Structural Basis of Protein–Protein Interactions between a trans-Acting

Jun 5, 2018 - Figure 1. DSZS trans-acting AT and module 1 ACP domain (ACP1): ... ACP, acyl carrier protein; DH, dehydratase; ER, enoylreductase; KR, ...
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Structural Basis of Protein−Protein Interactions between a transActing Acyltransferase and Acyl Carrier Protein in Polyketide Disorazole Biosynthesis Akimasa Miyanaga,*,† Risako Ouchi,† Fumihiro Ishikawa,‡ Ena Goto,† Genzoh Tanabe,‡ Fumitaka Kudo,† and Tadashi Eguchi*,† †

Department of Chemistry, Tokyo Institute of Technology, 2-12-1, O-okayama, Meguro-ku, Tokyo 152-8551, Japan Faculty of Pharmacy, Kindai University, 3-4-1 Kowakae, Higashi-Osaka, Osaka 577-8502, Japan

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ABSTRACT: Acyltransferases (ATs) are responsible for the selection and incorporation of acyl building blocks in the biosynthesis of various polyketide natural products. The transAT modular polyketide synthases have a discrete trans-acting AT for the loading of an acyl unit onto the acyl carrier protein (ACP) located within each module. Despite the importance of protein−protein interactions between ATs and ACPs in transAT assembly lines, the dynamic actions of ACPs and transacting ATs remain largely uncharacterized because of the inherently transient nature of ACP−enzyme interactions. Herein, we report the crystal structure of the AT−ACP complex of disorazole trans-AT polyketide synthase. We used a bromoacetamide pantetheine cross-linking probe in combination with a Cys mutation to trap the transient AT−ACP complex, allowing the determination of the crystal structure of the disorazole AT−ACP complex at 2.03 Å resolution. On the basis of the cross-linked AT−ACP complex structure, ACP residues recognized by trans-acting AT were identified and validated by mutational studies, which demonstrated that the disorazole AT recognizes the loop 1 and helix III′ residues of disorazole ACP. The disorazole AT−ACP complex structure presents a foundation for defining the dynamic processes associated with trans-acting ATs and provides detailed mechanistic insights into their ability to recognize ACPs.

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INTRODUCTION Polyketide synthases (PKSs) are responsible for the biosynthesis of various polyketide natural products, such as erythromycin, avermectin, and rifamycin.1 Bacterial modular type I PKSs contain multiple catalytic modules, each of which is basically responsible for a single round of polyketide chain elongation. 1−3 Each module minimally consists of a ketosynthase (KS) domain, an acyltransferase (AT) domain, and an acyl carrier protein (ACP) domain. The AT domain recognizes a specific acyl starter or extender unit and catalyzes its transfer onto the 4′-phosphopantetheine arm of the ACP to form a thioester conjugate. The resulting ACP-bound acyl building blocks are used for the condensation reaction catalyzed by the KS domain to extend a polyketide chain. Modular type I PKSs are further divided into two distinct classes: cis-ATs and trans-ATs. The cis-AT modular PKSs typically contain an AT domain in every module. Each AT domain transfers an acyl unit onto the cognate ACP domain located within the same module. On the other hand, the transAT PKSs have modules that lack AT domains and instead employ a discrete trans-acting AT.4−6 The trans-acting AT transfers the same extender unit onto each ACP domain. © XXXX American Chemical Society

The structural diversity of polyketides can be partly derived from the use of various acyl starter and extender units.7−9 ATs usually exhibit a strict specificity toward a single acyl-CoA or acyl-ACP.10 Because the acyl unit specificity of the AT largely affects the polyketide backbone structure, ATs are attractive targets for altering the substrate specificity to obtain biologically active polyketide analogs.10 The importance of proper AT−ACP interactions during the acyl transfer reaction has been previously demonstrated.11−14 For example, biochemical analyses of domains from module 3 and module 6 of 6deoxyerythronolide B synthase (DEBS) showed that cognate AT−ACP pairs have at least 10-fold greater specificity for each other than for noncognate pairs.11 These studies indicated that AT discriminates its cognate ACP from other ACPs through protein−protein interactions. The specific AT−ACP interactions are believed to be important for kinetically efficient polyketide chain elongation. However, mechanistic insights into ACP recognition remain limited because the structural determinations of AT−ACP complexes are hampered by the Received: April 18, 2018 Published: June 5, 2018 A

DOI: 10.1021/jacs.8b04162 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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Journal of the American Chemical Society

