A.1.1 Subcloning of pyc from Aspergillus nidulans
The subcloning of the pyc gene from Aspergillus nidulans into the pET-17b vector for recombinant protein expression was completed in the lab of Dr. Sarawut Jitrapakdee (Department of Biochemistry - Mahidol University, Bangkok, Thailand).
During protein crystallization trials (see Protein Crystallization), it was discovered the holoenzyme of AnPC does not yield crystals capable of diffracting X-rays to a
resolution suitable for structure determination. After aligning the protein sequence of
AnPC against other species-specific PC enzymes, it was realized AnPC contains a N-
terminal sequence (42 amino acid) leading into the BC domain, which is conserved only in PC from Aspergillus niger. In an attempt to enhance crystal lattice packing and X-ray diffraction quality, the N-terminal portion of AnPC (ΔN-AnPC) was removed by deleting a 126-nucleotide sequence using a single cloning step as previously described (Makarova, Kamberov, & Margolis, 2000)with the AnPC pET-17b vector as the cloning template. The primers used for cloning ΔN-AnPC are as follows: (forward) 5’-CCA CCA CCA TCA TCA CCA CCA CCA CAT GCA GTT CCA GAA GAT CCT CGT CG-3’ and (reverse) 5’-CGA CGA GGA TCT TCT GGA ACT GCA TGT GGT GGT GGT GAT GAT GGT GGT GG-3’.
A.1.2 Protein Expression and Purification
AnPC and ΔN-AnPC were expressed and purified in a similar manner. AnPC in a pET-
17b-(His)9 plasmid was co-expressed in E. coli BL21Star(DE3) with E. coli biotin protein
ligase A (BirA) on vector pCY216. AnPC was expressed in M9 minimal media
containing 200 mg/L ampicillin and 30 mg/L chloramphenicol. Cultures were grown at 37°C to an Optical Density (600 nm) of 0.8 – 0.9 and immediately supplemented with D-
biotin and MnCl2 to a final concentration of 4 mg/L and 80 µM, respectively. The cells
were placed on ice for ~ 10 minutes and then were induced with IPTG and L-arabinose to 1 mM and 25 mM, respectively. Following induction, cultures were grown at 16°C for 48 hours prior to harvesting.
The AnPC proteins were purified to homogeneity using Ni2+
-affinity and anion exchange chromatography. Harvested cells were re-suspended in “Buffer A” containing
20 mM Tris-HCl (pH 7.5), 5 mM imidazole, 200 mM NaCl, 10 mM MgCl2, 0.5 mM
EGTA, 6 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 µM pepstatin A, 5 µM E-64, and 200 µg/mL lyzozyme. Cells were disrupted by sonication and the cell lysate was cleared by centrifugation at 10°C prior to loading onto a 10 mL Ni2+
-NTA Profinity resin column (Bio-Rad Life Sciences; Hercules, CA). The protein was eluted from the column in Buffer A, with a gradient from 20 mM to 250 mM imidazole. Purified protein samples were pooled and dialyzed overnight in “Buffer B”
containing 20 mM triethanolamine (pH 8.0), 50 mM NaCl, 3 mM MgCl2, 1 mM EGTA,
and 2 mM DTT at 4°C. Protein was loaded on a 10 mL volume of Q-sepharose Fast Flow resin (GE Healthcare) and was subsequently eluted form the resin in Buffer B using a
linear gradient from 50 mM to 1 M NaCl. Fractions were pooled and dialyzed against a
storage buffer consisting of 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 10 mM MgCl2, 5%
(v/v) glycerol and 2 mM DTT. An Amicon stirred cell with a 100 000 molecular weight cutoff filter was used to concentrate the AnPC preparations to a range of 10 – 15 mg/mL. Concentrated protein was flash frozen in liquid nitrogen and stored at -80°C. AnPC and ΔN-AnPC protein concentration were determined spectrophotometrically using the calculated molar extinction coefficient of 94 660 M-1
cm-1
and 93 920 M-1
cm-1
, respectively at 280 nm (Gasteiger et al., 2005).
