analytical conditions (Supplementary Figure S4). Thus, Lys7 was found to be a crucial residue in homodimeric structure formation. We speculated that mutation of Lys led to perturbation of homodimeric MonBI interactions via Ser-LysAsp due to decreasing the binding affinity. In the Lsd19 crystal structure, similar Ser-Lys-Asp interaction found in MonBI homodimeric structure was observed between Lsd19A and Lsd19B (Supplementary Figure S5). This enabled us to find the corresponding residues Lys, Thr, and Asp in the amino acid sequence of MonBII (Supplementary Figure S1). On the basis of these observations, heterodimer formation of MonBI and MonBII was considered to occur via the KSD motif. To assess this hypothesis, mutants (MonBII-K8A and MonBII-D120A) were constructed (Supplementary Figure S3). MonBI/MonBII assay with 5 were then independently conducted using MonBI mutants (K7A and S138A) or MonBII mutants (K8A and D120A). Formation of 7 was observed in the reaction with BI-S138A/BII and BI/BII-K8A as in the case of wild type pair (Supplementary Figure S6; lane 2 and lane 3). In contrast, accumulation of THF-intermediate 6 was observed in the reaction with BI-K7A/BII and BI/BII-D120A (Supplementary Figure S6; lane 1 and lane 4), revealing that BI-K7A and BII-D120A lack the ability to interact with the corresponding partner, and this resulted in loss of the activity to catalyze the second cyclization. These results suggested that, in two interdomain interactions between MonBI and MonBII, a single interaction formed by MonBII-Thr119/MonBI-K7A/ MonBII-Asp120 is found to be crucial in the formation of the catalytically active MonBI/MonBII complex. Experimental Evidence of Heterodimeric Form of MonBI/MonBII. Biochemical and indirect structural evidence prompted us to employ physicochemical analysis of the unique MonBI/MonBII interaction. Assuming the ability to form MonBII homodimer was due to the presence of the key K(S/ T)D motif, we initially conducted gel filtration analysis of MonBII. Unexpectedly, MonBII was eluted in the void volume (Supplementary Figure S4), suggesting that MonBII exists as a soluble aggregate under analytical conditions. Addition of MonBI to MonBII solution did not affect its chromatographic behavior (data not shown). Next, we carried out surface plasmon resonance (SPR) analysis to observe binding of MonBII to immobilized MonBI at various concentrations (1.9−62 μM). A dose-dependent increase in response showed interaction of MonBII with MonBI under the analytical conditions used here (Supplementary Figure S7A). The data could be convincingly fit to a simple one-site binding model (Langmuir isotherm), and the dissociation constant KD(MonBI/MonBII) value, calculated from the association rate constant kon (1.58 × 102 M−1 s−1) and dissociation rate constant koff (3.49 × 10−3 s−1), was determined as 2.21 × 10−5 M. To examine the effect of mutation on the KSD motif toward homodimeric MonBI/ MonBI interaction, similar SPR analysis was conducted with either MonBI or MonBI-K7A mutant as an analyte. In each case, a dose-dependent response was observed. However, the data could not fit the simple binding model (Supplementary Figure S7B,C), implying that the BI/BI interaction may involve complicated states in contrast to the corresponding BI/BII interaction.