Figure 1. DSZS trans-acting AT and module 1 ACP domain (ACP1): (A) domain organization of DSZS PKS modules; (B) DSZS AT reaction with ACP1. Abbreviations are the following: AT, acyltransferase; ACP, acyl carrier protein; DH, dehydratase; ER, enoylreductase; KR, ketoreductase; KS, ketosynthase; KS0, ketosynthase-like transacylase; MT, methyltransferase. Light gray circles indicate inactive domains. 30:1) to yield a PMB-protected bromoacetamide pantetheine analog (20 mg, 94%). 1H NMR (400 MHz, CDCl3): δ 7.43 (d, J = 8.8 Hz, 2H), 7.18 (br, 1H), 7.01 (br, 1H), 6.93 (d, J = 8.8 Hz, 2H), 6.60 (br, 1H), 5.47 (s, 1H), 4.09 (s, 1H), 3.83 (s, 3H), 3.81 (s, 2H), 3.72 (d, J = 10.8 Hz, 1H), 3.66 (d, J = 12.8 Hz, 1H), 3.55 (m, 2H), 3.45−3.34 (m, 4H), 2.44 (t, J = 6.2 Hz, 2H), 1.11 (s, 3H), 1.09 (s, 3H). 13C NMR (125 MHz CDCl3): δ 172.0, 169.8, 166.7, 160.3, 130.1, 127.6, 113.8, 101.4, 83.9, 78.5, 55.4, 40.8, 39.3, 36.3, 35.0, 33.1, 28.8, 21.9, 19.2. The PMB-protected bromoacetamide pantetheine analog (20 mg, 0.041 mmol) was dissolved in 80% AcOH aq (0.41 mL) and stirred at room temperature for 3 h. After the solvent evaporated, the residue was purified by silica gel chromatography (chloroform/methanol = 20:1) to yield bromoacetamide pantetheine analog 1 (13 mg, 81%). 1 H NMR (500 MHz, D2O): δ 3.90 (s, 1H), 3.83 (s, 2H), 3.47−3.37 (m, 3H), 3.31 (d, J = 11.3 Hz, 1H), 3.29−3.24 (m, 4H), 2.41 (t, J = 6.6 Hz, 2H), 0.84 (s, 3 H), 0.80 (s, 3H). 13C NMR (125 MHz, D2O): δ 175.1, 174.3, 170.2, 75.9, 68.4, 39.2, 38.6, 38.4, 35.5, 35.4, 28.1, 20.5, 19.1. High resolution fast atom bombardment (HR-FAB)-MS (positive mode): m/z calculated for C13H25BrN3O5, 382.0978 ([M + H]+); observed, 382.0981. Synthesis of Pantetheineamides 2−5. Iodoacetamide pantetheine analog 2 was synthesized using a method similar to that used for analog 1. From PMB-protected pantetheine amine (70 mg, 0.18 mmol) and N-succinimidyl iodoacetate (57 mg, 0.20 mmol), the PMB-protected iodoacetamide pantetheine analog (87 mg, 87%) was obtained. From this analog (87 mg, 0.16 mmol), iodoacetamide pantetheine analog 2 (48 mg, 69%) was obtained. 1H NMR (500 MHz, D2O): δ 3.90 (s, 1H), 3.68 (s, 2H), 3.48−3.39 (m, 3H), 3.31 (d, J = 11.3 Hz, 1H), 3.29−3.25 (m, 4H), 2.42 (t, J = 6.6 Hz, 2H), 0.84 (s, 3H), 0.81 (s, 3H). 13C NMR (125 MHz, D2O): δ 175.1, 174.2, 172.3, 75.9, 68.4, 39.1, 38.6, 38.4, 35.5, 35.4, 20.5, 19.1, −2.3. HR-FAB-MS (positive mode): m/z calculated for C13H25IN3O5, 430.0839 ([M + H]+); observed, 430.0830. Chloroacetamide pantetheine analog 3 was also synthesized using a similar method. From PMB-protected pantetheine amine (130 mg, 0.34 mmol) and N-succinimidyl chloroacetate (72 mg, 0.37 mmol), the PMB-protected chloroacetamide pantetheine analog (148 mg, 95%) was obtained. From this analog (148 mg, 0.32 mmol), chloroacetamide pantetheine analog 3 (106 mg, 97%) was obtained. 1 H NMR (500 MHz, D2O): δ 4.07 (s, 2H), 3.92 (s, 1H), 3.49−3.39 (m, 3H), 3.34−3.27 (m, 5H), 2.43 (t, J = 6.6 Hz, 2H), 0.86 (s, 3H), 0.82 (s, 3H). 13C NMR (125 MHz, D2O): δ 175.2, 174.3, 170.0, 75.9, 68.4, 42.3, 39.2, 38.6, 38.5, 35.5, 35.3, 20.5, 19.1. HR-FAB-MS

transient protein−protein interactions among them. We have recently reported the complex structure of the standalone AT VinK with the standalone ACP VinL from the vicenistatin pathway, in which VinK transfers a dipeptidyl starter substrate from VinL to the ACP domain of the VinP1 PKS loading module.14,15 The VinK−VinL complex structure provides the first structural insights into the AT−ACP interactions. However, it remains unclear whether the molecular details of the VinK−VinL interactions could be applied to understand the other AT−ACP interactions because various types of AT− ACP pairs are used in polyketide biosynthesis. Protein−protein interactions between trans-acting ATs and ACPs are important for the regioselective incorporation of acyl units in trans-AT PKS reactions. Because the trans-acting AT is not covalently connected to the PKS module, the trans-acting AT must accurately discriminate cognate ACP domains from other ACP domains. The AT−ACP interactions in the transAT PKS assembly lines have been analyzed by mutational and docking studies using trans-acting AT and the module 1 ACP domain (ACP1) from disorazole synthase (DSZS) (Figure 1).16 Nevertheless, the ACP recognition mechanism in transAT PKS assembly lines is not understood in detail because of the lack of structural information for AT−ACP complex. Herein, we report the crystal structure of the DSZS AT−ACP1 complex. We captured the transient DSZS AT−ACP1 complex in its functional state using a synthetic pantetheineamide crosslinking probe in combination with a Cys mutation, which enables the structural determination of the DSZS AT−ACP1 complex. The structure of the DSZS AT−ACP1 complex provides the first detailed insights into the protein−protein interactions between a trans-acting AT and ACP in trans-AT PKS assembly lines.

