Crystal structure determination of a chimeric FabF by XRD

Crystal structure determination of a chimeric FabF by XRD 1 2 first-author 2 2 corresp Corresponding author. E-mail address: ycxu@simm.ac.cn Corresponding author. E-mail address: ycxu@simm.ac.cn ycxu@simm.ac.cn College of Science, Shanghai University, 99 Shangda Road, Shanghai 200444, China College of Science, Shanghai University, 99 Shangda Road, Shanghai 200444, China CAS Key Laboratory of Receptor Research, Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), Shanghai 201203, China CAS Key Laboratory of Receptor Research, Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), Shanghai 201203, China Beta-ketoacyl-acyl-carrier-protein synthase II, an important enzyme in biosynthesis of bacterial fatty acid, is an attractive target in antibacterial drug design. Platensimycin (PTM), produced by Streptomyces platensis , has a strong, broad-spectrum Gram-positive antibacterial activity by selectively targeting to FabF but exhibits no inhibition to the FabF from Streptomyces platensis (spFabF). To study the self-resistance mechanism within the PTM-producing strain and provide hint for development of novel antibiotics, it is imperative to solve the structure of spFabF and elucidate the difference between spFabF and other FabFs which are not resistant to PTM. To this end, we constructed four chimeric FabFs based on the sequence of spFabF and its homologous protein after the expression of wide-type spFabF was failed. The crystal structure of one chimera, js 200 FabF, of 91.2% sequence identity to spFabF, was solved. A structure comparison of js 200 FabF with a PTM-bound FabF suggested that three loops nearby the catalytic site might play key roles in preventing the binding of PTM to spFabF. The results provide an encouraging basis for further studies on the self-resistance mechanism and structure-based design of novel antibiotics targeting FabFs. FabF Chimera X-ray diffraction Crystal structure determination. 1. Introduction 1 Fatty acids are essential building blocks used in assembly of many important bacterial components such as phospholipids, lipoproteins and lipopolysaccharides. Fatty acid synthases (FAS) play crucial roles in biosynthesis of bacterial fatty acids [ 1 1 - 5 5 1-5 ] . While Type I FASes are found in animals, fungi, and lower eukaryotes; Type II enzymes are found in prokaryotes, plastids, and mitochondria. Both types are attractive targets for antibiotics [ 6 6 ] . Beta-ketoacyl-acyl-carrier-protein synthases (KAS) are important members of the FAS(II) family, including KAS I (FabB), KAS II (FabF) and KAS III (FabH). FabH catalyzes the initiation of fatty acid elongation by condensing malonyl-ACP with acetyl-CoA. FabB and FabF, which catalyze acyl-ACP reactions with malonyl-ACP, can produce long-chain acyl-ACPs [ 7 7 ] . FabFs catalyze unique condensation reactions that are essential steps for chain elongation in fatty acid biosynthesis, hence the importance for antibacterial drug discovery. Platensimycin (PTM), produced by Streptomyces platensis ( S. platensis ), has potent, broad-spectrum Gram-positive antibacterial activity and exhibits no cross-resistance to other key antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus ( S. aureus ), vancomycin-resistant Enterococci , vancomycin-intermediate S. aureus , and linezolid- resistant and macrolide-resistant pathogens [ 8 8 ] . The antibacterial effect of PTM was achieved by selectively targeting to FabF/B in the synthetic pathway of fatty acids [ 8 8 - 10 10 8-10 ] . Recently, it was reported that FabF from the PTM-producing S. platensis resisted PTM [ 11 11 ] , but the underlying molecular mechanism is not clear yet. In this work, aimed at providing structural interpretation for the difference between the FabF from S. platensis (spFabF) and other FabFs which are susceptible to PTM, we tried to solve the crystal structure of spFabF. A chimeric protein design strategy was applied to obtain the soluble protein of spFabF. X-ray diffraction of multiple crystals of the chimeric FabF, namely js 200 FabF, was performed on Beamline BL17U1 of Shanghai Synchrotron Radiation Facility (SSRF), with the resolution of 3.15 Å. The structural difference between js 200 FabF and the PTM-bound FabF from E . coli (ecFabF) was discussed for the resistance of spFabF to PTM. 2. Experimental section 1 2.1 Protein expression and purification. 