Acylated-acyl carrier protein stabilizes the Pseudomonas aeruginosa WaaP lipopolysaccharide heptose kinase

Phosphorylation of Pseudomonas aeruginosa lipopolysaccharide (LPS) is important for maintaining outer membrane integrity and intrinsic antibiotic resistance. We solved the crystal structure of the LPS heptose kinase WaaP, which is essential for growth of P. aeruginosa. WaaP was structurally similar to eukaryotic protein kinases and, intriguingly, was complexed with acylated-acyl carrier protein (acyl-ACP). WaaP produced by in vitro transcription-translation was insoluble unless acyl-ACP was present. WaaP variants designed to perturb the acyl-ACP interaction were less stable in cells and exhibited reduced kinase function. Mass spectrometry identified myristyl-ACP as the likely physiological binding partner for WaaP in P. aeruginosa. Together, these results demonstrate that acyl-ACP is required for WaaP protein solubility and kinase function. To the best of our knowledge, this is the first report describing acyl-ACP in the role of a cofactor necessary for the production and stability of a protein partner.

Expression and purification of WaaP for crystallography E. coli BL21(DE3)/pLysS was freshly transformed with pET21b-waaP [PT7::PawaaP-his6]. The fresh transformants were inoculated in SelenoMet medium base (Molecular dimensions) supplemented with 100 µg/mL carbenicillin (Cb), 34 µg/mL chloramphenicol (Cm), and 8 µg/mL methionine. After overnight incubation, the cultures were centrifuged and the pellets were resuspended in fresh medium described above with methionine replaced with selenomethionine. Cells were grown to the OD600 of 0.6 and cooled to 25°C prior to addition of 1 mM IPTG and grown for 4 hours at 25°C. The cells were harvested by centrifugation at 250 rpm and resuspended (5 mL per gram cell pellet) in buffer A (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 50 mM arginine, 50 mM glutamic acid, 1 mM TCEP) supplemented with 20 mM imidazole, 1 mM PMSF, and complete Protease Inhibitor Cocktail Tablets (1 tablet/50 mL -Roche Biochemicals).
The cell suspension was homogenized on ice using a Polytron Mechanical Homogenizer (1x 30 sec) and then lysed with a micro-fluidizer (Microfluidics). The lysate was clarified by ultracentrifugation (138,000 x g, 60 min, 4°C) and passed through a batch-gravity column containing 5 mL volume equivalents of pre-washed IMAC sepharose fast flow resin (GE Healthcare). The resin was washed with 5 column volumes (CV) of lysis buffer before bound protein was eluted in 5 CV buffer A with imidazole. WaaP-His containing fractions were pooled and adjusted to 100 mM NaCl by dilution. At this stage, 1 mM EDTA added to all subsequent purification steps. WaaP-His was further purified from contaminants by ion exchange chromatography using Mono-Q and CM-sepharose columns followed by size-exclusion chromatography using a Superdex75 column (GE Healthcare). WaaP-His containing fraction were pooled and stored at 3 mg/mL in buffer A.

Crystallization and structure determination
WaaP-His in buffer (20 mM Tris-HCl pH 8.0, 100mM NaCl, 500 mM ammonium acetate, 1 mM TCEP) was mixed with equal volumes of crystallization buffer (100 mM HEPES-NaOH pH 7.4, 5% Jeffamine M-600) and crystals were grown via hanging drops at 4°C over the course of 48 hours. Crystals were flash frozen using 20% ethylene glycol as a cryo-protectant and data collected at 100K with monochromatic X-rays at a wavelength of 0.9791Å using a Dectris Pilatus 6M detector on the PXII-X10SA beamline at the Swiss Light Source, Paul Scherrer Institut, Villigen, Switzerland. Data were integrated and scaled using the XDS package 3 and selenium sites identified using the SHELX software package 4 . Data diffracted to 2.5Å, albeit with a high Wilson B-factor of 69.5 Å 2 , and a space group of P3121 and unit cell dimensions of a=92.023 Å, b=92.023 Å, c=99.172Å. The electron density map generated using this information combined with solvent flattening restraints was of such quality that ~80% of the protein structure could be readily identified. Extra density and an unaccounted for selenium position led to the discovery of E. coli ACP protein bound to WaaP with an extended lipid attachment.
Model building and refinement were carried out using COOT 5 and PHENIX 6