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EXPERIMENTAL SECTION

Synthesis of Pantetheineamide 1. p-Methoxybenzylideneacetal (PMB)-protected pantetheine amine17 (16 mg, 0.043 mmol) and N-succinimidyl bromoacetate (11 mg, 0.047 mmol) were dissolved in CH2Cl2 (0.22 mL). The solution was then stirred at room temperature for 15 h. After the solvent evaporated, the residue was purified by silica gel chromatography (chloroform/methanol = B

DOI: 10.1021/jacs.8b04162 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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Journal of the American Chemical Society (positive mode): m/z calculated for C13H25ClN3O5, 338.1483 ([M + H]+); observed, 338.1482. trans-3-Chloroacrylamide pantetheine analog 4 and cis-3-chloroacrylamide pantetheine analog 5 were synthesized from PMBprotected pantetheine amine according to a previously reported protocol.17 Preparation of Recombinant Proteins. The artificial dszsat gene that was optimized for overexpression in Escherichia coli was synthesized by Eurofins Genomics (Tokyo, Japan). The construct of DSZS AT protein comprised the AT region (residues 1−286) of DisD, as reported previously.16 The synthesized dszsat gene was cloned into the expression vector pCold I using the NdeI and XhoI restriction sites to form pCold I-dszsat. E. coli BL21(DE3) cells harboring pCold I-dszsat were grown at 37 °C in Luria−Bertani broth containing ampicillin (50 μg/mL). When the optical density at 600 nm reached 0.6, protein expression was induced by the addition of 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and the cells were cultured for an additional 18 h at 15 °C. The harvested cell pellets were suspended in buffer [50 mM Tris-HCl (pH 7.2) and 10% (v/v) glycerol] and lysed by sonication. The supernatant was directly applied to a TALON affinity column (Clontech). Bound DSZS AT protein was eluted with elution buffer [50 mM Tris-HCl (pH 7.2), 150 mM imidazole, and 10% (v/v) glycerol]. The purified DSZS AT protein was desalted using a PD-10 column (GE Healthcare) and then concentrated using an Amicon Ultra centrifugal filter (Merck Millipore). The artificial dszsacp1 gene that was optimized for overexpression in E. coli was also synthesized by Eurofins Genetics. The construct of DSZS ACP1 protein consisted of 90 amino acid residues and corresponded to the ACP domain of DisA module 1. DSZS ACP1 residues 1−90 are identical to residues 1809−1898 of DisA. The amino acid residues of DSZS ACP1 are numbered according to a previous paper.16 The synthesized dszsacp1 gene was cloned into the expression vector pET28b using the NdeI and XhoI restriction sites to form pET28b-dszsacp1. E. coli BL21(DE3) cells harboring pET28bdszsacp1 were grown at 37 °C in Luria−Bertani broth containing kanamycin (30 μg/mL). When the optical density at 600 nm reached 0.6, protein expression was induced by the addition of 0.2 mM IPTG, and the cells were cultured for an additional 18 h at 15 °C. The harvested cell pellets were suspended in buffer [50 mM Tris-HCl (pH 7.2) and 10% (v/v) glycerol] and lysed by sonication. The supernatant was directly applied to a TALON affinity column. Bound DSZS ACP1 protein was eluted with elution buffer [50 mM Tris-HCl (pH 7.2), 150 mM imidazole, and 10% (v/v) glycerol]. The purified DSZS ACP1 protein was then desalted and concentrated using a PD-10 column and an Amicon Ultra centrifugal filter, respectively. To prepare the cross-linked complex on a large scale for crystallization, the N-terminal His-tag of DSZS ACP1 was removed by a thrombin protease treatment. CoaA, CoaD, and CoaE were prepared as recombinant proteins, each of which is fused to a maltose-binding protein. The plasmids encoding CoaA, CoaD, and CoaE18 were provided by Prof. M. D. Burkart. These Coa proteins were expressed and purified using a previously described protocol.18 The Sfp protein from Bacillus subtilis was prepared as a His-tagged protein. E. coli BL21(DE3) cells harboring the sfp plasmid19 were grown at 37 °C in Luria−Bertani broth containing ampicillin (50 μg/mL). When the optical density at 600 nm reached 0.6, protein expression was induced by the addition of 0.2 mM IPTG, and the cells were cultured for an additional 20 h at 15 °C. The harvested cell pellets were suspended in buffer [50 mM Tris-HCl (pH 7.5) and 10% (v/v) glycerol] and lysed by sonication. The supernatant was directly applied to a TALON affinity column. Bound Sfp protein was eluted with elution buffer [50 mM Tris-HCl (pH 7.5), 200 mM imidazole, and 10% (v/v) glycerol]. The purified Sfp protein was then desalted and concentrated using a PD-10 column and an Amicon Ultra centrifugal filter, respectively. Site-Directed Mutagenesis. pCold I-dszsat was used in the construction of DSZS AT mutants. pET28b-dszsacp1 was used in the construction of DSZS ACP1 mutants. Site-directed mutagenesis was performed with the following oligonucleotides and their comple-