2 The wide-type of full-length spfabF with fusion proteins including TRX, MBP and GST, and four chimeras as well, constructed on the basis of spfabF and its homologous genes Streptomyces albus J1074 fabF (safabF) ( Fig. 1 Fig. 1 ) were subcloned into the vector pET-28a with a TEV protease cleavage site following a six-His tag added at the N-terminus. Restriction sites BamHI and XhoI were used. The DNA sequence of the wide-type spfabF and safabF were synthesized by the company of Generay Biotech ( Shanghai ). The primers used for gene amplification are listed in Table 1 Table 1 . 1 1 js 50 fabF primer 1 1 N-J50 forward primer 1 1 CGCGGATCCATGAGCCCGACCAATCGTACC 1 1   1 1 N-J50 reverse primer 1 1 CTGCCTGTGCTGCAATACGAACCGGCAGGTCCGCATAACGTTC 1 1   1 1 C-S50 forward primer 1 1 GAACGTTATGCGGACCTGCCGGTTCGTATTGCAGCACAGGCAG 1 1   1 1 C-S50 reverse primer 1 1 CCGCTCGAGTTAAACGGTACGAAATGCCAG 1 1 js 100 fabF primer 1 1 N-J100 forward primer 1 1 N-J50 forward primer 1 1   1 1 N-J100 reverse primer 1 1 CACCAATACCGCTTGCAATAACTGTTCCCAGACGTTCCGGGCT 1 1   1 1 C-S100 forward primer 1 1 AGCCCGGAACGTCTGGGAACAGTTATTGCAAGCGGTATTGGTG 1 1   1 1 C-S100 reverse primer 1 1 C-S50 reverse primer 1 1 js 200 fabF primer 1 1 N-J200 forward primer 1 1 N-J50 forward primer 1 1   1 1 N-J200 reverse primer 1 1 CTGCAATCGGCAGCGGATGAATTGCTGCTTCCGTGCCACCTGC 1 1   1 1 C-S200 forward primer 1 1 GCAGGTGGCACGGAAGCAGCAATTCATCCGCTGCCGATTGCAG 1 1   1 1 C-S200 reverse primer 1 1 C-S50 reverse primer 1 1 sj 200 fabF primer 1 1 N-S200 forward primer 1 1 CGCGGATCCATGAATGCAACCAATCGTAC 1 1   1 1 N-S200 reverse primer 1 1 CAACAACCGGCAGCGGATGAATTGCTGCTTCAGTACCACCTGC 1 1   1 1 C-J200 forward primer 1 1 GCAGGTGGTACTGAAGCAGCAATTCATCCGCTGCCGGTTGTTG 1 1   1 1 C-J200 reverse primer 1 1 CCGCTCGAGTTAAACGGTACGAAATGCCAG The recombinant plasmids were transformed into E. coli BL21 (DE3) cells [ 12 12 ] . Cells harboring this expression plasmid were grown in LB containing 50 μg/mL kanamycin to the mid-log phase at 37℃. Subsequently, the culture was induced by 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubated for 14–16 hours at 16℃. The cell pellets were harvested, re-suspended, and lysed with the buffer consisting of 20 mM Tris pH 8.0, 10% (v/v) glycerol, 500 mM NaCl, 5 mM BME (β-mercaptoethanol), and 20 mM imidazole. The lysate was centrifuged. The supernatant was loaded on a 10-ml Ni-NTA Sepharose column ( GE Healthcare ). The column was washed with buffer A containing 20 mM Tris pH 8.0, 10% (v/v) glycerol, 500 mM NaCl, 5 mM BME, and 80 mM imidazole, until no further protein was detected by the Bio-Rad protein-assay kit, and then eluted with buffer B containing 20 mM Tris pH 8.0, 10% (v/v) glycerol, 500 mM NaCl, 5 mM BME, and 250 mM imidazole. The eluted fraction was concentrated and further loaded on the Superdex TM 200 20/300 GL column ( GE Healthcare ) which is equilibrated in a gel filtration buffer consisting of 20 mM Tris pH 8.0, 100 mM NaCl, 10% (v/v) glycerol, 1 mM DL-Dithiothreitol (DTT). The eluted protein was concentrated to 10 mg/ml for crystallization. 2.2 Protein crystallization 2 Screening of the initial crystallization conditions was carried out using the sparse matrix approach with a series of kits including Index, Crystal Screen, Crystal Screen 2, PEG/Ion, PEG/Ion 2, SaltRx, SaltRx 2, PEGRx, PEGRx 2 ( Hampton Research ), and Wiz1-4 ( Emerald BioSystems ). Crystallization was performed at 4℃ and 20℃ using a sitting-drop vapour- diffusion method by mixing equal volumes of the protein solution (1 μL) and reservoir solution (1 μL). Once the crystal was grown, the vapour-diffusion method in hanging drops was utilized to optimize the condition. The perfluoropolyether PFO-X175/08 (Hampton Research) [ 13 13 ] was used as a cryoprotectant for all of the crystals. The crystals were picked up with cryo-loops and flash-frozen in liquid nitrogen. 