Construction of the E. coli waaP deletion strain
The pKOV-EcwaaP::Gm plasmid was inserted onto the MC1061 chromosome using transformation and colonies were selected on L agar supplemented with 10 µg/mL gentamicin (Gm) and 20 µg/mL Cm. Sucrose selection was used to isolate colonies with double crossover events as described previously 7 .
WaaP kinase activity assay A radioactive filter binding assay was used to monitor the kinase reaction. The LPS substrates were prepared from E. coli MC1061 and the waaP::Gm strain as described Laemmli sample buffer and incubated at 95°C for 5 min. Fifteen microliter of each sample was loaded onto a SDS gradient gel (BioRad 4-20%) and separated by electrophoresis. The gel was dried on a Whatman paper and exposed for 72 hours to Amersham hyperfilm ECL.
Expression and purification of E. coli holo-ACP E. coli His-TEV-ACP was expressed in BL21-AI without and with co-expression of E.
Expression and purification of V. harveyi acyl-ACP synthase Acyl-ACP synthase AasS from V. harveyi was expressed and purified from BL21-AI carrying pJ414-AasS-His6 as described above with modifications. Briefly, Protease inhibitors and universal nuclease (Thermo Fisher) were added to the cell suspension the cells were lysed by sonication. His-tagged AasS was purified using a cOmplete™ His-Tag Purification Column (Roche) with a linear gradient from 0 to 500 mM imidazole in buffer (50 mM HEPES-NaOH pH 7.5, 500 mM NaCl, 10 % glycerol, 1 mM TCEP).
In vitro protein synthesis PURExpress in vitro protein synthesis Kit (New England Biolabs) was used following the instruction with modifications. A PCR product containing a T7 promoter, codon optimized waaP-flag, and a T7 terminator, was amplified using pETite-waaP-FLAG as a template and primers NK218 and NK219. The reaction was performed at 37°C for 2. Cell-based functional assay for WaaP variants in P. aeruginosa and E. coli The WaaP functional assays in P. aeruginosa and in E. coli were performed using growth and EDTA MIC of P. aeruginosa waaP-controlled expression strain (CDR0031) 1 and novobiocin (Nov) MIC of E. coli ΔwaaP 9 , respectively. CDR0031 and E. coli ΔwaaP were transformed with plasmids expressing WaaP wild type and variants (pAK1900-waaP and derivatives). Susceptibility of P. aeruginosa CDR0031-derived strains to EDTA was determined using a broth serial dilution assay. Strains were streaked on LB agar supplemented with 0.2% arabinose and Cb 100 µg/mL and incubated at 37°C overnight. Single colonies were used to prepare standardized cell suspension (approximately 1.5 x 10 8 colony forming units (CFU)/mL) with the BBL Prompt inoculation system (BD). The cell suspensions were diluted 1,000 fold in LB. The diluted cell suspensions were added to two-fold serial dilutions of EDTA ranging the final concentrations from 0 to 10 mM in LB in 96-well microtiter plates that were dispensed with a Janus liquid handler (Perkin Elmer). The microtiter plates were incubated overnight at 37°C and were monitored for bacterial growth with a Spectromax microtiter plate reader (Molecular Devices) at 600 nm as well as by visual observation. EDTA MIC was defined as the lowest concentration of EDTA at which less than 10% of the OD600 in the control well (full growth) was measured. All measurements were performed in a minimum of biological triplicate. Likewise Nov MIC for E. coli ΔwaaP strains expressing WaaP variants was determined using concentrations of Nov ranging from 0 to 128 µg/mL.