A.1.3 Reductive Alkylation of ΔN-AnPC
ΔN-AnPC crystallizes in the space group P4 with unit cell dimensions of a=b=331 Å, c=202 Å and α=β=γ=90°. Due to the high solvent content (77% assuming 2 tetramers of ΔN-AnPC in the asymmetric unit), poor diffraction resolution (~7 Å), increased
susceptibility to radiation damage, and a lack of diffraction improvement using various dehydration techniques, an alternate crystal form was required for structure
determination. Primary amines of ΔN-AnPC were therefore reductive methylated using borane dimethylamine and formaldehyde as previously described (Rayment, 1997). Prior to reductive methylation, protein was purified as described in Protein Expression and Purification with the Tris-HCl and Trienthanolamine buffers of the protocol being replaced with HEPES. The reductive methylation reaction proceeded for 24 hours at 4°C and was quenched with excess glycine for 1 hour. The purified methylated protein was
dialyzed against 10 mM HEPES (pH 7.5), 50 mM NaCl, 10 mM MgCl2, 2 mM DTT, and
storage at -80°C.
A.1.4 Protein Crystallization
Reductive methylated ΔN-AnPC (rmΔN-AnPC) was crystallized using the batch crystallization method under oil. A protein solution consisting of 11.5 mg/mL rmΔN-
AnPC, 15 mM pyruvate and 5 mM AMP-PNP was mixed at a 1:1 ratio with a precipitant
solution comprised of 9.2% (w/v) PEG 8000, 80 mM BisTris (pH 6.0), and 390 mM tetramethylammonium chloride (TMACl). A seed stock was created using the seed bead kit from Hampton Research (Aliso Viego, CA). Briefly, a single rmΔN-AnPC crystal was pulverized in 500 µL of precipitant solution and 0.5 µL of the seed solution was added to the crystallization drop immediately following mixing. The drop was covered with paraffin oil and cuboid shaped crystals (300 µm × 250 µm × 200 µm) formed within 2-3 weeks (Figure A1). After 2-3 months, the crystals equilibrated for 5-10 minutes in a synthetic mother liquor consisting of 10% (w/v) PEG 8000, 70 mM BisTris (pH 6.0), 220 mM TMACl, 5% (v/v) glycerol, 15 mM pyruvate and 2.5 mM AMP-PNP. Crystals were subsequently transferred to a cryoprotectant consisting of 26% (w/v) PEG 8000, 70 mM BisTris (pH 6.0), 220 mM TMACl, 14% (v/v) glycerol, 15 mM pyruvate, and 2.5 mM AMP-PNP and flash cooled in liquid nitrogen.
A.1.5 Data Collection and Structure Determination
X-ray diffraction data were collected at the Advanced Photon Source (APS), beamline LS-CAT 21-ID-D on a Rayonix MarMosaic 300 CCD detector. The X-ray wavelength
Figure A1. Protein crystal of rmΔN-AnPC in a cryo-loop at beamline APS 21-ID-D.
was tuned to 0.976 Å. Diffraction images were processed with the HKL2000 suite (Otwinowski & Minor, 1997). The structure was solved by molecular replacement using the program Phaser (McCoy et al., 2007). To obtain a molecular replacement solution, four biotin carboxylase (BC) domains (residues 36-489) and four pyruvate
tetramerization + caroxyltransferase (PT+CT) domains (residues 495-1091) from wild- type SaPC enzyme (pdb i.d. 3BG5) were used as the search models. Following molecular replacement, translation/liberation/screw (TLS) refinements were performed using REFMAC (Murshudov, Vagin, & Dodson, 1997; Winn, Isupov, & Murshudov, 2001; Winn, Murshudov, & Papiz, 2003). Each domain was treated as a rigid TLS group. All BC and CT domains in the asymmetric unit were restrained using non-crystallographic symmetry during the entire refinement process. The program AutoBuild assisted with building amino acid side chains prior to several rounds of manual building with COOT
(Adams et al., 2010; Emsley & Cowtan, 2004; Terwilliger et al., 2008). Data collection and processing statistics are summarized in Table A1.
A.1.6 Enzyme Assays.
The reaction conditions for the assays performed in order to evaluate various properties of AnPC wt or ΔN-AnPC are included for each figure or table of data.