2.3 Data collection and processing ( Table 2 Table 2 ) 2 2 1 Data collection 1 1   1 1   1 1 Wavelength (Å) 1 1 0.97923 1 1   1 1 Temperature (K) 1 1 100 1 1   1 1 Resolution (Å) 1 1 50–3.15(3.26–3.15) 1 1   1 1 Total No. of reflections 1 1 1326684 1 1   1 1 No. of unique reflections 1 1 24808 1 1   1 1 Redundancy 1 1 10.5 1 1   1 1 Completeness (%) 1 1 94.4 1 1   1 1 Mean I/σ 1 1 12.7 1 1   1 1 R merge 1 1 0.174 1 1   1 1 Space group 1 1 P 3 2 21 2 1 Unit cell parameters 1 1   1 1 a (Å) 1 1 160.27 1 1   1 1 b (Å) 1 1 160.27 1 1   1 1 c (Å) 1 1 95.57 1 1   1 1 α , β , γ , (°) 1 1 90, 90, 120 2 1 Refinement 1 1   1 1 R work 1 1 0.1958 1 1   1 1 R free 1 1 0.2790 2 1 R.m.s.d from ideality 1 1   1 1 Bonds (Å) 1 1 0.013 1 1   1 1 Angles (deg) 1 1 1.476 2 1 Ramachandran plot 1 1   1 1 Allowed regions (%) 1 1 95.1 1 1   1 1 Disallowed regions (%) 1 1 4.9 X-ray diffraction data were collected at 100 K on Beamline BL17U1 at SSRF [ 14 14 ] , with the wavelength of 0.97923 Å and the crystal-to-detector distance of 250 mm. A total of 360 frames were collected with 0.5°oscillation and 1 s exposure time per frame. The data were processed with the HKL-2000 software package [ 15 15 ] . Molecular replacement was performed with Phaser [ 16 16 ] using the structure of Thermus thermophilus FabF (PDB code 1J3N) [ 17 17 ] as a search model. The structure was refined with the Phenix code [ 18 18 ] . Using the Coot code [ 19 19 ] , water molecules were fitted into to the initial F o -F c map. 3. Results and Discussion 1 3.1 Design of chimeric FabFs 2 The expression and purification of the wide-type spFabF with different fusion proteins in E. coli , as described in Section 2, led to the aggregates of the protein. A chimeric protein design strategy was subsequently applied. Sequence alignment suggested that saFabF might be of the highest sequence identity to spFabF (76.4%). The chimeric FabFs were thus constructed with the combination of spFabF and saFabF. In general, the N-terminal region of saFabF was used in the chimeric proteins because that its sequence was more identical than the C-terminal to spFabF. The first 200 amino acid residues of saFabF showed 81.5% sequence identity to the corresponding residues of spFabF. Four chimeras, namely js 50 FabF, js 100 FabF, js 200 FabF, and sj 200 FabF, were designed ( Fig. 1 Fig. 1 ). Taking js 200 FabF as an example, the first 200 amino residues from the N-terminal of saFabF and the last 200 residues from the C-terminal of spFabF were combined ( Fig. 1 Fig. 1 ). The resulted js 200 FabF had 91.2% sequence identity to the wide-type of spFabF. The chimeric proteins were also expressed in E. Coli. The overexpressed proteins were purified by a Ni-NTA affinity chromatography together with a gel-filtration chromatography to obtain the homogeneous soluble proteins. The gel- filtration chromatographic experiment suggested that only js 200 FabF might have proteins of low molecular weight, while the other three were aggregates. A further characterization of the low molecular weight samples with the analytical size exclusion chromatography showed that a single peak eluting at about 13.49 mL, which is usually corresponding to a protein of molecular weight around 90 kDa ( Fig. 2 Fig. 2 ). It is thus conjectured that js 200 FabF was a comparatively stable dimer in solution. The SDS-PAGE showed that the proteins are quite pure (purity over 95%). 3.2 Screening of crystallization conditions for js 200 FabF 2 After the pure and dimeric js 200 FabF were concentrated to 10 mg/mL, a crystallization condition screening was carried out using multiple screening kits. Rod crystals of the protein were obtained under conditions containing salts (the most), or PEGs (fewer) as a precipitating agent. However, these crystals had the low-resolution diffraction properties. The crystals from the condition No. 46 of Crystal Screen 2 consisting of 0.1 M NaCl, 0.1 M BICINE pH 9.0, 20% (v/v) PEG MME 550 ( Fig. 3a Fig. 3a ) and the condition No. 30 of PEGRx consisting of 0.1 M HEPES pH 7.5, 12% PEG 3350 ( Fig. 3b Fig. 3b ) were in bar-shapes. The crystal with the best diffraction was obtained using a reservoir solution consisting of 0.1 M Bis-Tris pH 5.5, 25% PEG 3350, 0.2 M ammonium sulfate ( Fig. 3c Fig. 3c ). The crystals grown at 4℃ often showed better diffraction quality than those grown at 20 ℃. However, the crystal growth at 4℃ took about half a year before it was ready for XRD experiment. Besides, the six-His tag at the N-terminal of js 200 FabF showed no apparent effects on crystallization and diffraction. 3.3 Structure determination 2 Crystals of js 200 FabF were flash-cooled in liquid nitrogen with a cryoprotectant and examined by XRD. An example of the XRD results is shown in Fig. 4 Fig. 4 . The data processing indicated that it belonged to the cubic space group P 3 2 21, with unit-cell parameters of a =160.27 Å, b =160.27 Å, and c = 95.57 Å. The calculated Matthews coefficient (V M ) was 2.32 Å 3 /Da with 47.01% solvent content, assuming the presence of six molecules in the asymmetric unit [ 20 20 ] . The structure of js 200 FabF was finally solved with the best resolution of 3.15 Å. PTM is a potent inhibitor of FabF from E. coli (ecFabF) and the crystal structures of the mutant ecFabF(C163Q and C163A) in complex with PTM are the only known structure of FabF bound with PTM [ 8 8 , 21 21 ] . Although the sequence identity of js 200 FabF to ecFabF was 39.8%, the superimposition of two structures in Fig. 5(a) Fig. 5(a) revealed that the topology of js 200 FabF was similar to ecFabF. In addition, according to the complex structure of PTM bounded ecFabF(C163Q), the binding site for PTM in js 200 FabF should be located at the C-terminal region, and the sequence was completely identical to the C-terminal of spFabF ( Fig. 5b Fig. 5b ). This means that js 200 FabF can be regarded as spFabF in studying the PTM binding to spFabF. Exploring the binding pocket of PTM in ecFabF(C163Q) and spFabF, we found that the three loops (residues 211-215, 277-281 and 407-411) adopted different conformations as compared with them in ecFabF(C163Q), resulting that the assumed binding pocket of PTM in spFabF was greatly occupied by these loop residues and the superimposed PTM had many bumping with the neighboring residues of spFabF ( Figs. 5c and 5d Figs. 5c and 5d ). Therefore, the different conformations of three loops may account for the resistance of spFabF to PTM. 4. Conclusion 1 We constructed four chimeric FabFs based on spFabF from the PTM-producing S. platensis and its homologous protein in an attempt to solve the structure of spFabF and interpret its resistance to PTM which is a potent and selective inhibitor of bacterial FabFs. One of the chimeras, js 200 FabF, could be expressed in E. Coli and purified with a pure and stable dimer protein for crystallization. The protein crystal best diffracted was grown in the condition containing 0.1 M Bis-Tris pH 5.5, 25% PEG 3350, and 0.2 M ammonium sulfate. The solved structure shown that js 200 FabF has a similar topology to other FabFs such as ecFabF. As compared to the structure of ecFabF in complex with PTM, three loops nearby the catalytic site change their conformations, reduce the volume of the assumed binding pocket and thus prevent the binding of PTM to spFabF. 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(Science Press), Shanghai Institute of Applied Physics, the Chinese Academy of Sciences, ChineseNuclear Society and Springer Nature Singapore Pte Ltd. 2017 This is a post-peer-review, pre-copyedit version of an article published in Nuclear Science and Techniques. The final authenticated version is available online at: http://dx.doi.org/10.1007/s41365-017-0281-0 http://dx.doi.org/10.1007/s41365-017-0281-0 . 2017 Nuclear Science and Techniques 09 28