mentary oligonucleotides: DSZS AT S86A, 5′-CTCGCGGGTCACGCGTTAGGGGAGTTCTCTG-3′; DSZS AT S86C, 5′CTCGCGGGTCACTGCTTAGGGGAGTTCTCTG-3′; DSZS AT R111G, 5′-CCCTGGTGAAGAAAGGCGGTGAACTTATGG-3′; DSZS AT Q239C, 5′-GCGGCACTGTCCGAGTGCATTGCGAGTCC-3′; DSZS ACP1 D45A, 5′-CCTGGGTGTGGCCTCAGTGGCTCTGCAG-3′; DSZS ACP1 F69A, 5′-CGACTCTGCTGGCTGAGAACCCGAATATCC-3′; and DSZS ACP1 E70A, 5′CGACTCTGCTGTTTGCGAACCCGAATATCC-3′. The mutations were confirmed by determining the nucleotide sequences. The plasmids were introduced into E. coli BL21 (DE3) cells, and the mutated proteins were prepared as described above. Cross-Linking Reaction with 1,2-Bismaleimidoethane. To obtain the holo-ACP protein, DSZS ACP1 was coexpressed with the sf p gene and purified as described above. 15 μM DSZS AT protein was mixed with 50 μM DSZS ACP1 protein and 0.3 mM 1,2bismaleimidoethane (BMOE) in a buffer containing 40 mM potassium phosphate (pH 7.0) and 10% (v/v) glycerol and then incubated on ice. At 5, 10, and 60 min, 10 μL samples of the reaction mixture were taken, and the reaction was quenched by the addition of SDS−PAGE loading buffer. Cross-Linking Reaction with Pantetheineamides. For DSZS AT−ACP1 cross-linking assays, apo DSZS ACP1 protein was mixed with each pantetheineamide, DSZS AT, and related enzymes in onepot enzymatic reactions. First, 150 μM of each pantetheineamide was mixed with 5 mM ATP, 7.5 mM MgCl2, 0.5 μM CoaA, 0.7 μM CoaD, 0.6 μM CoaE, 2.5 μM Sfp, and 100 μM His-tagged DSZS ACP1 protein in a buffer containing 50 mM NaH2PO4/K2HPO4 (pH 7.5) and 10% (v/v) glycerol (total volume 37.5 μL). The modified reaction was carried out at 28 °C for 3 h. The cross-linking reaction was then initiated by adding 37.5 μM the DSZS AT wild-type, S86A mutant or S86C mutant directly into the reaction mixture, which was then incubated at 20 °C. The total volume was increased to 50 μL, and the concentration of DSZS ACP1 was diluted to 75 μM by the addition of the DSZS AT solution. For the mutational analysis with probe 1, the concentrations of DSZS AT and ACP1 were adjusted to 20 μM and 50 μM, respectively. At 10, 30, 60, and 180 min, 10 μL samples of the reaction mixture were taken, and the reaction was quenched by the addition of SDS−PAGE loading buffer. The production rate of the DSZS AT−ACP complex was estimated using a Gel Doc EZ Imager (Bio-Rad). Standard errors were calculated from three independent experiments. Before the addition of DSZS AT, the complete conversion of apo DSZS ACP1 protein (wild-type and mutants) to modified DSZS ACP1 protein was monitored by liquid chromatography-electrospray ionization (LCESI)-MS analysis (LCMS-2020, Shimadzu). Observed molecular weights were calculated from multicharged states by deconvolution (LabSolutions LCMS Multi-Charged Ion Analysis Software, Shimadzu). LC−ESI-MS data are summarized in Table S1. Preparation and Purification of the Cross-Linked DSZS AT− ACP1 Complex. To prepare the cross-linked complex on a large scale, the DSZS ACP1 protein that was modified with pantetheineamide 1 was first prepared. Briefly, 150 μM pantetheineamide 1 was mixed with 5 mM ATP, 7.5 mM MgCl2, 0.5 μM CoaA, 0.7 μM CoaD, 0.6 μM CoaE, 2.5 μM Sfp, and 100 μM His-tag free DSZS ACP1 protein in a buffer containing 50 mM NaH2PO4/K2HPO4 (pH 7.5) and 10% (v/v) glycerol (total volume 2.5 mL). After 3 h at 28 °C, the modified ACP1 protein was desalted using a PD-10 column and then purified using a Resource Q column (GE Healthcare). Next, 100 μM modified DSZS ACP1 protein was mixed with 50 μM DSZS AT S86C mutant in a buffer containing 50 mM NaH2PO4/K2HPO4 (pH 7.5) and 10% (v/v) glycerol (total volume 2.0 mL). After 15 h at 20 °C, the generated cross-linked DSZS AT−ACP1 complex was desalted and concentrated using an Amicon Ultra centrifugal filter to replace the buffer. The new buffer contained 5 mM Tris-HCl (pH 8.5) and 10% (v/v) glycerol. The cross-linked DSZS AT−ACP1 complex was then purified using a Resource Q column. Crystallization, Data Collection, and Structural Determination. Crystals of the DSZS AT−ACP1 complex were grown using sitting-drop vapor diffusion by mixing the protein solution [6.4 mg/ C

DOI: 10.1021/jacs.8b04162 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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Figure 2. Chemical structures of the pantetheineamide probes used in this study: (A) structures of pantetheineamide probes 1−5; (B) proposed cross-linking reaction to generate a covalent DSZS AT−ACP1 complex. mL in 5 mM Tris-HCl (pH 8.5) and 10% (v/v) glycerol] with an equal volume of reservoir solution [2.0 M ammonium sulfate and 5% (v/v) isopropanol] at 20 °C. Prior to X-ray data collection, crystals were soaked in a cryoprotectant solution [2.0 M ammonium sulfate, 1% (v/v) isopropanol, and 25% (v/v) glycerol] and flash-frozen in liquid nitrogen. Diffraction data were collected in the BL-5A beamline at the Photon Factory (Tsukuba, Japan) and processed with XDS software.20 The initial phase was determined by molecular replacement using the Molrep program21 with the DSZS AT structure (PDB code 3SBM) as the search model. The protein model building of the DSZS AT−ACP1 complex was carried out automatically with the ARP/wARP program22 and subsequently inspected by Coot.23 Refmac 24 was used to refine the structure. The structural representations were prepared with PyMOL (DeLano Scientific LLC, Palo Alto, CA, USA). The geometries of the final structure were evaluated using the program MolProbity.25 The interface area between DSZS AT and ACP1 was analyzed by the PISA server.26 The atomic coordinates and structure factors have been deposited in the Protein Data Bank (PDB code 5ZK4).