Native mass spectrometry analysis is an emerging method to analyze intact protein complexes with high sensitivity and resolution.18,19 It allows detection of relatively weak protein− protein interactions. The major advantage of this technique is that the observed molecular mass directly reflects the protein complex, which allows us to determine the exact multimeric state of the complex. Initially, overexpressed MonBI and MonBII were independently analyzed by native ESI-MS spectrometry. The deconvoluted mass spectrum of MonBI mainly gave two molecular ion peaks at m/z 17644 and 35288 (Figure 4B). Each molecular mass corresponds to a monomeric structure lacking N-terminal methionine (MonBIΔMet) and its dimeric form, respectively. Next, the mass spectrum of MonBII afforded two major peaks at m/z 17359 and 17491, corresponding to monomeric states of MonBII lacking the Nterminal methionine (MonBIIΔMet) and MonBII, respectively. In addition, three minor peaks corresponding to dimeric structures (MonBIIΔMet) 2, MonBIIΔMet/MonBII, and (MonBII)2 were also observed at m/z 34717, 34850, and 34984, respectively (Figure 4C). Applying the same analytical conditions, a 1:1 mixture of MonBI and MonBII was then analyzed. The deconvoluted mass spectrum gave two apparent peaks at m/z 35006 and 35136 corresponding to heterodimeric forms MonBIΔMet/MonBIIΔMet and MonBIΔMet/MonBII, respectively. Several peaks corresponding to monomeric, homodimeric, and heterodimeric structures were also observed. These results represent the first evidence of heterodimer formation of polyether EH with a 1:1 stoichiometry (Figure 4A). Currently, the crystal structure of MonBI/MonBII heterodimer is not available. However, experimental evidence including enzymatic reactions with wild-type and mutant EHs, native ESI-MS data, SPR data, and the crystal structure of MonBI dimer revealed that MonBI/MonBII heterodimeric structure is closely related to that of Lsd19 consisting of Lsd19A and Lsd19B domains. Thus, MonBI/MonBII can be regarded as functionally relevant alternatives of dissected Lsd19B/Lsd19A domains. In the case of MonBI/MonBII complex-catalyzed cyclization, MonBI seems to be temporally inactive during the MonBII-catalyzed cyclization. Considering this result, we reexamined our previous data on the crystal structure of Lsd19, which was found in complex with substrate analogue in Lsd19A active site and with non-enzymatic reaction product in Lsd19B active site. We can now speculate that the Lsd19 crystal structure is a snapshot of the catalytically active form of Lsd19A and temporally inactive form of Lsd19B whose active site is filled with the cyclization product that is abundant in the acidic crystallization buffer. Thus, MonBI/MonBII system would provide a detailed mechanism of controlling catalytic activity on multiple cyclizations in both monomeric and dimeric forms. Allosteric Regulation in Polyether Skeletal Construction. Enzymatic epoxide opening cascades in monensin biosynthesis catalyzed by the MonBI/MonBII pair has been of interest for a long time. Although previous gene disruption experiments revealed enzymatic control of epoxide opening cascades,6 the detailed function of MonBI/MonBII pair, especially in multiple catalysis, remained unknown. Our biochemical data using synthetic analogue 5 unambiguously established that MonBII catalyzes two rounds of cyclization and that MonBI acts as an auxiliary protein to support MonBII via protein−protein interaction. Requirement of MonBI in the second reaction (6 to 7) suggested that the conformation of 566

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Conclusion. Epoxide opening cascade for construction of the polyether skeleton is a key transformation in biosynthesis of natural polyether metabolites. Recently, we established the basic catalytic mechanism and regioselective control of lasalocid epoxide hydrolase (EH) Lsd19 consisting of two fused EH domains. In this study, we investigated another intriguing issue, i.e., how a small number of EH(s) catalyze multistep epoxide opening reactions, and we unveiled the novel reaction mechanism of monensin epoxide hydrolase MonBI and MonBII, which are structurally relevant to Lsd19, utilizing allosteric regulation of two EH components. Efficient conversion of newly synthesized substrate showed that a pair of EHs was required for two-step epoxide opening reactions. Experiments with a combination of wild-type EH and catalytically inactive mutant EH demonstrated the importance of heterodimer formation of MonBI and MonBII. Although direct evidence of this weak interaction was difficult to obtain by conventional analytical techniques, native ESI-MS spectrometry unambiguously revealed molecular ion peaks of heterodimers, and the dissociation constant of MonBI/MonBII was measured by surface plasmon resonance. The X-ray crystal structure of MonBI dimer identified the K(S/T)D motif essential for the interaction, which was confirmed by enzymatic reactions with the corresponding mutant. Among polyether EHs, there are two options, a fused-type such as Lsd19 and a separated type such as MonBI and MonBII that are equilibrium mixtures of monomeric, homodimeric, and heterodimeric states. One of the reasons why nature creates the optional reversible interactions is that flexibility of the EH complex likely adds novel catalytic properties to polyether EHs, such as conformational changes of the EH active site to repeatedly accept the intermediate with structurally similar but several reactive sites in a single molecule. The presence of a pair of homologous EHs possessing conserved KSD motif in gene clusters of all ionophore polyethers indicates that the same allosteric regulation strategy may be chosen for creating individual polyether scaffolds. To our knowledge, allosteric regulation of homologous enzymes is rare. Therefore, sequential epoxide opening reaction is an intriguing target for studying novel enzymatic reaction mechanisms.