Immunoblotting to monitor expression levels of WaaP variants
Overnight cultures of P. aeruginosa CDR0031 and E. coli ΔwaaP carrying pAK1900-waaP plasmids expressing wild type WaaP-His6 and variants were inoculated in LB supplemented with 0.2% arabinose, and 100 µg/mL Cb for the P. aeruginosa strains or 30 µg/mL Cb for the E. coli strains, respectively. The fresh culture was grown at 37°C to mid-exponential phase (OD600 = 0.4 -0.6). Six milliliter of the P. aeruginosa CDR0031 cultures and 1 mL of the E. coli ΔwaaP cultures were pelleted and resuspended in 50 mM MOPS-NaOH pH 7.0 to a final volume of 100 µL for E. coli and 120 µL for P.
aeruginosa. An equal volume of 2x Laemmli sample buffer was added and the samples were boiled. The volumes normalized by cell density (OD600) were loaded onto two 6-12% Bis-Tris SDS-PAGE gels and PAGE was run concurrently. One gel was stained with Coomassie Brilliant Blue Gels to confirm the equivalent protein levels of samples.
The other gel was transferred to nitrocellulose membranes using the iBlot system (Thermo Fisher) and anti-His WaaP was detected with the iBind system (Thermo Fisher) using mouse monoclonal anti-His primary antibody (THE TM His-tag Antibody, GenScript, A00186) and IRDye 800 CW donkey anti-mouse IgG.

WaaP-His pulldown in P. aeruginosa
Cells were grown with 1% inoculum from overnight culture of P. aeruginosa CDR0031 carrying pAK1900-waaP or pMM-waaP into 6 L LB supplemented with either 100 µg/mL Cb for pAK1900-waaP or 50 µg/mL Cm and 1 mM IPTG for pMM-waaP. Cells were grown at 37°C with shaking to OD600 of 1.0, harvested, and frozen at -20°C. Two procedures were used to resuspend and lyse cells. One was resuspending the cell pellets in 5 mL BugBuster (Millipore Sigma) per gram of cell paste followed by cell lysis

Deacylation of acyl-AcpP
The WaaP-His elution was treated with 500 mM DTT overnight at room temperature to reduce the thioester bond between an acyl chain and the phophopentheine moiety of holo-AcpP. After the sample was centrifuged to remove precipitates, the supernatant was run on LC-MS as described above.

Trypsin protein digestion and MS analysis
The WaaP-His pulldown elution was diluted 20 fold with 100% ethanol and incubated overnight at -80°C. The precipitated protein was pelleted by centrifugation and   Protein levels of WaaP variants expressed in exponentially growing cells of P. aeruginosa CDR0031 (a) and E. coli ΔwaaP (b). Cells were collected and subjected to SDS-PAGE and anti-His western blot analysis, where loading amounts were normalized to OD600. SDS-PAGE verified that the approximately same amounts of total proteins were loaded onto each western blot. Cells expressing wild type WaaP-His and purified WaaP-His protein (12.5, 25, 50, and 100 ng) were loaded for comparative controls. Gels shown are representatives of biological duplicates. These results are summarized in Fig  2b, 4b, 5b and Supplementary Table 2.
Supplementary Figure 4. In vitro synthesis of soluble WaaP only in the presence of acyl-ACP.
WaaP-FLAG was expressed from a DNA fragment containing PT7::waaP-FLAG using an in vitro transcription and translation system in the absence of ACP or in the presence of apo-ACP (unmodified ACP), holo-ACP (phosphopentheinylated ACP), palmitoyl-ACP, or free palmitic acid. Sypro Orange stained protein gel (left) and the uncropped immunoblot with anti-FLAG antibody (right) are shown. WaaP was produced in every reaction sample at similar level (left side of both gels). After ultracentrifugation, WaaP was only present in the supernatant when expressed in the presence of acyl-ACP (right side of both gels).
showing lower expression from pAK1900-waaP. (c,d) Confirmation of acyl-AcpP peaks using thioester cleavage of the acyl chain by reduction with DTT. Peaks in the 9140-9220 mass range in the WaaP-His pull down sample from CDR0031 carrying pMM-waaP before (c) and after treatment of DTT (d) were shown. The trace in d shows a loss of acyl-AcpP species with a concomitant increase in holo-AcpP after overnight incubation with DTT.