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BMOE method combined with a Cys mutation could not exclude nonspecific cross-linking in this case. Burkart and co-workers developed a one-pot chemoenzymatic strategy using a set of synthetic probes27 to investigate the transient protein−protein interactions of ACP with KS,28,29 thioesterase (TE),30 and dehydratase31,32 in fatty acid synthase and PKS. We used this strategy to investigate the cross-linking reaction between DSZS AT and ACP1. Here, we designed short halogenated acyl amide pantetheine analogs, including bromoacetamide 1 (Figure 2A), based on the chemical structure of the α-bromopalmitamide pantetheine probe used for the cross-linking reaction between TE and ACP30 because both AT and TE form acyl−enzyme intermediates by attacking thioester bonds. In the AT reaction, the hydroxy group of the catalytic Ser residue is activated by a His residue and nucleophilically attacks the carbonyl carbon of malonyl-CoA to form the transient malonylated AT intermediate (Figure 1B). The thiol group of the phosphopantetheine moiety of ACP then nucleophilically attacks the carbonyl carbon of the malonylated Ser residue to produce malonyl-ACP. Therefore, we expected the catalytic Ser86 residue of DSZS AT wild-type to nucleophilically attack the electrophilic pantetheineamide loaded onto the DSZS ACP1 protein to form a covalent linkage. Concurrently, we constructed the DSZS AT S86C mutant as Khosla and coworkers used the S86C mutant for the cross-linking experiments with 1,3-dibromoacetone. In fact, the catalytic Cys residues of KS and TE have been used for cross-linking reactions with ACPs.28,30 The halogenated pantetheineamides were expected to be appropriate electrophiles that react with the soft nucleophile thiol (Figure 2B). To evaluate the crosslinking ability of the nucleophilic substitution at the α position, we designed bromoacetamide (1), iodoacetamide (2), and chloroacetamide (3) pantetheine analogs. These pantetheineamides 1−3 were synthesized from a PMB-protected pantetheine amine17 by coupling with the corresponding Nsuccinimidyl haloacetates, followed by acidic deprotection (Figure S2A). In addition, we prepared the trans-3chloroacrylamide (4) and cis-3-chloroacrylamide (5) pantetheine analogs as described previously28,29 to examine the reactivity at the β position. We hypothesized that pantetheineamides 4 and 5 would irreversibly react with a DSZS AT residue through Michael addition−elimination reactions.

RESULTS

Cross-Linking Reaction. Khosla and co-workers reported that attempts to cocrystallize DSZS AT with DSZS ACP1 resulted in obtaining only crystals of the DSZS AT protein.16 Therefore, they examined a cross-linking reaction between DSZS AT and ACP1 proteins to trap the transient DSZS AT− ACP1 complex for crystallization.16 They showed that the DSZS ACP1 protein could be cross-linked to the DSZS AT S86C mutant using bifunctional electrophilic reagents such as 1,3-dibromoacetone. Although this method seems to be useful for trapping the transient DSZS AT−ACP1 complex, the yield of the cross-linked complex was not sufficient for crystallization. Because we previously succeeded in the structural determination of the VinK−VinL complex using a BMOE cross-linking reagent,14 we first investigated the cross-linking reaction between DSZS AT and ACP1 using BMOE. We introduced a Cys mutation at the position of Gln239 in addition to the R111G mutation based on the VinK−VinL complex structure (Figure S1A) and then used the mutant for the cross-linking reaction. However, both the DSZS AT wildtype and R111G/Q239C mutant produced a cross-linked complex, suggesting that the cross-linking reaction occurred at an undesired position (Figure S1B). The presence of some Cys residues, such as Cys247, located on the surface of DSZS AT might have caused the undesired cross-linking. Thus, the D

DOI: 10.1021/jacs.8b04162 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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Journal of the American Chemical Society Synthetic pantetheineamide probes 1−5 were enzymatically loaded on the apo DSZS ACP1 protein in a one-pot reaction as described previously (Figure S2B).17 CoaA, CoaD, and CoaE proteins from the CoA biosynthetic pathway were used for the conversion of each pantetheineamide to a CoA derivative. Sfp,33 which is a highly promiscuous phosphopantetheinyl transferase, was used to attach the pantetheineamide moiety of the CoA derivative to the Ser46 residue of the apo DSZS ACP1 protein. The formation of modified DSZS ACP1 proteins was confirmed by LC−ESI-MS analysis (Figure S3 and Table S1). The DSZS AT wild-type, S86A mutant, or S86C mutant was subsequently added directly to the reaction mixture, and the reaction products were analyzed by SDS− PAGE. As a result, DSZS ACP1 modified by probe 1 reacted with the DSZS AT S86C mutant to form a cross-linked complex but not with the DSZS AT wild-type or S86A mutant (Figure 3A). Thus, the specific cross-linking reaction appears to have occurred at the position of the mutated Cys86. The nucleophilicity of the catalytic Ser residue of the DSZS AT wild-type is likely insufficient for cross-linking. The Ser residue is a rather hard nucleophile and appears to be less suitable for soft electrophiles such as bromoacetamide group, explaining the different reactivity level between the DSZS AT wild-type and S86C mutant. DSZS ACP1 modified with probe 2 was also cross-linked with the DSZS AT S86C mutant (Figure 3B), albeit to a lesser extent than probe 1. The low yield may be caused by hydrolysis of the reactive iodoacetamide functionality during the preparation of modified ACP1 protein. DSZS ACP1 modified with probe 2 was slightly cross-linked even with the DSZS AT S86A mutant, suggesting that probe 2 might be too reactive, resulting in an unexpected cross-linking reaction. DSZS ACP1 modified with probe 3 produced only a small amount of the cross-linked complex with the DSZS AT S86C mutant (Figure 3C). The electrophilicity of the chloroacetamide group might not be sufficient enough for the nucleophilic substitution of the mutated Cys residue. In contrast, the DSZS ACP1 protein modified with 3chloroacrylamide-type probe 4 or 5 resulted in no detectable cross-linked complex with the AT S86C mutant (Figure S4). Thus, the β position of the acyl amide pantetheine probes 4 and 5 may be far from the mutated Cys residue of the DSZS AT. The proper positioning of the reactive site of the electrophilic pantetheineamide probes at the AT active center appears to be critical for the cross-linking reaction. Alternatively, the electrophilicity of 3-chloroacrylamide moiety may be unsuitable for the nucleophilic addition of Cys residue. Crystal Structure of DSZS AT−ACP1 Complex. Next, a large scale cross-linking reaction using probe 1 was performed, and the DSZS AT−ACP1 complex was purified for crystallization (Figure S5A). The DSZS AT−ACP1 complex existed as a monomer in solution (Figure S5B). After crystallization attempts, we succeeded in determining the crystal structure of the DSZS AT−ACP1 complex at 2.03 Å resolution (Figure 4A and Table S2). In the complex structure, two DSZS AT−ACP complex molecules are present in the crystallographic asymmetric unit (Figure S6). The overall architecture of DSZS ACP1 is similar to that of other structurally characterized ACPs, including the ACP domain of DEBS module 2 (PDB code 2JU1; 31% sequence identity; 1.8 Å rmsd for Cα atoms)34 and VinL (PDB code 5CZD; 17% sequence identity; 2.2 Å rmsd for Cα atoms).14 DSZS ACP1 has a helix bundle fold with three main helices (helices I, II, and III) and one small distorted helix III′ as observed in most