MonBII for initial reaction (5 to 6) does not accept 6 for the second cyclization. Thus, conformational change of MonBII by MonBI interaction most likely affects the arrangement of catalytic residues and/or the binding of 6. As MonBI does not directly affect MonBII catalysis, we speculated that MonBI is an allosteric regulator of MonBII to catalyze multiple cyclization reactions. The relatively high dissociation constant (KD = 2.21 × 10−5 M) seems to match the allosteric regulation. Further experimental results would provide insight into the detailed conformational change of MonBII in multiple cyclization reactions. Taking into consideration the previous finding on cavity sizes of EH and chain length of the substrate,13 a putative mechanism can be envisioned for the epoxide opening cascades (Supplementary Scheme S2). First, MonBII itself selectively recognizes I-1 from a hemiketal mixture of I-1 and I-1′ and catalyzes cyclization to give I-2. Conformational change triggered by MonBI interaction allows MonBII to catalyze the second cyclization from I-2 to I-3 in the MonBII active site. The intermediate I-3 is then transferred to MonBI, which has a smaller active site than MonBII, and serves as a substrate for the third cyclization to afford I-4. The structural resemblance of I-1 and I-2 with substrate analogues 5 and 6 supports the biosynthetic proposal. The mechanism of spiroketal construction is different from those of avermectin and reveromycin biosynthesis.20,21 As there is no direct evidence such as in vitro enzymatic cyclization of triepoxide intermediate 3, other mechanisms cannot be excluded (Supplementary Scheme S2). Including those of monensin and lasalocid, five biosynthetic gene clusters of ionophore polyethers, nanchangmycin (nan),22 nigericin (nig),23 and salinomycin (sal)24,25 have been characterized to date. All of these gene clusters contain EH gene(s) encoding two essential EH domains as either a fused system (lsd19 and nanI) or a separated system (monBI/monBII, nigBI/nigBII, salBI/salBII/salBIII). The key K(S/T)D motif for MonBI/MonBII interaction is highly conserved in those EHs (Supplementary Figure S1), suggesting that allosteric control of EH activity by EH/EH interaction is a common feature in skeletal construction of polyethers, especially in multiple cyclizations. Monensin and nigericin have one spiroketal moiety, while nanchangmycin and salinomycin have two. All EH reactions include 5-exo cyclization accompanying spiroketal formation. Interestingly, one of EH (NanIA and SalBIII) in nanchangmycin and salinomycin is proposed to be catalytically inactive due to mutation of a highly conserved acidic amino acid pair. Allosteric regulation by functionally inactive EH, which was experimentally confirmed in the reaction with MonBI-E64A and MonBII, indicated that both EHs participate in multiple cyclizations and possibly affect selection of a hemiketal isomer for spiroketalization. According to this hypothesis, skeletal constructions of nanchangmycin, nigericin, and salinomycin are proposed as shown in Supplementary Scheme S3. For multiple catalysis of polyether EH, dissection of the subunit may offer flexibility of the EH active site, which accepts several intermediates with different sizes of linear chain, and this may allow construction of complicated polyether skeletons. Detailed analysis of heterodimeric EH complex is required for further elucidation of its novel function. The allosteric regulation of EH found in this study might be applied to the biosynthesis of oxasqualenoids,26 annonaceae acetogenins,27 and possibly ladder-type polyethers.28

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METHODS

General. Cell disruption was dealt with an ultrasonic disrupter UR200P (TOMY SEIKO). Analysis of the samples during protein purification was performed using SDS-polyacrylamide gel electrophoresis, and the proteins were visualized by using Coomassie brilliant blue staining. Protein concentration was determined by the Bradford method with bovine serum albumin as a standard. Enzymatic Reactions. Typical conditions are as follows. A reaction mixture (final volume 50 μL of MOPS buffer, pH 7.0) containing 100 μM of the substrate, 0.5 μM of MonBI and/or MonBII, and 10% (w/v) glycerol was incubated at 37 °C. The reaction was quenched by the addition of methanol, and the resultant mixture was vortexed and centrifuged at 12,000 rpm. The supernatant was directly analyzed by LC−MS equipped with Develosil XG30-C30 (Nomura Chemical, ϕ 2.1 × 50 mm) at the following conditions: 78% (v/v) methanol in water at a flow rate of 0.2 mL/min. Gel Filtration Chromatography. Gel filtration chromatography was carried out using a Superdex 200 10/300 column (GE healthcare). The gel filtration buffer contained 10 mM HEPES (pH 8.0) and 200 mM sodium chloride. Gel filtration standard (Bio-Rad Laboratories), containing bovine thyroglobulin (670 kDa), bovine γ-globulin (158 kDa), chicken ovalbumin (44 kDa), and horse myolgobin (17 kDa), was used as calibration standard (Supplementary Figure S3). 567

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Crystallization and X-ray Data Collection. Purification of MonBI was performed following the procedure previously reported.15 To perform a crystallization trial, the buffer of the purified protein was changed to condition with 10 mM Tris-HCl and 100 mM NaCl (pH 7.5) using dialysis and concentrated to 10 mg mL‑1 using a concentrating filter (Amicon Ultra-15, 3 kDa cutoff; Millipore). Screening for the crystallization of MonBI was performed using JCSG core suite I−IV (Qiagen), Index Screen, Crystal Screen Lite, Crystal Screen Cryo, PEG Ion Screen (Hampton Research) and Wizard Screen I and II (Emerald Biosciences), by the sitting-drop vapordiffusion method in 96-well plates. Drops of 400 nL of each sample were mixed with an equal volume of reservoir solution and equilibrated against 0.1 mL of reservoir solution at 293 K. Crystals of MonBI were obtained from JCSG core III suite no. 94 (2.0 M ammonium chloride, 10% (v/v) PEG6000). Several conditions were further screened by the hanging-drop method using 24-well VDX plates (Hampton Research) by mixing 1.5 μL of protein solution and 1.5 μL of reservoir equilibrated against 0.5 mL of reservoir solution at 293 K. The best conditions for the MonBI crystals were 0.1 M potassium phosphate (pH 6.0), 2.25 M sodium chloride, 15% (v/v) PEG6000. Se-Met-substituted MonBI (Se-MonBI) for the experimental phasing was also crystallized with almost identical condition as the native MonBI. Prior to diffraction data collection, crystals were cryoprotected by transfer into a solution containing 25% (v/v) glycerol for a few seconds and flash-cooled. An X-ray absorption spectrum of a Se-MonBI crystal was collected around the Se K absorption edge by measuring the fluorescent signal perpendicular to the beam during a wavelength scanning performed at BL32XU of SPring-8 followed by MAD data set (0.93000 Å for the remote and 0.9797 Å for the edge) collection using MX225HE (Rayonix) detector. The native crystal was collected at beamline BL41XU using a MX225HE (Rayonix) detector. The data sets were integrated, merged, and scaled using HKL-2000.29 Se-MonBI diffracted up to 2.1 Å, and the space group is P212121 with unit cell dimensions of a = 38.44, b = 56.47 Å and c = 106.90 Å. The asymmetric unit contains two molecules, corresponding to a VM value of 2.09 Å3 Da−1 and a solvent content of 40.6%. Native crystal diffracted up to 2.0 Å with the space group P212121 (a = 38.76, b = 56.61 Å and c = 106.97 Å). Details of the data collection and processing statistics are given in Supplementary Table S1. Structure Determination and Refinement. The MAD data were further scaled using the program in the CCP4 program suite.30 Out of 10 selenium atom sites, 4 sites were initially located using the program Shelx.31 After the initial experimental phases were calculated from 4 selenium sites using SOLVE,32 the electron density was modified with RESOLVE.33,34 The phases were extended to 2.0 Å resolution using the native data with DM35 followed by model building using ARP/wARP36 and coot.37 The molecular dynamic refinement was performed using the program phenix 38 with a 31.4−2.0 Å resolution region of native data, and 5% of the data were selected for the calculation of the Rfree factor in order to monitor refinement. The final structure of MonBI was refined to an Rfree factor of 26.2% and an R factor of 21.8% with a root-mean-square deviation of 0.004 Å in bond length and 1.14° in bond angles. The statistics of phasing and structure refinement statistics are also summarized in Supplementary Table S1. The volume of the cavities was calculated using POCASA39 with a 2 Å probe size and a 1 Å unit grid. All of the figures containing protein structures were generated using PyMOL.40 Construction of Mutants. Mutation was introduced into pColdImonBI using respective primers as shown in Supplementary Table S2. The amplified monBI-K7A gene was purified with a GeneElute Plasmid Miniprep Kit. The purified fragments were then treated with T4 Polynucleotide Kinase for phosphorylation of 5′-OH termini of the polynucleotides and religated using Ligation high Ver.2 (TOYOBO). Other mutant plasmids (MonBI-S138A, MonBII-K8A, and MonBIID120A) were constructed using a PrimeSTAR Mutagenesis Basal Kit (Takara) according to the manufacture’s protocol. Escherichia coli BL21-Gold(DE3) competent cells were thereafter transformed with the ligation mixture for overexpression. The accuracy of the DNA

sequences of each mutated plasmid was confirmed by DNA sequencing. The mutant was expressed and purified as described for the native protein.15 Purification of each mutant was analyzed by SDSpolyacrylamide gel electrophoresis (Supplementary Figure S3). Surface Plasmon Resonance Analysis. The binding affinity was measured with a Biacore 2000 surface plasmon resonance spectroscope (GE Healthcare). Ligand protein (MonBI) was immobilized on a CM5 sensor chip using an Amine Coupling Kit (GE Healthcare) according to the manufacture’s protocol. All measurements were performed using running buffer (HBS-P buffer). Kinetic analysis for the interaction of MonBI with MonBII was carried out by injection of MonBII at different concentrations in running buffer at a flow rate of 20 μL min−1. Kinetic constants were determined using the curve-fitting facility of the BIAEVALUATION 4.0 program (GE Healthcare) to fit the rate equations derived from the simple 1:1 Langmuir binding model (A + B ↔ AB). Native MS Analysis. MS data of MonBI, MonBII, and MonBI/ MonBII mixed solution (20 μM each) were acquired using a Bruker maXis impact mass spectrometer. The mass spectrometer was operated using the following parameters: capillary, −4.5 kV; offset, −500 V; nebulizer pressure, 1.0 bar; dry gas flow, 3.0 L min−1; dry gas temperature, 200 °C; scan range, m/z 500−6000.

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ASSOCIATED CONTENT

S Supporting Information *

Synthetic procedures, 1H and 13C NMR spectra for all new compounds. This material is available free of charge via the Internet at http://pubs.acs.org. Accession Codes

Coordinates and structure factors are available from the Protein Data Bank with accession code 3WMD.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by MEXT research grant on innovative area 22108002 to H. Oikawa. We thank N. Iwasaki and N. Shimura for measurement of the native ESI-MS spectra.

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REFERENCES

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