Structural, Kinetic and Proteomic Characterization of Acetyl Phosphate-Dependent Bacterial Protein Acetylation

<div><p>The emerging view of N^ε-lysine acetylation in eukaryotes is of a relatively abundant post-translational modification (PTM) that has a major impact on the function, structure, stability and/or location of thousands of proteins involved in diverse cellular processes. This PTM is typically considered to arise by the donation of the acetyl group from acetyl-coenzyme A (acCoA) to the ε-amino group of a lysine residue that is reversibly catalyzed by lysine acetyltransferases and deacetylases. Here, we provide genetic, mass spectrometric, biochemical and structural evidence that N^ε-lysine acetylation is an equally abundant and important PTM in bacteria. Applying a recently developed, label-free and global mass spectrometric approach to an isogenic set of mutants, we detected acetylation of thousands of lysine residues on hundreds of <i>Escherichia coli</i> proteins that participate in diverse and often essential cellular processes, including translation, transcription and central metabolism. Many of these acetylations were regulated in an acetyl phosphate (acP)-dependent manner, providing compelling evidence for a recently reported mechanism of bacterial N^ε-lysine acetylation. These mass spectrometric data, coupled with observations made by crystallography, biochemistry, and additional mass spectrometry showed that this acP-dependent acetylation is both non-enzymatic and specific, with specificity determined by the accessibility, reactivity and three-dimensional microenvironment of the target lysine. Crystallographic evidence shows acP can bind to proteins in active sites and cofactor binding sites, but also potentially anywhere molecules with a phosphate moiety could bind. Finally, we provide evidence that acP-dependent acetylation can impact the function of critical enzymes, including glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, and RNA polymerase.</p></div

<i>In vitro</i> acetylation of LpdA using acP as the acetyl group donor is sensitive to acP concentration and time of incubation.

<p>Coomassie stain of SDS-polyacrylamide gel and anti-acetyllysine Western immunoblot analysis of acP (5, 10, 15, 20 mM) incubated with 1.25 µM LpdA for various lengths of time (5, 10, 15, and 30 min, 1, 2, 3, 4, 5, and 7 hours) at 37°C. Acetylation signals were quantified using AlphaView and normalized to the signal in the absence of acP.</p

<i>In vitro</i> acetylation of RNAP with AcP.

<p>Purified RNAP σ⁷⁰ holoenzyme was incubated with increasing concentration of acP for 1 hour at 30°C. After incubation, proteins were resolved by SDS-PAGE and transferred to a PVDF membrane. Protein acetylation was detected using a cocktail of two polyclonal anti-acetyllysine antibodies was used (Cell Signaling Technology) at a 1∶200 dilution and (ImmuneChem) at a 1∶500 dilution. Acetylation signals were quantified using AlphaView and normalized to the signal in the absence of acP.</p

Crystal structure of glyceraldehyde-3-phosphate dehydrogenase (GapA) from <i>E. coli</i> in native (PDB ID: 1S7C) and acP-modified (PDB ID: 4MVJ) forms.

<p><b>A</b>) Surface representation of GapA with the locations highlighted in red of up-regulated acetylated lysine residues in the <i>ackA</i> mutant as determined by mass spectrometry. Front and back views of the protein are shown and NAD⁺ (shown with sticks) is bound at the active site pocket. <b>B</b>) Electron density map surrounding acetylated K46 in the GapA crystal structure. The F_o-F_c omit map in blue mesh is contoured at the 3 sigma level. Residues are shown as sticks and water is represented as a sphere. <b>C</b>) Electron density map surrounding acetylated K249 in the GapA structure. The F_o-F_c omit map in blue mesh is contoured at the 2 sigma level. <b>D</b>) Electron density map surrounding acetylated K257 in the GapA structure. The F_o-F_c omit map in blue mesh is contoured at the 2 sigma level. Oxygens, nitrogens, and carbons are shown in red, blue and light blue, respectively. <b>E, F</b>) Overlay of the K249 and K257 residue of GapA (PDB ID: 4MVJ) in its acetylated and non-acetylated form, respectively. Phosphate is present in the non-acetylated form and binds in the same location as the acetyl group of the acetylated residue. The protein with the non-acetylated lysine and phosphate bound is shown in orange, the protein with the acetylated lysine is in teal, oxygen atoms are in red and nitrogens are in blue.</p

Mass spectrometric workflow.

<p><b>A</b>) <i>E. coli</i> strains MG1655 (WT), AJW5052 (<i>ackA</i>), AJW2785 (<i>pta ackA</i>), AJW5037 (<i>cobB</i>), and AJW5164 (<i>yfiQ</i>) were aerated at 37°C in TB7 (3 independent biological replicates) or TB7 supplemented with 0.4% glucose (4 independent biological replicates) and harvested when the OD₆₁₀ reached 1.0. For mass spectrometric analysis, the harvested cells were lysed and the protein lysates were proteolytically digested with trypsin, followed by affinity enrichment for acetyllysine (Ac-Lys)-containing peptides using a polyclonal anti-acetyllysine antibody. Enriched Ac-Lys peptides for each strain/condition were analyzed by high-resolution label-free LC-MS/MS (3 technical replicates each) for acetyl site identification, and these Ac-Lys sites were subsequently subjected to MS1- and MS2-based quantification methods. <b>B</b>) Skyline MS1 Filtering for acetylated peptide NLDAG<b>Kac</b>AGVEVDDR (^AcK284) obtained from dihydrolipoyl dehydrogenase (LpdA) to determine quantitative differences between the <i>ackA</i> mutant (strain AJW5052) and its WT parent (strain MG1655). MS1 ion chromatograms and corresponding peak areas demonstrating one of four biological replicates is shown for WT and <i>ackA</i> mutant (3 technical MS replicates acquired); precursor ions were extracted for M at <i>m/z</i> 750.87⁺⁺, M+1 at <i>m/z</i> 751.37⁺⁺, M+2 at <i>m/z</i> 751.87⁺⁺. <b>C</b>) Independent confirmation and validation of potential candidates and sites for acetyllysine regulation using SWATH MS2 Filtering (SWATH MS2): exemplified for NLDAG<b>Kac</b>AGVEVDDR (^AcK284): 1 biological replicate (3 technical replicates acquired) shown with extracted ion chromatograms and corresponding peak areas for fragment ions y₁₂ at <i>m/z</i> 1273.60⁺, y₁₁ at <i>m/z</i> 1158.57⁺, y₁₀ at <i>m/z</i> 1087.54⁺, y₈ at <i>m/z</i> 860.41⁺, y₇ at <i>m/z</i> 789.37⁺, and y₅ at <i>m/z</i> 633.28⁺.</p

Analysis of the amino acid composition and position relative to acetylated lysines.

<p><b>A</b>) Using WebLogo, a consensus sequence logo was generated showing the amino acid composition in positions −10 to +10 relative to 541 <i>ackA</i>-sensitive lysines (i.e., robustly, significantly and reproducibly more acetylated in the <i>ackA</i> mutant relative to WT (<i>ackA</i>/WT ratio >2 with a p-value<0.05 in at least 3 out of 4 biological replicates). Using WebLogo, consensus sequence logos were generated showing the amino acid composition in positions −1 and +1 relative to <b>B</b>) <i>ackA</i>-sensitive lysines and <b>C</b>) <i>ackA</i>-insensitive lysines from 4 proteins (LpdA, PflB, AceE, GroL). The relative frequencies were determined by comparing the frequency of each amino acid in positions -1 and +1 adjacent to the <b>D</b>) 45 <i>ackA</i>-sensitive lysines and <b>E</b>) the 131 <i>ackA</i>-insensitive lysines to the overall frequency of each amino acid in all 4 proteins (LpdA, PflB, AceE, GroL).</p

Mass Spectrometric Analysis, 0.4% Glucose.

<p>4 biological replicates in 3 technical MS replicates.</p><p>2661 unique acetyl Lys sites, 787 acetyl proteins.</p

Anti-acetyllysine Western immunoblot analyses.

<p><b>A</b>) Pta-AckA pathway schematic. <b>B</b>) <i>E. coli</i> WT (strain MG1655) and isogenic <i>ackA</i> mutant (strain AJW2012), each aerated at 37°C in TB7 and harvested at 5 time points, when the OD₆₁₀ reached 0.5 or 1.0, and then at 8, 24 and 32 hours. <b>C</b>) <i>E. coli</i> WT (strain MG1655) and isogenic <i>pta ackA</i> mutant (strain AJW2785), each aerated at 37°C in TB7 and harvested at 5 time points, when the OD₆₁₀ reached 0.5 or 1.0, and then at 8, 24 and 32 hours. <b>D</b>) <i>E. coli pta</i> mutant (strain AJW3699) aerated at 37°C in TB7 (-ace) or in TB7 supplemented with 10 mM acetate (+ace) and harvested at 8 and 24 hours. <b>E</b>) <i>E. coli</i> WT (strain AJW678) and isogenic <i>ackA</i> (strain AJW1939) and <i>pta ackA</i> (strain AJW2013) mutants, each aerated at 37°C in TB7 supplemented with 0.4% glucose and harvested at 5 time points, when the OD₆₁₀ reached 0.5 or 1.0, and then at 8, 24 and 32 hours. <b>F</b>) <i>E. coli</i> WT (strain MG1655) and isogenic <i>ackA</i> (strain AJW5052) and <i>cobB</i> mutants (strain AJW5037), each aerated at 37°C in TB7 and harvested at 5 time points, when the OD₆₁₀ reached 0.5 or 1.0, and then at 8, 24 and 32 hours. <b>F</b>) <i>E. coli</i> WT (strain MG1655) and isogenic <i>yfiQ</i> mutant (strain AJW5164), each aerated at 37°C in TB7 supplemented with 0.4% glucose and harvested at 5 time points, when the OD₆₁₀ reached 0.5 or 1.0, and then at 8, 24 and 32 hours.</p

Distribution of acetylation sites in <i>E. coli</i> proteins.

<p>Mass spectrometric analysis of the strains grown in the conditions shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094816#pone-0094816-g002" target="_blank">Figure 2</a> confidently identified 2730 unique lysine acetylation sites across 806 unique acetylated <i>E. coli</i> proteins. <b>A</b>) The frequency of individual proteins relative to the number of acetyllysines per protein. <b>B</b>) The estimated protein copy number per <i>E. coli</i> cell <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094816#pone.0094816-Ishihama1" target="_blank">[39]</a> relative to the number of acetylation sites per protein. The exponential regression trendline is indicated (R²  =  0.13), the y-axis is presented as a logarithmic scale.</p

Crystal structure of <i>E. coli</i> triose phosphate isomerase (TpiA) determined in the presence and absence of acP.

<p><b>A</b>) Cartoon and stick representation of acP bound in the active site of TpiA. The acP ligand is surrounded by the F_o-F_c omit map that was contoured at the 3 sigma level. Side-chain and main-chain interactions with acP are shown as gray dashed lines. Oxygens are shown in red, nitrogens in blue, phosphate in orange, carbons of acP in yellow, and carbons of the protein in light blue. <b>B</b>) Overlay of the crystal structure of the <i>E. coli</i> acP-bound TpiA protein (PDB ID: 4MVA, cyan) with the crystal structure of TpiA from <i>Saccharomyces cerevisiae</i> (PDB ID: 1NEY, gray). The <i>S. cerevisiae</i> structure has the substrate 1,3-dihydroxyacetone phosphate (13P) bound in its active site. K11 is shown in each structure. Nitrogen atoms are blue, oxygens are red, the carbon atoms of acP are cyan and carbon atoms of 13P are gray. An arrow indicates the movement for loop closure between open (cyan) and closed (gray) forms of the protein. <b>C</b>) Surface representation of TpiA (PDB ID: 4MVA) with the locations of up-regulated acetylated lysine residues in the <i>ackA</i> mutant highlighted in red. AcP is bound in the active site.</p