Figure 3. Cross-linking reaction of DSZS AT with DSZS ACP1 using synthetic pantetheineamide probes 1−3. After DSZS ACP1 was modified with each pantetheineamide using a one-pot chemoenzymatic strategy, DSZS AT wild-type, S86A mutant, or S86C mutant was added into the reaction mixture. SDS−PAGE after the reaction of DSZS AT with DSZS ACP1 for 10, 60, and 180 min is shown. Covalent cross-linking between DSZS AT and ACP1 results in gel shifts of the AT bands from 33 kDa (DSZS AT) to 46 kDa (DSZS AT−ACP1 cross-linked complex) on SDS−PAGE gel. The bands at 60−70 kDa are CoaA, CoaD, and CoaE proteins. Pantetheineamides 1 (A), 2 (B), and 3 (C) were used to modify DSZS ACP1 protein for the cross-linking reaction.

known ACP structures.35 The DSZS ACP1 structure contains three disordered loop regions (Met1−Gln6, Ile26−Ala32, and Ala87−Ala90). The N- and C-terminal regions of DSZS ACP1 are located on the side opposite from the DSZS AT-binding region (Figure 4A), implying that AT-binding causes no steric hindrance with other DSZS PKS (DisA) catalytic domains. The electron density of probe 1 was clearly observed in the substrate-binding tunnel of DSZS AT (Figure 4B). Probe 1 was covalently connected between the Ser46 of DSZS ACP1 and the mutated Cys86 of DSZS AT, as expected. The side chain of Cys86 rotated away from His191 probably because of the formation of the covalent linkage with probe 1 (Figure S7). E

DOI: 10.1021/jacs.8b04162 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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Thr57 in the acetate-bound DSZS AT structure (PDB code 3SBM), the side-chain of Gln9 rotates away from Thr57 for the formation of the hydrogen bond with the sulfate ion in the DSZS AT−ACP1 complex structure. The reorientation of Gln9 side chain was also reported in the ligand-free structure for which data were collected at room temperature using X-ray free-electron laser (PDB code 6APK), with the citrate-bound (PDB code 6APF) and the malonate-bound (PDB code 6APG) structures.36 The interface between DSZS AT and ACP1 comprises ∼600 Å2. The small contact area is consistent with the transient nature of the enzyme−ACP interactions.14,37 DSZS AT forms contacts with two surface regions of DSZS ACP1, which are the loop 1 region (Phe40−Asp45) and the helix III′ region (Pro65−Glu70). In the loop 1 region, Asp45 of DSZS ACP1 forms a salt bridge with Lys180 of DSZS AT (Figure 5). Phe40

Figure 5. Binding interface between DSZS AT and ACP1. DSZS AT and ACP1 are shown in green and cyan, respectively. Salt bridges and hydrogen bonds are shown as blue broken lines.

of DSZS ACP1 forms hydrophobic contact with Ile127 of DSZS AT. The helix III′ region of DSZS ACP1 interacts with the C-terminal helix (α13; Val269−Ala281) of DSZS AT. The side chain of Glu70 of DSZS ACP1 forms a salt bridge with Arg278 of DSZS AT. The main-chain carbonyl of Glu70 of DSZS ACP1 also interacts with Arg279 of DSZS AT. Phe69 of DSZS ACP1 forms hydrophobic contacts with Gly272, Ala275, and Gln276 of DSZS AT. Pro65 and Thr66 of DSZS ACP1 also form hydrophobic contacts with Ile268 and Thr271 of DSZS AT. Mutational Analysis. Among the DSZS ACP1 residues that interact with the DSZS AT, Asp45, Phe69, and Glu70 of ACP1 are highly conserved in seven ACP domains (ACP1−7) of the DSZS PKS modules (Figure 6), suggesting that these three residues are commonly important for recognition by DSZS AT. Therefore, we constructed the DSZS ACP1 D45A, F69A, and E70A mutants to evaluate the roles of these residues. After we confirmed that each DSZS ACP1 mutant was completely modified by probe 1 with CoaA, CoaD, CoaE, and Sfp (Table S1 and Figures S8 and S9), we carried out the one-pot cross-linking assay using the DSZS AT S86C mutant. The DSZS ACP1 D45A and F69A mutants showed significantly reduced cross-linking efficiency levels (54 ± 2% and 58 ± 3% of wild-type, respectively), although the DSZS ACP1 E70A mutant showed only a slightly reduced crosslinking efficiency (88 ± 5% of wild-type) (Figure S10). This

Figure 4. Structure of the DSZS AT−ACP1 complex. DSZS AT and ACP1 are shown in green and cyan, respectively. (A) Overall structure of the DSZS AT−ACP1 complex. Ser46 of DSZS ACP1, the phosphopantetheine analog moiety derived from probe 1, and Cys86 of the DSZS AT S86C mutant are shown as sticks. The N- and Ctermini of these DSZS proteins are denoted as N and C, respectively. (B) Substrate-binding tunnel region in the DSZS AT−ACP1 complex. An F0 − Fc electron density map contoured at 3.0σ was constructed before incorporation of the phosphopantetheine analog moiety.

The narrow active-site cavity of DSZS AT might explain why DSZS AT did not react with 3-chloroacrylamide-type probe 4 or 5. DSZS AT might be unable to accommodate the rigid and bulky 3-chloroacrylamide moiety near the Cys86 residue. The modified phosphopantetheine arm derived from probe 1 exhibits several interactions with DSZS AT. The β-alanine moiety is stacked between Gly10 and Leu184 of DSZS AT. The dimethyl group of the pantoic acid moiety forms hydrophobic contacts with Val269 of DSZS AT. The phosphate group interacts with Gln156 of DSZS AT. A sulfate ion presumably derived from the crystallization buffer is bound in the substrate malonate-binding pocket (Figure S7). The sulfate ion forms a bidentate salt bridge with the side chain guanidine group of Arg111 and a hydrogen bond with the side chain of Gln9. Although Gln9 forms a hydrogen bond with F

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Figure 6. Sequence comparison of DSZS ACP1 with other ACPs. DSZS_ACP2a, DSZS_ACP3, DSZS_ACP4, DSZS_ACP5, DSZS_ACP6, and DSZS_ACP7 are ACP domains of DSZS modules 2, 3, 4, 5, 6, and 7, respectively. DEBS_ACP3 and DEBS_ACP6 are ACP domains of DEBS modules 3 and 6, respectively. Kirr_ACP4 and Kirr_ACP5 are ACP domains of kirromycin synthase modules 4 and 5, respectively. The secondary structural elements of DSZS ACP1 are indicated above the sequence. The DSZS ACP1 residues that are involved in the interaction with DSZS AT are indicated with cyan circles. The VinL residues that are involved in the interaction with VinK are indicated with green circles. The 4′phosphopantetheine arm attachment site in DSZS ACP1 and VinL is indicated with red circles.

fold higher activities, respectively.16 Although Gln76 and Arg79 were previously proposed to be placed near Lys14 of DSZS AT based on the computational docking analysis,16 the complex structure clearly showed that Gln76 and Arg79 are located near Leu284 and Val285 of DSZS AT (Figure S11). The mutation of these polar residues might remove unfavorable interactions with Leu284 and Val285 of DSZS AT. Structural Comparison with the VinK−VinL Complex Structure. The VinK−VinL complex structure (PDB code 5CZD) was the first crystal structure of AT−ACP complex.14 The ACP Asp residue that is adjacent to the catalytic Ser residue also is important for the interaction between VinK and VinL. In the VinK−VinL complex structure, VinL Asp35, which corresponds to DSZS ACP1 Asp45, is recognized by Arg299 of VinK. However, the orientation of ACP1 in the DSZS AT−ACP1 complex structure is rotated approximately 180° from that of VinL in the VinK−VinL complex structure (Figures 7 and S12). The position of helix II is significantly different between these two complex structures. In the VinK− VinL complex structure, VinK recognizes the helix II region of VinL through several interactions. Glu47 of VinL forms a salt bridge with Arg153 of VinK. Thr39 and Leu43 of VinL form hydrophobic interactions with Met206 of VinK. However, the helix II region of DSZS ACP1 forms almost no interactions with DSZS AT. Although the helix II has catalytic Ser46 at the N-terminus, the C-terminus of helix II is located on the side opposite from the AT-binding site. In addition, the position of helix III′ is different between the two AT−ACP complex structures. The helix III′ region of VinL is located close to the

result was consistent with the previous alanine-scanning mutational study of DSZS ACP1 in which the D45A and F69A mutants exhibited approximately 50% decreases in their transacylation activity levels.16 Thus, DSZS AT appears to recognize Asp45 and Phe69 of DSZS ACP1 in the acyl transfer reaction by forming a salt bridge and a hydrophobic interaction, respectively.

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DISCUSSION Comparison with the Previous Mutational Study. In this study, we have succeeded in the structural determination of DSZS AT−ACP1 complex of disorazole trans-AT PKS. The determined DSZS AT−ACP1 complex structure enables us to understand the accurate binding interface between DSZS AT and ACP1, which was difficult to be elucidated by sequence alignment and biochemical analyses. Although the sequence alignment analysis showed that the helix II and helix III′ residues are relatively conserved in DSZS ACP1−7 (Figure 6), the helix II residues are not recognized by DSZS AT in the complex structure. In the previous alanine-scanning mutational study, the DSZS ACP1 L63A, L68A, L77A, and L81A mutants also showed significant decreases in their transacylation activity levels.16 However, it is unlikely that these Leu residues are involved in the interaction with DSZS AT because they are completely buried in the hydrophobic core of DSZS ACP1 protein in the crystal structure (Figure S11). The mutation of these Leu residues might disrupt the folding or stability of DSZS ACP1 protein. On the other hand, the DSZS ACP1 Q76A and R79A mutants were shown to have 2.5-fold and 2G

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Figure 7. Structural comparison of the DSZS AT−ACP1 complex (A) and the VinK−VinL complex (B). The large subdomain and small subdomain of AT are shown in green and yellow green, respectively. The α13 helix of DSZS AT is shown in cyan. The helix I, helix II, helix III′, helix III, and loop region of ACP are shown in yellow, orange, magenta, red, and gray, respectively. The 4′-phosphopantetheine arm and the catalytic Ser of ACP are shown as sticks.

small subdomain of VinK, while the helix III′ region of DSZS ACP1 is located close to the large subdomain of DSZS AT. Thus, the ACP-binding mode of the DSZS AT−ACP1 complex structure is strikingly different from that of the VinK−VinL complex structure. It is not obvious why their ACP-binding modes are different. Although cross-linking strategies for these two AT−ACP complexes were different, it is unlikely that the different cross-linking strategies gave the different ACP-binding modes because the position of catalytic Ser residue of DSZS ACP1 is almost the same as that of VinL. The ATs and ACPs have some structural differences. For example, VinL has an additional four residues in the helix III′ region (Figure 6). In addition, DSZS AT has a long C-terminal helix (α13) for the interaction with the helix III′ region of DSZS ACP1, while VinK lacks the corresponding long Cterminal helix (Figures 7 and S13). These structural deviations might account for the different ACP-binding modes. AT might differentiate between the binding modes of an acyl acceptor ACP, like DSZS ACP1, and an acyl donor ACP, like VinL. Sequence Comparison with Other PKS ACPs. In kirromycin biosynthesis, the trans-AT KirCII recognizes the module 5 ACP domain of kirromycin PKS (Kirr ACP5) for the transfer of the ethylmalonyl extender unit, while the trans-AT KirCI recognizes other kirromycin PKS ACP domains, such as the module 4 ACP domain (Kirr ACP4) for the transfer of the malonyl extender unit.13,38 DSZS AT has a modest activity for Kirr ACP4 (6% of the activity for DSZS ACP1) but shows no activity against Kirr ACP5.12 This ACP specificity might be attributed to the sequences of Kirr ACP4 and Kirr ACP5. Asp45 and Phe69 of DSZS ACP1 are conserved in Kirr ACP4, whereas Phe69 of DSZS ACP1 is replaced by Gly in Kirr ACP5 (Figure 6). Asp45, Phe69, and Glu70 of DSZS ACP1 are highly conserved in the ACP domains of cis-AT PKSs, such as

DEBS (Figure 6). This might explain why DSZS AT shows only a 5- to 7-fold preference for its cognate DSZS ACPs compared with DEBS ACPs.12 Because of the relatively promiscuous ACP specificity, ACP-based interactions might not be the only factor affecting the docking of trans-acting AT in the trans-AT assembly line. For the acyltransfer reaction, trans-acting AT might also interact with other regions of PKS modules. For example, the region between the KS and the downstream catalytic domains was hypothesized to facilitate the docking of trans-acting AT to the PKS module (Figure S14). 39−41 In the case of the disorazole PKS, the enoylreductase (ER) domain that is fused to the C-terminus of DSZS AT might also be involved in the interaction with the PKS module. To completely understand the docking of the trans-acting AT in trans-AT-containing PKS assembly lines, it is necessary to determine the structure of the whole trans-AT PKS module complexed with the trans-acting AT.

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CONCLUSION In this study, we established a mechanism-based cross-linking method using a bromoacetamide pantetheine probe in combination with a Cys mutation to trap the transient AT− ACP complex, enabling the structural determination of the DSZS AT−ACP1 complex of disorazole trans-AT PKS. This cross-linking method could be useful for studying other AT− ACP interactions. The DSZS AT−ACP1 complex structure provides the first detailed insights into the binding interface between trans-acting AT and ACP in trans-AT assembly lines. The ACP-binding mode in the DSZS AT−ACP1 complex is significantly different from that in the previously reported VinK−VinL complex, suggesting that the AT−ACP-binding interface might depend on the types of AT−ACP pairs. Nevertheless, the DSZS AT−ACP1 complex structure could H

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be useful for the prediction of a model for other trans-AT− ACP complex interactions. Furthermore, the DSZS AT−ACP1 complex structure might provide clues for PKS engineering to optimize protein−protein interactions between AT and ACP for production of unnatural polyketide compounds.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.8b04162. MS data, crystallographic results, cross-linking results, SDS−PAGE, comparison of structures, HPLC results, structures, and NMR spectra (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*[email protected] *[email protected] ORCID

Akimasa Miyanaga: 0000-0003-2219-6051 Fumihiro Ishikawa: 0000-0002-8681-9396 Genzoh Tanabe: 0000-0002-7954-8874 Fumitaka Kudo: 0000-0002-4788-0063 Tadashi Eguchi: 0000-0002-7830-7104 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Michael D. Burkart (University of California, San Diego) for providing the CoaA, CoaD, and CoaE expression constructs. This work was performed with the approval of the Photon Factory Program Advisory Committee (Proposal 2016G624) and was supported in part by the Promotion of Science Grants-in-Aid for Scientific Research (C) 17K07747 (to A.M.), the Ministry of Education, Culture, Sports, Science and Technology Grants-in-Aid for Scientific Research in Innovative Areas 16H06451 (to T.E.), and the Uehara Memorial Foundation (to F.K.).

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REFERENCES

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DOI: 10.1021/jacs.8b04162 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX