Acetylome with Structural Mapping Reveals the Significance of Lysine

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Acetylome with structural mapping reveals the significance of lysine acetylation in Thermus thermophilus Hiroki Okanishi, Kwang Kim, Ryoji Masui, and Seiki Kuramitsu J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr400245k • Publication Date (Web): 01 Aug 2013 Downloaded from http://pubs.acs.org on August 5, 2013

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Journal of Proteome Research

Acetylome with structural mapping reveals the significance of lysine acetylation in Thermus thermophilus Hiroki Okanishi1, Kwang Kim 1,*, Ryoji Masui1, 2, and Seiki Kuramitsu1, 2

1

Department of Biological Sciences, Graduate School of Science, Osaka University, 1-1

Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan 2

RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.

Keywords: Acetylome/ Mass spectrometry/ Protein acetylation/ Tertiary structure/ Thermus thermophilus

1 ACS Paragon Plus Environment


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ABSTRACT Lysine acetylation in proteins has recently been globally identified in bacteria and eukaryotes. Even though acetylproteins are known to be involved in various cellular processes, its physiological significance has not yet been resolved.

Using a proteomics approach in

combination with immunoprecipitation, we identified 197 lysine acetylation sites and 4 Nterminal acetylation sites from 128 proteins in Thermus thermophilus HB8, an extremely thermophilic eubacterium.

Our analyses revealed that identified acetylproteins are well

conserved across all three domains of life and are mainly involved in central metabolism and translation.

To further characterize functional significance, we successfully mapped 172

acetylation sites on their 59 authentic and 54 homologous protein structures.

Although

percentage of acetylation on ordered structures was higher than that of disordered structure, no tendency of acetylation in T. thermophilus was detected in secondary structures. However, the acetylated lysine was situated near the negatively charged glutamic acid residues. In tertiary structure analyses, 58 sites of 103 acetylations mapped on 59 authentic structures of T. thermophilus were located within considerable distance that can disrupt electrostatic interactions and hydrogen bonding networks on protein surface, demonstrating the physiological significances of the acetylation that can directly alter protein structure. In addition, we found 16 acetylation sites related to Schiff-base formation, ligand binding, protein-RNA and proteinprotein interaction that involve the potential function of the proteins. The structural mapping of acetylation sites provides new molecular insight into the role of lysine acetylation in the proteins.


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INTRODUCTION Acetylation of ε-amino groups of lysine residues is one of the most prevalent posttranslational modifications detected in eukaryotes and bacteria

1-3

.

Recently, protein

phosphorylation, another common post-translational modification, has been extensively studied in eukaryotes using various phosphopeptide-enrichment techniques and mass spectrometry in combination with nano-scale liquid chromatography (nano-LC) 4. Phosphoproteome studies have also been performed in many prokaryotes, including Escherichia coli 5, Bacillus subtilis 6, Mycobacterium tuberculosis 7, Streptococcus pneumonia

8

and Thermus thermophilus HB8 9.

Until now, genome-wide acetylation profiles have been reported for several species using immunopurification with an anti-acetyllysine antibody and mass spectrometric analyses

10-25

.

The identified acetylproteins are involved in various cellular processes, especially central metabolism, suggesting a close link between acetylation and diverse biological processes. However, previous studies have concentrated in the identification of acetylproteins and the monitoring of acetylation ratio under different culture conditions

11, 14

. Even though functional

classification of acetylproteins, motif search and secondary structure analysis near acetylation site were also examined in the previous reports, the precise functions of lysine acetylation in the majority of cases are still obscure. In eukaryotes, protein acetylation regulates various cellular processes. This is exemplified by many proteins whose cellular activities are regulated by acetylation, in particular histone 26. The acetylation of histone neutralizes the positive charge of lysine residues resulting in a decrease in binding affinity for the negatively charged phosphate groups of DNA. Acetylation may regulate some other proteins by single- or multiple-site acetylation in bacteria

27, 28

.

Acetyl-CoA

synthetase is one of a few proteins known to be regulated by protein lysine acetylation in bacteria



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. Acetyl-CoA synthetase is regulated by a single acetylation of a lysine residue near the active

site. It is also reported that six lysine residues in the C-terminal region of CheY are subject to acetylation and this multiple-site modification inhibits the interaction of CheY with CheZ, CheA and FliM 30, 31. Previously, a few acetylome study has used structural information to deduce the likely function of several acetylation sites even though limited number of proteins was investigated 19, 22

17,

. Because a good indication of protein function can be obtained from structural information,

we reasoned that structural investigation of whole-cell acetylproteins would provide valuable information concerning the cellular roles of this intriguing post-translational modification. Thermus thermophilus HB8 is an extremely thermophilic eubacterium

32

and its complete

genome sequence has been published in the public database (AP008226, chromosome; AP008227, pTT27; AP008228, pTT8) 33. The 2.2 Mb genome of T. thermophilus HB8 contains 2,238 open reading frames (ORFs).

Because T. thermophilus HB8 has an optimal growth

temperature of 70°C, the proteins produced in this bacterium are very stable and therefore suitable for structural and functional analyses

34

. We have studied this bacterium as a model

organism for atomic biology in the Structural-Biological Whole-Cell project

35

. This project

aims to understand all biological phenomena in the cell of T. thermophilus HB8 based on structural, transcriptional and translational information of ORFs. Hitherto, structures of about 20% of T. thermophilus HB8 proteins have been determined

36

. In addition, there has been an

enormous increase in the number of protein structures appearing in the Protein Data Bank (PDB, http://www.rcsb.org/pdb/home/home.do) over the last decade

37

.

This vast accumulation of

protein structural information can be used to predict likely mechanisms of enzymatic function such as ligand binding, conformational changes and protein-protein interactions. In particular,


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abundant structural information of T. thermophilus HB8 proteins will allow us to globally analyze acetylproteins from this organism. In this study, we identified 128 acetylation proteins of T. thermophilus HB8 and determined the 197 lysine acetylation sites. We used two different types of anti-acetyllysine antibodies against different antigens to enrich for the acetylated peptides, followed by nano-LC combined with mass spectrometric detection to identify the acetylation sites. Based on the structural information of T. thermophilus HB8 proteins and their homologs, we were able to map most of the acetylation sites on the structures and then propose the likely regulatory function of this posttranslational modification.

MATERIALS AND METHODS Media and Culture Conditions Tryptone was purchased from Nihon Pharmaceuticals (Tokyo, Japan).

Yeast extract was

purchased from Nippon Seiyaku (Tokyo, Japan). Phytagel was purchased from Sigma-Aldrich (St Louis, MO). Chemical compounds were purchased from Wako (Tokyo, Japan). A single colony of T. thermophilus HB8 strain (ATCC27634) on a TT plate (0.4% tryptone, 0.2% yeast extract, 0.1% NaCl, 1.5 mM MgCl2, 1.5 mM CaCl2 and 1.5% Phytagel) was inoculated into TT broth (0.4% Tryptone, 0.2% yeast extract, 0.1% NaCl, 0.4 mM MgCl2 and 0.4 mM CaCl2) and cultivated overnight at 70°C with vigorous shaking. A 6-mL aliquot of the T. thermophilus HB8 seed culture was used to inoculate 600 mL of TT broth. The cells were then cultured at 70°C until they reached stationary phase (O.D600 = 2.5) before harvesting by centrifugation at 7,000 × g for 5 min. The cell pellet was finally washed with cold phosphate-buffered saline (PBS).



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Preparation of Crude Extracts and Tryptic Digests The cell pellet was suspended in 4 mL of lysis buffer (PBS containing 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF) at pH 7.5) and disrupted using an ultrasonic disruptor, UD-201 (TOMY, Tokyo, Japan). The lysates were stored at –80°C until further use. The cell lysates were thawed and 20 mg of proteins were mixed with 4 volumes of cold acetone and then kept at –20°C for 2 h. The mixture was centrifuged at 10,000 × g for 10 min and then the precipitate was dried to eliminate traces of acetone after washing with 100% cold acetone. The pellets were suspended in 6 M guanidine hydrochloride. The concentration of protein was determined by the method of Bradford (Bio-Rad, Hercules, CA) prior to heat denaturation at 90°C. A 10-mg sample was reduced with 5 mM DTT at 50°C for 30 min and then alkylated at cysteine residues with 10 mM of iodoacetamide at room temperature in the dark for 30 min. The reaction was stopped by adding dithiothreitol (DTT) up to 10 mM. After 4-fold dilution of the mixture with 50 mM ammonium bicarbonate buffer (pH 8.0), the sample was digested with TPCK trypsin (Thermo Scientific, Rockford, IL) using an enzyme:substrate ratio of 1/100 (w/w) at 37°C overnight. The sample was further digested with the same trypsin (1/100 (w/w)) and buffer at 37°C overnight after heat treatment at 90°C for 5 min. The digested peptides were desalted using a Sep-Pak Plus C18 Cartridge (Waters, Milford, MA) and the eluent was lyophilized by Freezone 4.5 (Labconco, Kansas City, MO) overnight.

Enrichment of Acetylpeptides Antibody against acetylated bovine serum albumin (BSA) was generated by the previously reported method

38

. BSA was chemically acetylated with acetic anhydride. The degree of

chemical acetylation of primary amine was confirmed by ninhydrin reaction (~90% of amino


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groups were acetylated). Generation of rabbit anti-acetyllysine antiserum (homemade) was carried out by Kitayama Labes Co. (Nagano, Japan). A 50-µL aliquot of anti-acetyllysine antiserum (homemade) or 200 µL of anti-acetyllysine polyclonal antibody (ImmuneChem, Burnaby, BC, Canada) was conjugated to protein A agarose (Santa Cruz, Santa Cruz, CA) in 500 µL of PBS (pH 7.5) by gently shaking using an RT-30 mini (TAITEC, Tokyo, Japan) at 4°C for 4 h. The conjugated beads were then washed three times with 1.0 mL of NETN buffer (50 mM Tris-HCl [pH 8.0] containing 100 mM NaCl, 1.0 mM EDTA and 0.5% Nonidet P40). Tryptic digests were mixed with the conjugated beads and gently shaken at 4°C for 6 h. The tryptic digests were suspended in NETN buffer, mixed with the conjugated beads and gently shaken at 4°C for 6 h. The beads were washed three times with 1.0 mL of ETN buffer (50 mM Tris-HCl containing 100 mM NaCl and 1.0 mM EDTA at pH 8.0). Acetylated peptides were eluted four times with 100 µL of 0.1% trifluoroacetic acid and the eluates were pooled. Totally, 14 immunoprecipitations were carried out using both antiacetyllysine antibodies. The eluted peptides were further desalted using a Sep-PAK Plus C18 Cartridge (Waters) and then lyophilized by Freezone 4.5 for 3 h. The dried sample was dissolved in 20 µL of 0.1% formic acid prior to mass spectrometry analysis.

Nano-LC and MS/MS Analyses Ten µL of enriched acetylated peptides was injected into the SPE trap column NS-MP-10 BioSphere (100 µm i.d. and 20 mm length; Nanoseparations, Nieuwkoop, The Netherlands) at a flow rate of 8 µL/min by the autosampler in Proxeon EASY-nLC (Bruker Daltonics, Billerica, MA) and subsequently washed at a flow rate of 8 µL/min for 10 min with solvent A (0.1% formic acid in water). The peptides were separated at a flow rate of 120 nL/min using an 7 ACS Paragon Plus Environment


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Acclaim PepMap100 analytical column (C18, 5 µm, 100 Å, 50 µm i.d. and 250 mm length; Dionex, Breda, The Netherlands) with the following gradient: 5% solvent B for 5 min, 5 to 40% solvent B (0.1% formic acid and acetonitrile) for 30, 60 or 120 min, 40 to 90% solvent B for 5 min and 90% solvent B for 20 min. The eluted peptides were introduced into the micrOTOF-Q II mass spectrometer (Bruker Daltonics) by ESI typically at a capillary voltage of –1.3 kV and drying gas temperature of 180°C.

The micrOTOF control software version 3.0 controlled

MS/MS analysis by automatically switching the processes between MS scan and MS/MS fragmentation with three most abundant ions within m/z range from 300 to 3,000. MS/MS fragmentation of precursor ions by Ar gas was set to 20–40 eV depending on their charge-states and m/z values. Each sample was examined twice for identification of acetylation sites on peptides using nano-LC and Q-TOF MS.

Data Processing and Mascot Analysis MS and MS/MS spectral data produced from 28 times nano-LC-MS/MS runs were processed by DataAnalysis 4.0 software (Bruker Daltonics). The peak lists containing m/z of precursor ions with that of their product ions were generated by the Compound-Auto MS(n) option of the DataAnalysis 4.0 software. Fifty non-deconvoluted peaks over the intensity threshold 150 and charge deconvoluted peaks in each MS/MS spectrum were exported into peak list files. The spectra were searched against our in-house T. thermophilus HB8 database, containing 2,238 protein sequence entries from GenBank accession numbers, AP008226, AP00827, and AP00828 of the complete genome sequence using Mascot search engine (version 2.3; Matrix Science, London, UK). The acetylated peptides were identified using a mass tolerance of ±0.05 Da for precursor and product ions and allowed a maximum of 6 mis-cleavage sites for trypsin. The


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carbamidomethylation of cysteine was selected as a fixed modification.

The oxidation of

methionine, deamidation of asparagine and glutamine, acetylation of lysine and acetylation of protein N-terminus were selected as variable modifications. Only peptides in the confidence range of 99% probability (P value < 0.01) in Mascot ion score were assumed to be identified. The identification results and MS/MS spectral data (ProteomeXchange accession, PXD000184; PRIDE accession number, 28759-28786) were deposited in ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) and PRIDE database (http://www.ebi.ac.uk/pride/). We filtered out the possibility that a C-terminal lysine was acetylated because the acetylated lysine is not cleaved by trypsin. The false discovery rate (FDR) was determined using Mascot decoy database search (Supplemental Table S1).

In silico Analysis Functional annotation was based on the Clusters of Orthologous Groups of proteins (COGs) database

39

. In addition, to compensate for lack of functional information, KEGG Orthology

and the InterPro database

41

40

were additionally employed for proteins belonging to ‘Function

unknown’ and ‘General function prediction only’ groups to complement protein functional information. N-terminal acetylation site were excluded for in silico analyses. Normalized amino acid sequence frequency around the acetylated lysine was analyzed by using IceLog program

42

. Ten amino acids on both sides of acetylated lysine residues were

subjected to the frequency analysis and plotted by percent difference (P value < 0.05). Secondary structure assignments of an acetylation site or its corresponding sites were derived from the DSSP assignments on PDB entry 43. Orthologs of the proteins that were acetylated in T. thermophilus HB8 were searched using



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Basic Local Alignment Search Tool (BLAST, http://blast.ncbi.nlm.nih.gov/) 44. Blastp (proteinprotein BLAST) and NCBI non-redundant (nr) protein sequences were selected for program and database, respectively. The amino acid sequences of acetylproteins that were identified in T. thermophilus HB8 and their orthologs were obtained from the NCBI protein database. The orthologous proteins of 11 model organisms were determined by using two criteria of BLAST search result that showed either less than 1.00E-10 of E-value or larger than 24% of Max identity. The multiple amino acid sequence alignments were performed in the Multiple Sequence Comparison by Log-Expectation (MUSCLE) web server

45

. Information of acetylation in the

orthologs was obtained from previously reported acetylome studies on Homo sapiens Mus musculus

10, 18

, Drosophila melanogaster

16

, Arabidopsis thaliana

17

, S. enterica

10, 13, 15

14

,

and E.

coli 11, 12, and the UniProt Knowledgebase 46.

Localization of Acetylation Sites on the Tertiary Structure The tertiary or quaternary structures of acetylproteins were obtained from the PDB and the positions of acetylated lysines were mapped on the structure. The structures of homologous proteins with at least 25% identity were also used when either (i) no structural data was available for the T. thermophilus HB8 proteins in the PDB, or (ii) only a truncated structure lacking the region containing the acetylation site(s) was available. The PyMOL Molecular Graphics System (version 1.4.1; http://www.pymol.org) was employed to present the structural results of this study. We used the following criteria to speculate on the likely function of acetylation based on previous reports: hydrogen bond, 3 Å; electrostatic interaction between lysine and acidic amino acid residues, 6 Å; electrostatic interaction between lysine and the phosphate group of nucleic acids, 6 Å.


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RESULTS Identification of Acetylated Peptides in T. thermophilus HB8 We used immunoaffinity purification and a mass spectrometric approach, which was combined with nano-LC, to identify acetylproteins in T. thermophilus HB8 and their site of modification. Both homemade polyclonal anti-acetyllysine antibody and pan-anti acetyllysine polyclonal antibody of ImmuneChem were used to enrich acetyllysine-containing peptides in whole-cell protein tryptic digests.

The enriched acetylated peptides were separated by nano-LC and

introduced into Q-TOF MS by ESI to generate fragmentation with Ar gas. The number of identified acetylpeptides and FDR result against each antibody in this study were summarized in Supplemental Table S1. Figure 1 represents a MS/MS spectrum of acetylated peptides, GGADFLKacGETEAK of deoxyribose-phosphate aldolase (TTHA1186). Each acetylpeptide identified by Mascot had a score over the threshold corresponding to 99% confidence (p < 0.01) at FDR < 1.0%, which was calculated by a decoy option in the Mascot software (version 2.3). A score with a difference of >5 of the identified acetylpeptides with the other peptides in the same spectrum was also used as a cut-off value to increase confidence and to remove uncertain acetylation sites. The mascot score, delta mass, mass measurement accuracy, charge state, retention time and experimental number of nanoLC-MS/MS run for identified acetylpeptides were tabulated in Supplemental Table S2. Trypsin cannot cleave peptide bonds on the C-terminus of lysine residues neutralized by acetylation. Thus, we limited acetylation to only internal lysines of the peptide. These procedures enabled the precise location of all acetylation sites on identified peptides in this study. We identified 197 lysine acetylation sites and 4 N-terminal acetylation sites on 128 proteins



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(Table 1). Using different types of anti-acetyllysine antibodies, we succeeded in increasing the number of identified acetylpeptides and reducing antibody-related bias.

A total of 165

acetylation sites were identified with the homemade antibody and a further 78 sites with the commercial antibody (Fig. 2A). Forty six acetylated peptides were commonly identified by these two antibody preparations. Multiple-lysine acetylation on a protein occurred at 100 positions in 26 different proteins (Fig. 2B and Table 1). Most notably, the acetylation on 6 proteins occupied ~27% (53/197 sites) of total lysine acetylation sites identified in this study. Among the 6 proteins, GroEL (TTHA0271) had 12 lysine acetylation sites. The acetylation was identified at 6 lysines of SufD (TTHA1840) and hypothetical protein (TTHA1485), respectively. ClpB (TTHA1487) was acetylated at 5 lysine residues. In addition, six other proteins had four acetylation sites each.

Conservation of Acetylproteins in All Three Domains of Life A recent study of the E. coli acetylome revealed that many acetylproteins have homologs in H. sapiens, suggesting that acetylation tends to occur on proteins conserved between E. coli and H. sapiens

12

. However, until now, there has been no systematic examination of orthologs of

acetylproteins in all three domains of life. In this study, we searched the orthologs of 127 lysineacetylated proteins of T. thermophilus HB8 by a protein BLAST search against nine representative organisms: Deinococcus radiodurans, E. coli, B. subtilis, Sulfolobus solfataricus, Halobacterium salinarum, Saccharomyces cerevisiae, A. thaliana, M. musculus and H. sapiens. As a result of a global homology search using the amino acid sequence of target proteins, we identified 688 orthologs of the 118 acetylproteins of T. thermophilus HB8 in the protein database (Supplemental Table S3).

This result shows that 93.7% of the identified lysine-acetylated


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proteins have orthologs (Fig. 3A).

We classified acetylproteins of T. thermophilus HB8

depending on the existence of their orthologous proteins in the identified acetylproteins. Interestingly, 22% of total acetylproteins (28/127 proteins) belong to the ‘completely conserved’ category, which has orthologous proteins in all nine organisms.

The percentage of ‘well

conserved’ proteins containing 6 to 8 orthologs and ‘conserved’ proteins containing 3 to 5 orthologs were 33.1% (42/127 proteins) and 24.4% (31/127 proteins) of total acetylproteins, respectively. Only 13.4% (17/127 proteins) of acetylproteins of T. thermophilus HB8 were grouped as ‘poorly conserved’ proteins with 1 to 2 orthologs. These results indicate that the acetylproteins identified in T. thermophilus HB8 are highly conserved in other organisms. We further examined whether the lysine residues that were acetylated in 118 proteins of T. thermophilus HB8 are conserved in 688 orthologs of the other nine organisms. As a result, we found that 384 lysine residues corresponding to the acetylation sites in T. thermophilus HB8 were conserved in the orthologs, raising the possibility that the same sites might be acetylated in the other organisms (Supplemental Table S2).

Comparison of Acetylomes between T. thermophilus and Other Organisms To further compare acetylproteins among different species, we selected some of the previous acetylome studies. We investigated the orthologs of acetylproteins of T. thermophilus HB8 in previously reported acetylproteins of E. coli 16

11, 12

, S. enterica 14, A. thaliana 17, D. melanogaster

, M. musculus, 10, 18 and H. sapiens 10, 13, 15. Figure 3B and Supplemental Table S2 indicates that

70 acetylproteins in T. thermophilus HB8 had orthologous lysine-acetylated proteins in E. coli (34 proteins), S. enterica (17 proteins), A. thaliana (2 proteins), D. melanogaster (29 proteins), M. musculus (19 proteins) and H. sapiens (47 proteins). We further examined whether lysine



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acetylation occurred at the same sites in orthologous proteins. A total of 17 acetylation sites in T. thermophilus HB8 were the same in the six organisms (Supplemental Table S2). The acetylation of 5 sites from 5 proteins of E. coli, 3 sites from 3 proteins of S. enterica, 2 sites from 2 proteins of D. melanogaster, 1 site from 1 protein of A. thaliana and 9 sites from 8 proteins of H. sapiens were conserved in T. thermophilus HB8. These results suggest that the lysine residues of these 17 proteins may play a critical role in their function.

Local Sequence Context around Acetylation Site We investigated the local sequences around 197 acetylated lysines in T. thermophilus HB8. Based on structural studies of domain of histone acetyltransferase (HAT) that recognizes less than 20 amino acids in length for peptide substrates 47, we examined the context of amino acids sequence from –10 to +10 position around acetyllysine.

Sequence windows centered on

acetylated lysines were compared with non-acetylated sequences to remove unwanted bias due to a general predisposition of certain amino acids close to lysine residues. Remarkably, negatively charged amino acids, mainly glutamic acid, were located at most positions from –5 to +4 and +7 (Fig. 4). Thus, lysine residues in close proximity to glutamic acid in the primary amino acid sequence may be preferentially acetylated in T. thermophilus HB8. In particular, glutamic acid was occupied at the –1 position and positively charged amino acid, arginine, was usually excluded at the position. Even though glutamic acid was disappeared and aspartic acid still preferred at the –2 position, bulky amino acids, glutamine and tryptophan were typically occupied and arginine was usually excluded at the position. Intriguingly, the result of eukaryotes showed similar aspect that bulky residues, phenylalanine and tyrosine, are mostly located at the – 2 positions in H. sapiens and D. melanogaster

16

. The profile of surrounding amino acid


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sequence in E. coli supported our hypothesis that acidic amino acids frequently appeared nearby acetylated lysine and bulky amino acids were located at the –2 position (Supplemental Fig. S120A). However, the acetylated lysine was surrounded by positively charged lysines at most positions in S. enterica (Supplemental Fig. S120B). Although similar distribution of basic amino acid, lysine, was detected in H. sapiens and D. melanogaster 16, S. enterica also showed slightly increased frequency of acidic amino acids at the –3 and –1 positions. Our observation with the sequence profile of E. coli suggests that acetylation neutralizes the positive charge of the lysine, which is located near the negatively charged glutamic acid and may therefore hamper electrostatic interaction between these two amino acids.

Secondary and Tertiary Structures around Acetylated Lysines Generally, acetylation of lysine residue on protein affects the protein structures due to the change of positive net charge to zero. However, extensive analysis for the structural alteration by lysine acetylation using structural information has seldom been reported under molecular level. To understand the significance of acetylation on proteins, we carried out a structural analysis of all acetylproteins. First, we examined the lysine acetylation sites on its tertiary or quaternary structures. We obtained structural data of 59 T. thermophilus HB8 proteins from the PDB for 103 acetylation sites and, except for the sites situated in disordered regions, successfully mapped 100 of the acetylation sites on the structures (Fig. 5 and Supplemental Table S4). As for T. thermophilus HB8 proteins whose structures have not yet been determined, we used structural data from homologous proteins that displayed over 25% amino acid sequence identity based on a protein BLAST search (Supplemental Table S5).

Approximately 89% (113 of 127

acetylproteins) of structural information of lysine-acetylated proteins could be obtained from the



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entry of 54 homologous and 59 authentic structures (Supplemental Table S4). Except for 3 acetylated lysine residues located in disordered regions, all of the acetylation sites and the corresponding sites were mapped onto protein structures.

Further details are presented in

Supplemental Figure S1 to S119. The mapped position of acetylated lysine residues on the structures shows that this post-translational modification mainly occurs on the surface of the protein, indicating that most lysine acetylation has an effect on their surface properties as we expected. On the other hands, dozens of acetylation sites were located near ligand binding sites or enzyme active sites (Table 2 and Supplemental Table S4). Additionally, the acetylation of several ribosomal proteins and elongation factors indicates this modification alter their interaction with ribosomal RNA. To assess the effect of acetylation on surface lysine residue on protein structures, we further examined the distance between the ε-amine of acetylated lysine residues and spatially nearby amino acids, nucleic acids and ligands (Table 2).

In the structures of 59 proteins of T.

thermophilus HB8, 47 of 103 lysine acetylation sites were located within 6 Å from glutamic acid or aspartic acid residues in the same protein or subunit. This result strongly suggests that the side chains of the lysine and acidic residues could form an electrostatic interaction. Also, 11 of the 103 mapped sites were located within 3 Å from oxygen atoms of spatially adjacent amino acids in the same subunit, which suggests the lysines subject to acetylation could form a hydrogen bond. An electrostatic interaction or hydrogen bond in a protein can contribute to its stability 6448, 495. Furthermore, a recent report proposes a model of regulation for E. coli RNase R in which acetylation breaks an electrostatic intrasubunit interaction, leading to a conformational change that inhibits protein-protein interaction

50

. Taken together, 56.3% (58/103 residues) of

the acetylated lysine residues that were identified in this study are able to form electrostatic


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interactions or hydrogen bonds within a single polypeptide (Fig. 5 and Table 2). We further investigated secondary structures of lysine acetylation sites using authentic (103 sites in 59 proteins) and homologous (69 sites of 54 proteins) structures (Supplemental Fig. S122). Approximately 50.6% (87/172 sites) of the acetylation sites were located at regions of ordered secondary structure. Among them, 73 acetylated lysines were located in an α-helix and 14 sites were in a β-sheet. However, 17 acetylation sites were found in the termini of an α-helix and 5 such sites were found at the termini of a β-sheet (i.e., together accounting for 12.8% of the 172 acetylation sites). The remained ~36.6% (63/172 sites) of acetylation sites were located in unstructured regions of the protein such as loops. These results seemed to be the acetylation preferred ordered structures to disordered structure in proteins. However, a distribution of secondary structures containing non-acetylated lysine in 10 representative proteins of T. thermophilus showed similar result with that of acetylated lysine, indicating no relation between acetylation and secondary structures (Supplemental Fig. S122).

Functional Classification of Acetylproteins To better understand the general function of lysine acetylation, we investigated what kinds of proteins are often acetylated in T. thermophilus. We assigned a functional group to each acetylprotein, which yielded 11 distinct classes based on the COGs of proteins, the KEGG Orthology and the InterPro database (Table 1, Fig. 6A and B). The classification result showed that 48.8% (62/127 proteins) of lysine-acetylated proteins in T. thermophilus HB8 were categorized into ‘metabolism’. This group contained 16 enzymes involved in central metabolism (Fig. 7). Our finding is consistent with the results of six organisms whose acetylome analyses have already been reported

10-18

. Previous reports on the acetylome of E. coli and S. enterica



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showed that about half of the lysine-acetylated proteins were grouped as ‘metabolic enzyme’ 11, 12, 14

.

Moreover, similar results were also reported in A. thaliana

17

, M. musculus

10, 18

, D.

melanogaster 16 and H. sapiens 10, 13, 15. For detailed analysis, we further classified ‘metabolism’ into eight subgroups as shown in Fig. 6B. Of these, the ‘energy production and conversion’ subgroup has the largest number of acetylproteins. Nine enzymes involved in the TCA cycle, including components of the pyruvate dehydrogenase complex, 2-oxoglutarate dehydrogenase complex, succinyl-CoA synthetase complex and succinate dehydrogenase complex, were categorized into this subgroup, suggesting an intimate relationship between protein acetylation and energy metabolism (Fig. 7). The second largest acetylation group was the ‘translation’ group, which contained 20 proteins in all: 8 ribosomal proteins, 4 translation elongation factors, 3 aminoacyl-tRNA synthetases, 1 translation initiation factor and 1 amidotransferase. The class of ‘Post-translational modification, protein turnover, chaperones’ contained 9 proteins associated with protein fate and 5 proteins related to stress response. The proteins associated with protein refolding such as GroEL, GroES, DnaK, trigger factor, ClpB, and protein degradation, ClpA and FtsH were identified as acetylproteins in T. thermophilus HB8. Interestingly, GroEL and ClpB, SufB, SufC and SufD belonging to the class, ‘Post-translational modification, protein turnover, chaperones’ were highly acetylated (i.e., at multiple sites) as described above.

DISCUSSION First of all, we estimated amount of acetylprotein in T. thermophilus HB8 by using Western blot analyses (Supplemental Fig. S124A and B). The immunoblotting results indicated that the lysine acetylation in T. thermophilus was an infrequent event even though a few proteins were


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highly acetylated. With reference gels image of previous report

51

, we believe that highly

acetylated protein on Western blot image of 2DE gel are TTHA0271 (60 kDa GroEL), TTHA0232 (pyruvate dehydrogenase E2 component) and TTHA1272 (ATP synthase β subunit), respectively. Although the amount of acetylation was relatively low, we supposed that many proteins were acetylated in T. thermophilus HB8 because about 2% ~ 8% of proteins were acetylated in other bacteria

11, 12, 14, 25

. Using two different anti-acetyllysine antibodies, we

successfully enriched acetylated peptides and identified 197 lysine acetylations in 127 proteins of T. thermophilus HB8 (except only one N-terminal acetylprotein). Our findings reveal that 5.7% of all the ORFs in the complete genome sequence of T. thermophilus HB8 are subject to acetylation. The ratio of acetylproteins to non-acetylated proteins in the extremely thermophilic bacterium, T. thermophilus HB8 is not significantly different from those in E. coli (7.8%), S. enterica (4.2%) and Erwinia amylovora (2.6%)

11, 12, 14 24, 25

. Furthermore, the acetylproteins

identified in this study have 673 orthologous proteins present in all of three domains of life by examination of protein BLAST searches (Supplemental Table S3). Figure 3 shows that ~80% of acetylproteins of T. thermophilus HB8 have orthologous proteins in more than three organisms (i.e., so called ‘conserved protein’ in this study). In these orthologous proteins, 384 lysine residues are conserved at the sites corresponding to acetylated lysine residues in T. thermophilus HB8. The result demonstrates that the acetylated lysines (so called ‘conserved lysine’ in this study) are highly conserved throughout the three domains of life. In this analysis, a total of 17 lysine residues of T. thermophilus HB8 proteins were found to be acetylated at the same lysine residues in the other organisms (so called ‘conserved lysine acetylation’ in this study) (Supplemental Table S2). Because these acetylations are conserved in orthologous proteins of other organisms, we speculated that the acetylation of lysine might



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frequently occur on a specific structure or a certain motif where the lysine forms part of a characteristic amino acid sequence. In the case of eukaryotes (e.g., H. sapiens and D. melanogaster), amino acid sequence context around acetylation sites shows a degree of preference for certain amino acids at specific positions; namely phenylalanine at –2, glycine at -1 and tyrosine at +1

13

. Additionally, lysine

residues are often located close to the acetylation site i.e., at –4 to –6 and +3 to +6 13, 16. On the other hands, S. cerevisiae showed that acidic amino acids were often located nearby acetylated lysine in amino acid sequence context analysis

23

. Likewise, the frequency of amino acids

surrounding acetylation site in E. coli demonstrated that glutamic acid and aspartic acid were often located near acetylated lysine (Supplemental Fig. S120A). Consistently, the amino acid sequence context around acetylation sites in T. thermophilus HB8 proteins showed a preferential distribution of glutamic acid and aspartic acid residues near the acetylated lysine. This site preference is completely different from that of H. sapiens and D. melanogaster acetylomes, but appears related to that of the mitochondrial acetylome, in which aspartic acid residues are frequently found at the -3 to -4 and +3 to +4 positions

16

. Structural analysis of acetylproteins

often showed that the acetylated lysine (i.e., 48/103 sites or ~46.6% of cases) on its authentic tertiary or quaternary structure is situated within 6 Å of a spatially adjacent acidic amino acid (Fig. 5 and Table 2). We suggest that this blocking of electrostatic interaction by acetylation affect protein stability and possibly protein conformation. In T. thermophilus HB8, 50.6% of acetylation was found to occur in ordered structures, such as α-helix or β-sheet, whereas 36.6% was located in unstructured regions (Supplemental Fig. S122). By contrast, protein phosphorylation in T. thermophilus HB8 was found to preferentially occur in disordered structures 9. For several proteins, phosphorylation in an unstructured region


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was reported to induce a conformational change 52-55. The respective features of acetylation and phosphorylation in T. thermophilus HB8 are consistent with those in H. sapiens

16

. However, we

found no marked preference of secondary structure for acetylation from comparative analysis of the location of acetylated lysines with non-acetylated lysines in 10 representative acetylated proteins of T. thermophilus (Supplemental Fig. S122). Taken together, the common distribution of ordered or disordered structures for acetylation supports that acetylation generally occur on a surface of protein without structural preference, indicating that meaning of acetylation is different from that of phosphorylation which stimulates conformational change of protein to control enzymatic activity. In spite of no significance to the structural preference of acetylation, detection of acetylation in lots of metabolic enzyme, existence of many orthologs of acetylprotein and conservation of acetylated lysines between orthologs imply a presence of possibility for a different role of acetylation in protein. Moreover, ‘functional assigned lysine’ and ‘hydrogen bonding lysine’ were frequently acetylated rather than ‘electrostatic interaction related’ and ‘not assigned lysine’ in tertiary structure analysis (Supplemental Fig. S123).

Based on structural information and

preceding reports, we can deduce possible regulatory mechanisms resulting from lysine acetylation using representative members among the 127 lysine-acetylated proteins (Fig. 8 and Table 2). Here in, we introduce our finding to understand a significance of representative acetylation.

The crystal structure of deoxyribose-phosphate aldolase (TTHA1186) from T.

thermophilus HB8 (PDB ID, 1UB3) was reported with and without its substrate, and found that the enzyme forms a covalent Schiff-base intermediate at the active lysine (Lys-151) residue

56

.

In this study, the active lysine residue (Lys-151) of TTHA1186 was revealed to be acetylated in T. thermophilus HB8 (Fig. 8A and Supplemental Fig. S61). Our finding of acetylation at Lys-151



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in active site give an important clue to deduce possible mechanism to control its enzymatic activity. In T. thermophilus HB8 inorganic pyrophosphatase (TTHA1965) which is composed of a homohexamer in solution and catalyzes the hydrolysis of pyrophosphate to two phosphate ions was acetylated at three different lysine residues, Lys-30, Lys-41 and Lys-100. The structure of TTHA1965 (PDB ID, 2PRD) revealed that Lys-30 was situated at a distance of 3.2 Å from the bound sulfate ion in the active site (Fig. 8B and Supplemental Fig. S113). Because this sulfate ion mimics a phosphate ion

57

, the acetylation at Lys-30 is able to alter the structure of ligand

binding site. The acetylation at the same lysine residue of inorganic pyrophosphatase of Erwinia amylovora was recently reported 24 and amino acid substitution of the lysine of E. coli inorganic pyrophosphatase that corresponds to the Lys-30 of TTHA1965 lost enzymatic activity 58, 59. It is plausible that the Lys-30 could control the enzymatic activity by acetylation. Recently, interacting proteins with acetylated proteins were reported in E. coli with a complete interaction network of acetylated proteins by using STRING database 25. Even though this report provides useful information about what proteins are interact with acetylproteins, the detail mechanisms such as how acetylation sites affect the interaction under atomic level still remain unclear. Here, we present a possibility about intersubunit interactions with a distance for electrostatic interactions (Table 2).

We identified the acetylation in all components of 2-

oxoglutarate dehydrogenase complex in T. thermophilus HB8, which is involved in central metabolism as described above (Fig. 7). In the crystal structure of the complex (PDB ID, 2EQ7), the acetylation site, Lys-131 of E2 component (TTHA0288), was situated in the interface between E2 and E3 components. Because of 2.6 Å distance between Lys-131 of E2 component and Glu-425 of E3 component (TTHA0287), the lysine residue has strong potential to form an


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intersubunit electrostatic interaction E3 component (Table 2 and Supplemental Fig. S15). In the crystal structure of 2-oxoisovalerate dehydrogenase E1 component (α2β2) of T. thermophilus HB8 (PDB ID: 1UMC), one of the two acetylation sites, Lys-33, of the β subunit (TTHA0230), was located at the interface between the α and β subunits and might form an intersubunit electrostatic interaction (at a distance of 5.3 Å) with Asp-211 of the α subunit (Supplemental Fig. S8).

Consistently, putative adenine phosphoribosyltransferase (TTHA1613), which forms a

homodimer, was also acetylated in the dimer interface (PDB ID: 1VCH), where Lys-117, the acetylation site, and Asp-34 in different subunits were positioned at a distance of 5.8 Å from each other (Table 2 and Supplemental Fig. S88). We suggest that lysine acetylation of these sites blocks intersubunit interaction and inhibit oligomerization. Our study revealed that 14 proteins of T. thermophilus HB8 grouped as ‘Post-translational modification, protein turnover, chaperones’ were acetylated at 45 different sites (Fig. 6A). We found that GroEL, GroES, DnaK, trigger factor and ClpB were acetylated in T. thermophilus HB8 as well as in E. coli

11, 12, 25

.

In the structure of the GroEL-GroES complex of T.

thermophilus HB8 (PDB ID, 1WF4), 10 of the 12 acetylated lysine residues (Lys-74, 166, 169, 336, 337, 349, 388, 390, 429 and 430) of GroEL (TTHA0271) were located in close proximity to acidic amino acid residues (i.e., within 6 Å) on the same polypeptide (Fig. 8C, Table 2 and Supplemental Fig. S13), indicating the lysines are able to contribute to intrasubunit interaction. Another acetylation site, Lys-440, was positioned within 6 Å of glutamic acid from another subunit (Fig. 8C and Supplemental Fig. S13), which contributes to the intersubunit interaction. This result indicates that all these acetylation sites might participate in regulation of GroEL in a complicate manner. In this study, we also found an evidences about how lysine acetylation affect to an interaction



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between proteins and nucleic acids (Table 2). We identified the acetylation of 20 translationrelated proteins in T. thermophilus HB8 (Fig. 6A). One of the acetylation sites in elongation factor Tu (TTHA0251), Lys-53, was located near the phosphate group of tRNA at a distance of 5.7 Å (PDB ID, 2XQD, Fig. 8D, Table 2 and Supplemental Fig. S11). Lys-7 in 50S ribosomal protein L13 (TTHA1465), Lys-18 in 30S ribosomal protein S19 (TTHA1688) and Lys-102 in 50S ribosomal protein L2 (TTHA1689) were all found to be acetylated and in close proximity to rRNA at a distance of 4.8 Å (Supplemental Fig. S75), 5.2 Å (Supplemental Fig. S42) and 3.2 Å (Supplemental Fig. S10), respectively. Because of reasonable distance within 6 Å distance, the acetylation at these sites can decreases the binding affinity of the protein for the phosphate group of RNA. Elongation factor-P binds to the ribosome in the first step of peptide bond formation 60. The structure of elongation factor-P bound to 70S ribose in T. thermophilus HB8 (PDB ID, 3HUW) showed that Lys-29 of TTHA1125 was situated at a distance of 3.1 Å from the oxygen of the ribose of the P-loop of 23S rRNA (Fig. 8E and Supplemental Fig. S58). The previous study proposed that this lysine residue may contribute to correct positioning of the P-site of tRNA or additional stabilization of the initiator tRNA in the P-site through the interaction with the P-loop of 23S rRNA 60. Elongation factor-P (TTHA1125) was acetylated at Lys-29 in T. thermophilus HB8. The sites corresponding to acetylated lysines in T. thermophilus HB8 lacking structural information were mapped on homologous structures that have already been obtained based on a protein BLAST search from PDB. We identified some interesting proteins where acetylation occurred on the Schiff-base formation sites, near the bound ligand or intersubunit interaction site (Fig. 8F and G). The structural analysis results of homologous proteins were presented in


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‘Supplemental Figure S1 to S119’ and ‘Supplemental Table S4’ with that of authentic structures of T. thermophilus. In the genome of T. thermophilus HB8, (at least) one gene can be annotated as protein acetyltransferase and three genes as protein deacetylase. Presence of such a relatively small number of protein acetyltransferases and deacetylases is advantageous for identifying the interrelationship between the protein acetyltransferases and their targets. Therefore, we believe that T. thermophilus HB8 is suitable for further acetylome analysis, especially focusing on elucidating the structure-function relationships of acetylproteins.

CONCLUSIONS Here, we identified 197 lysine acetylation sites and 4 N-terminal acetylation sites from 128 proteins of T. thermophilus. The analysis of amino acid sequence context revealed that lysine surrounded by negatively charged amino acids was frequently acetylated.

This result was

consistent with those of E. coli and S. cerevisiae but not S. enterica. In addition, we examined significance of acetylation using secondary structure of 172 acetylation sites of 113 authentic and homologous protein structures, and tertiary structure of 103 acetylation sites of 59 authentic protein structures. Structural evidences with precise analysis under atomic level suggest that acetylation affect protein-protein interactions and overall protein stability by altering electrostatic interactions and hydrogen bonding networks.

Finally, we should note that 10% of lysine

acetylation occurs at the enzymatically important positions such as substrate binding sites, Schiff-base forming lysine residues and nucleic acid binding sites in T. thermophilus.

Supporting Information 25 ACS Paragon Plus Environment


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Supplemental Table S1 showed experimental outline of this study with technical replicates of two different types of anti-acetyllysine polyclonal antibodies and results of enrichment of acetylpeptides. Supplemental Table S2 contains general information of acetylpeptides identified in this study and UniProt protein IDs of orthologous acetylproteins of 6 representative organisms whose acetylome studies had been published. Supplemental Table S2 also contained MS/MS information (mascot score, charge state, delta mass and mass measurement accuracy) of each identified acetylpeptide. Supplemental Table S3 presented results of protein BLAST search in 11 model organisms to find homologs of proteins that is subject to acetylation in T. thermophilus HB8. Supplemental Table S4 showed structural information nearby acetylated lysine as well as molecular distance between the lysine and bound ligand or amino acid residue. Supplemental Table S5 contained BLAST search results of 59 homologous proteins which were not registered their authentic structure of T. thermophilus yet.

In Supplemental Figure S1 to S119, we

presented results of MS/MS spectrum, mascot search and multiple amino acid sequence alignment of each identified acetylpeptide, and stereoview of tertiary structure of acetylproteins with mapped acetylation site. Supplemental Figure S120 presented amino acid sequence context around acetylated proteins in E. coli (A) and S. enterica (B), respectively. Supplemental Figure S121 showed alignment result of amino acid sequence around acetylated lysine between acetylprotein of T. thermophilus and its homolog of other organism whose tertiary structure is registered in PDB. Supplemental Figure S122 showed distribution of secondary structures that containing acetylated lysine, and compared the result with that of ordinary lysine of 10 representative proteins. Supplemental Figure S123 was presented proportion of potential roles of acetylated lysine and ordinary lysine. Supplemental Figure S124 showed results of Western blot analysis of whole-cell lysate using anti-acetyllysine polyclonal antibodies. These material are


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available free of charge via the Internet at http://pubs.acs.org.

Corresponding author: *E-mail: [email protected]; [email protected], Tel: +81-6-6850-5434, Fax: +81-6-6850-5442

Notes The authors have declared no conflict of interest.

ACKNOWLEDGMENT

ABBREVIATIONS nano-LC, nano-scale liquid chromatography; PDB; Protein Data Bank; COGs, Clusters of Orthologous Groups; AMPPNP, adenylyl-imidodiphosphate



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FIGURE LEGENDS

Figure 1. A representative MS/MS spectrum of the acetylpeptide (GGADFLKacTSTGFGPR) from deoxyribose-phosphate aldolase (TTHA1186). In the MS/MS spectrum of m/z 776.94 at a retention time of 62.4 min, N-terminal fragment ions (b-type ions) and C-terminal fragment ions (y-type ions) that were detected are labeled.

Figure 2. Summary of acetylated proteins and peptides. (A) Venn diagram of acetylproteins identified using two different anti-acetyllysine antibodies.

(B) Distribution of multiple-site

acetylation in proteins. Gray bars indicate the number of proteins in each group based on the number of multiple-site acetylations.

White bars indicate the number of acetylation sites

contained in multiple-site acetylproteins.

Figure 3. (A) Classification of conserved acetylproteins. Each protein in the six groups has orthologs from nine representative species (D. radiodurans, E. coli, B. subtilis, S. solfataricus, H. salinarum, S. cerevisiae, A. thaliana, M. musculus and H. sapiens) across all three domains of life. Grouping was performed as follows; Completely conserved, 9 orthologs; Well conserved, 6 to 8 orthologs; Conserved, 3 to 5 orthologs; Poorly conserved, 1 to 2 orthologs; Novel, 0 orthologs. (B) The number of orthologous acetylproteins in six organisms that have previously been reported in acetylome studies.

Figure 4.

Analysis of amino acid sequence frequency around the acetylation sites.

frequency is represented by percent difference, P value = 0.05.


The

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Figure 5. Classification of potential roles of the acetylation sites based on structural mapping. Annotations of 103 acetylation sites were based on 59 authentic protein structures of T. thermophilus HB8. (A) Distribution of potential function of acetylated lysine residues. The numbers in bracket refer to the acetylation site.

Figure 6. Functional classification of acetylproteins of T. thermophilus HB8. (A) Functional distribution of 128 lysine-acetylated proteins based on COGs database and KEGG Orthology. (B) Distribution of functional subcategories in ‘metabolism’.

Figure 7. Acetylproteins in representative metabolic flows in T. thermophilus HB8. Proteins related to glycolysis/gluconeogenesis and TCA cycle were preferentially acetylated. Acetylproteins are highlighted in gray.

Figure 8. The representative structures of acetylation sites. Lysine acetylation sites and the corresponding sites in homologs are highlighted by magenta sticks.

Previously reported

phosphorylation sites (8) are highlighted by red sticks. The ligand, RNAs and amino acids that can interact with acetylated lysines in T. thermophilus HB8 are displayed by sticks. Blue dashed lines indicate possible connections between two atoms. (A) Deoxyribose-phosphate aldolase (TTHA1186) from T. thermophilus HB8 (PDB ID: 1UB3), which forms a Schiff base with 1hydroxy-pentane-3, 4-diol-5-phosphate (HPD). (B) Inorganic pyrophosphatase (TTHA1965) from T. thermophilus HB8 (PDB ID: 2PRD). A sulfate ion, which mimics a phosphate ion, is bound in the active site. (C) GroEL-GroES complex (TTHA0271 and TTHA0272) from T.



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thermophilus HB8 (PDB ID: 1WF4).

(D) Elongation factor Tu (TTHA0251) from T.

thermophilus HB8 (PDB ID: 2XQD).

(E) Elongation factor P (TTHA1125) from T.

thermophilus HB8 (PDB ID: 3HUW)).

(F) Bifunctional fructose 1,6-bisphosphate

aldolase/phosphatase from S. tokodaii (PDB ID: 3R1M), a TTHA0980 homolog. This enzyme forms a Schiff base with dihydroxyacetone phosphate (DHAP). (G) Rod shape-determining protein MreB from T. maritima (PDB ID: 1JCG), a TTHA1816 homolog. imidodiphosphate (AMPPNP) is bound.


Adenylyl-

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TABLE Table 1. List of identified acetylproteins in T. thermophilus HB8 †. ORF

Definition

Acetylpeptide sequence

Metabolism Energy production and conversion 1

TTHA0185

pyruvate dehydrogenase complex, pyruvate dehydrogenase E1 component

K429acLTEEDLK

2

TTHA0206

nicotinamide nucleotide transhydrogenase, alpha subunit 1

ELTEEEK235acR

3

TTHA0230

2-oxoisovalerate dehydrogenase, E1 component beta subunit

VVVLGEDVGK33acR SVK181acEEVPEEDYTLPIGK

4

TTHA0233

pyruvate dehydrogenase complex, dihydrolipoamide dehydrogenase E3 component

ALLHAAETLHHLK66acVAEGFGLK ALK163acVEEGLPK AGVK284acVDER VGK373acFPLAASGR

5

TTHA0278

MoxiLGEK413acIR

ATP-dependent phosphoenolpyruvate carboxykinase

LFQENFQK512acYASGVAK 6

TTHA0287

2-oxoglutarate dehydrogenase E3 component (dihydrolipoamide dehydrogenase)

VGK367acFPYSASGR

7

TTHA0288

2-oxoglutarate dehydrogenase E2 component (dihydrolipoamide succinyltransferase)

ILK131acEDVMR

8

TTHA0289

2-oxoglutarate dehydrogenase E1 component (2-oxoglutarate dehydrogenase)

VYYDLLQK797acR

9

TTHA0537

succinyl-CoA synthetase alpha chain

EGQFHTK25acQMLSYGTK AVEEIK112acALGSR LIGGNCPGIISAEETK133acIGIMPGHVFKR

10

TTHA0538

YGVPVPPGK36acVAYTPEEAKR

succinyl-CoA synthetase beta chain

VAYTPEEAK45acR LADTPQEAYEK84acAQAILGMNIK VAGTAEEEAK362acK 11

TTHA1117

iron-sulfur protein

SGNPWGIGQDK404acR

12

TTHA1145

electron transfer flavoprotein, beta subunit

VTLISEEIQEK217acSR

13

TTHA1272

V-type ATP synthase subunit B

LGVSK86acEMLGR



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

14

TTHA1343

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EALYEK120acGLDLIAK

citrate synthase

AGVLEK273acLAR EYQILK292acIVEEEAGK IVEEEAGK300acVLNPR 15

TTHA1454

succinate dehydrogenase, flavoprotein subunit

K321acLPDITEFSR

16

TTHA1965

inorganic pyrophosphatase

GSGNK30acYEYDPDLGAIKLDR GSGNKYEYDPDLGAIK41acLDR VVGLLLMEDEK100acGGDAK

Amino acid transport and metabolism 17

TTHA0270

carboxypeptidase

IGEWLEK69acVEGSPLVQDPLSDAAVNVR

18

TTHA0534

aspartokinase (aspartate kinase)

AVHQAFELDK404acA

19

TTHA1210

2-isopropylmalate synthase (LeuA)

K115acTEEEVLEMADR

20

TTHA1240

spermidine/putrescine ABC transporter, ATP-binding protein

IEQVGLPDEVYERPK245acTR

21

TTHA1524

serine hydroxymethyltransferase

GLTGK326acEAEER

22

TTHA1605

probable acylamino-acid-releasing enzyme

SEVLWEK539acSPLR

23

TTHA1634

peptide ABC transporter, peptide-binding protein

K395acAEALLAEMGWR

24

TTHA1755

acetylornithine/acetyl-lysine aminotransferase

AAELGPWFMEK320acLR

25

TTHA1852

oligoendopeptidase F

VLEEK553acVAQLER

Nucleotide transport and metabolism 26

TTHA0188

nucleoside diphosphate kinase

EK55acPFFPGLVR

27

TTHA0432

IMP dehydrogenase/GMP reductase

YFQDPEK411acGETEAK

28

TTHA1186

deoxyribose-phosphate aldolase

GGADFLK151acTSTGFGPR

29

TTHA1466

CTP synthase

LYGK462acEEVLER

30

TTHA1552

GMP synthase

LLFK359acDEVR

31

TTHA1613

putative adenine phosphoribosyltransferase

FAEK117acLLNQR

32

TTHA1742

orotate phosphoribosyltransferase

ALFAEK88acDGR

33

TTHB209

ribonucleoside-diphosphate reductase, alpha subunit

AEALTK49acVEGFGR

ribonucleoside-diphosphate reductase, alpha subunit

NAEIFK359acTIR

Carbohydrate transport and metabolism


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


34

TTHA0002

enolase

DGK252acYHLEGEGK

35

TTHA0481

oligo-1,6-glucosidase

NVAAQEK435acDPR

36

TTHA0579

sugar ABC transportor, ATP-binding protein

AEIAK178acLQR

37

TTHA0905

glyceraldehyde 3-phosphate dehydrogenase (GAPDH)

GVEVALINDLTDNK36acTLAHLLK LLDLPHK190acDLR EVTAEEVNAALK259acAAAEGPLK

38

TTHA0906

GPDPK67acYSLAPVGEALR

phosphoglycerate kinase

FEPGEEK122acNDPELSAR VSDK198acIGVIESLLPR SLVEEDRLDLAK244acDLLGR 39

TTHA0980

conserved hypothetical protein

YVGK233acDDPVAIVR

40

TTHA1773

fructose-1,6-bisphosphate aldolase

LAGIEEHVAVDEK148acDALLTNPEEAR

41

TTHA0122

manganese-containing pseudocatalase

ALESLTGVEMTK206acMLPIPK

42

TTHA1028

thiosulfate sulfurtransferase

NIPWAK207acAVNPDGTFK

Inorganic ion transport and metabolism

AVNPDGTFK216acSAEELR 43

TTHA1040

cation efflux system membrane protein

ALEVHDLK231acTR

44

TTHA1477


VIEWVK92acVLPGSK

45

TTHA1622

probable thiosulfate sulfurtransferase

SPEEFQGK146acVHPPCCPR

46

TTHA1628

iron ABC transporter, periplasmic iron-binding protein

GVALDPNLLPLEEALAK306acSPK

47

TTHA1662


ALK121acEEVAETLEK

Lipid transport and metabolism 48

TTHA0290

probable enoyl-CoA hydratase

AFSAGQDLTEFGDRK70acPDYEAHLR

49

TTHA0559

acetyl coenzyme A acetyltransferase (thiolase)

FGGVFK22acDVSPVDLGAHAMR

50

TTHA0890

putative 3-hydroxyacyl-CoA dehydrogenase

TK304acLELPELR

51

TTHA1246

methylmalonyl-CoA mutase

AIDPLGGSFYVEHLTDK370acLER

52

TTHA1248

acetyl-coenzyme A synthetase

LESVLK9acEER KVLEGDLPHPK72acWFVGGK

53

TTHA1262

GFGDDK255acYRPSPLLR

3-hydroxybutyryl-CoA dehydrogenase



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

54

TTHA1434

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VAEDALEEAK204acK

3-hydroxybutyryl-CoA dehydratase

Coenzyme transport and metabolism 55

TTHA0341

molybdopterin biosynthesis enzyme, MoaB

VGILTVSDK12acGFRGER

56

TTHA1642

S-adenosylmethionine synthetase

ISDAILDALIAQDK37acK

57

TTHB056

precorrin-3 C17-methyltransferase

GYALK264acYDLDTK

58

TTHB066

cobyric acid synthase/cobinamide kinase

DEEMAEK479acIR

Multifunctional 59

TTHA0006

1-deoxy-D-xylulose-5-phosphate synthase

KWVQDAEK202acLGK

60

TTHA0413

3-oxoacyl-[acyl carrier protein] synthase II

VVVTGLGALTPIGVGQEAFHK24acAQLAGK

61

TTHA0604

medium-chain-fatty-acid--CoA ligase

TSAGK524acFLKR

62

TTHA1211

probable ketol-acid reductoisomerase (IlvC)

SWEK55acAEAAGLR

Translation 63

TTHA0246

50S ribosomal protein L1

ALLEK14acVDPNKVYTIDEAAR

64

TTHA0251

translation elongation factor EF-Tu.B

AK3acGEFIR DYGDIDK53acAPEER GITINTAHVEYETAK75acR

65

TTHA0366

aspartyl/glutamyl-tRNA(Asn/Gln) amidotransferase subunit B

TK256acEEEADYR

66

TTHA0699

translation initiation factor IF-2

GLQEK59acLAEEER

67

TTHA0860

elongation factor Ts (EF-Ts)

YVSAEEIPAEELEK120acER

68

TTHA0861

30S ribosomal protein S2

NdeamiGIHIIDLQK46acTMEELER LK139acHELER

69

TTHA1067

isoleucyl-tRNA synthetase (isoleucine--tRNA ligase) (IleRS)

VLPNLK878acLLGR

70

TTHA1125

elongation factor P (EF-P)

MDGGLWECVEYQHQK29acLGR

71

TTHA1169

valyl-tRNA synthetase (valine--tRNA ligase) (ValRS)

VWQWK118acEESGGTILK

72

TTHA1465


TYVPK7acQVEPR

73

TTHA1485


acMIGK4acEEIHR AK45acDAIKALEVPGDLSER TAFLDEK107acAGR GGAGQDLFAK202acR


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FYK213acALAHELER LVLMGPEEHTK241acLFLGHLPK 74

TTHA1675


TK145acADVER

75

TTHA1684


KLSPVELEK23acLVR

76

TTHA1686


GLLEK45acELYSAGLAR

77

TTHA1688


KGVFVDDHLLEK18acVLELNAK

78

TTHA1689


IALLHYVDGEK102acR

79

TTHA1695

elongation factor G (EF-G)

acAVK4acVEYDLKR acAVKVEYDLK10acR ADQEK423acLSQALAR

80

TTHA1696


IIQEK53acTGQEPLKVFK

81

TTHA1785


MFFEK5acIAPYTYRIPR

82

TTHA1799


EAK110acYLLLR

Post-translational modification, protein turnover, chaperones Protein refolding 83

TTHA0271

EVELEDHLENIGAQLLK74acEVASK

60 kDa chaperonin (Protein Cpn60) (GroEL protein)

AVEAAVEK129acIK LIADAMoxiEK166acVGK VGK169acEGIITVEESK GK336acKEDIEAR GKK337acEDIEAR K349acELETTDSEYAR VGAATETELK388acEK VGAATETELKEK390acK AISAVEELIK429acK K430acLEGDEATGAK KLEGDEATGAK440acIVRR 84

TTHA0272

10 kDa chaperonin (Protein Cpn10) (GroES protein)

RIEEEPK26acTK

85

TTHA0614

trigger factor

ASYEALLK32acDLASR



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

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EALLQDLK67acER 86

TTHA1480

acMoxiLEK4acLWPFGR

small heat shock protein, HSP20 family

K15acAVEEALEK DAK102acKEGIEAR 87

TTHA1484

small heat shock protein, HSP20 family

FDPFK8acELEELQER

88

TTHA1487

ATP-dependent Clp protease, ATP-binding subunit ClpB

GLKEK347acYEVHHGVR LTEEIAK448acLR YGELPKLEAEVEALSEK510acLR LEEELHK558acR IDMTEYMEK630acHAVSR EHGVDLK245acADR

89

TTHA1491

chaperone protein DnaK (heat shock protein 70)

90

TTHA0403

cell division protein FtsH

DVAGHEEAK174acR

91

TTHA0542

ATP-dependent Clp protease, ATP-binding subunit (ClpA)

YIEK317acDAALER

Protein degradation

Stress response 92

TTHA1300

bacterioferritin comigratory protein, thiol peroxidase, putative

FAEK84acYGLNFPLLADPERK NLYGK114acEVEGVLR

93

TTHA1625

osmotically inducible protein OsmC

FLEIAEAAK120acEGCPVSR

94

TTHA1838

SufC protein (ATP-binding protein)

LALQAK108acLGR YLNEGFSGGEK149acK VVATGGPELALELEAK237acGYEWLK GYEWLKEK245acVK

95

TTHA1839

DAK93acSWEEVPEEIRR

SufB protein

YEDLFK161acEYFAK GLVK364acVMEGAR GLK427acEDEAAALIVR 96

TTHA1840

ALGEPAWLLEK25acR

SufD protein

LPYPSK41acKDENWR LPYPSKK42acDENWR


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VLLEGEK178acASAGR GAQK336acTDAYQANR GLAQELLVK402acAHLADVLSR Transcription 97

TTHA0139


HLK69acAEEGEEEA

98

TTHA0701

N utilization substance protein A (NusA)

SEQIPTEK182acYHPGQR AHEK213acLLEHLLK

99

TTHA1664

TDLDK194acLTLR

DNA-directed RNA polymerase alpha chain

VLHSLK270acEEGIESVR 100

TTHA1812

DNA-directed RNA polymerase beta' chain (RpoC)

FTQVVDQK1490acTLK

101

TTHA1813

DNA-directed RNA polymerase beta chain (RpoB)

LYHMVEDK1004acMHAR

Replication, recombination and repair 102

TTHA1016


AVQGENPALEK90acAR

103

TTHA1054

DNA polymerase I

LLWLYHEVEK437acPLSR

104

TTHA1349

DNA-binding protein HU (DNA-binding protein II)

AMVDALLAK37acVEEALANdeamiGSK

105

TTHB151


LALLLPEDHPEAQEPK371acTR

Signal transduction mechanisms 106

TTHA0843

FKEK64acLLHYR

serine protein kinase

LFDDQK615acDTIR 107

TTHA1046

PhoH-related protein

IIK318acAYEEAER

108

TTHA1864

S-layer protein-related protein

SLAALKEEK112acEALETR

Cell cycle control, mitosis and meiosis 109

TTHA1089

ccell division protein FtsZ

IQLGEK56acLTR

110

TTHA1816

rod shape-determining protein MreB

TAEELK210acIR

111

TTHA1893

S-layer protein precursor (P100 protein)

SDLVGLSDK279acVSK LEEQVAELNK292acVR AVSSFK423acFNDYLFANDNDSEPANPR

Cell wall/membrane biogenesis 112

TTHA1085

APNYHESLEALK196acEAMR

UDP-N-acetylmuramate--alanine ligase



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

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Multifunctional 113

TTHA0365

type IV pilus assembly protein, pilus retraction protein PilT

EGK302acTHQLR

114

TTHA0466

alcohol dehydrogenase

VIATAGSEDK203acLR ALGADETVNYTHPDWPK225acEVR

115

TTHA0472

peptide ABC transporter, ATP-binding protein

DLTK86acLSEAEMR

116

TTHA0474

acetoin utilization protein AcuB (acetoin dehydrogenase)

SPVLTVGPEATLEEAYK25acLLLER

117

TTHA0829

putative acetoin utilization protein, acetoin dehydrogenase

LVGLVTDK50acDLK

118

TTHA1233

short-chain dehydrogenases/reductases family protein

SYK209acEEGLLLTPEEAAR

119

TTHA1473


TEIPK102acVHPEDPLGK

120

TTHA1632*

121 TTHB182

peptide ABC transporter, permease protein

acATATASPKFPR


SALLGK72acPELGDER

General function prediction only 122

TTHA0660

oxidoreductase, short-chain dehydrogenase/reductase family

AGSLGEGLSLHPLDITDK66acEK

123

TTHA1483

conserved putative protein

ALWELK147acDYPK

124

TTHA1817

putative hydrolase (HD domain)

SLQEELK132acEER

Function unknown 125

TTHA0972

phenylacetic acid degradation protein PaaA

YDEK264acTGNWIHGPIPWDEFWK

126

TTHA1048


VGEGIVTTIGSANSHK189acEVLENPDR

127

TTHA1902


AFFHLLK490acGASPEELR

128

TTHB059

hypothetical protein

IAIGELLEK19acEAHDLLHER LAEAK48acTLPALEEALR

†

Table 1 listed 128 acetylated proteins with 198 acetylated peptides of both N-terminal acetylation and lysine acetylation. * Only N-terminal acetylation was detected in TTHA1632. Finally, N-ε−acetyllysine was detected in 197 lysine sites of 127 proteins in T. thermophilus.


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Table 2. List of molecular distance and potential function of each acetyllysine based on structural mapping.

TTHA0230

PDB I.D. 1UMC

TTHA0233

2EQ8

TTHA0246

2Y15

TTHA0251

2XQD

TTHA0270

1WGZ

TTHA0271

1WF4

TTHA0278

1XKV

Acetylprotein

TTHA0287 TTHA0288 TTHA0341 TTHA0413 TTHA0466

2IS8 1J3N 2EIH

TTHA0537

1OI7

TTHA0860 TTHA0861

1AIP

TTHA0905

1VC2

TTHA0906

1V6S

TTHA1028 TTHA1040 TTHA1125 TTHA1169

1UAR 3BYR 3HUW 1IVS

2EQ7

Acetylation site K33 K163 K373 K14 K3 K53 K75 K69 K74 K166 K169 K336 K337 K349 K388 K390 K429 K430 K440 K413 K512 K367 K131 K12 K24 K225 K112 K133 K120 K139 K36 K190 K259 K198 K244 K216 K231 K29 K118

Potential function

Counterpart

Intersubunit interaction Electrostatic interaction Electrostatic interaction Electrostatic interaction Electrostatic interaction Nucleic acid binding Electrostatic interaction Electrostatic interaction Electrostatic interaction Electrostatic interaction Electrostatic interaction Electrostatic interaction Electrostatic interaction Electrostatic interaction Electrostatic interaction Electrostatic interaction Electrostatic interaction Electrostatic interaction Intersubunit interaction Hydrogen bond Hydrogen bond Electrostatic interaction Intersubunit interaction Electrostatic interaction Electrostatic interaction Electrostatic interaction Electrostatic interaction Hydrogen bond Electrostatic interaction Electrostatic interaction Hydrogen bond Electrostatic interaction Electrostatic interaction Electrostatic interaction Hydrogen bond Hydrogen bond Ligand binding Nucleic acid binding Electrostatic interaction

D211 E166 E211 E32 E372 tRNA D205 E68 E61 D162 E138 E338 E342 E352 E207 E386 E426 E426 E432 S258 N509 E209 E425 E17 E20 D222 D189 M137 E121 E143 Y57 D181 E263 D197 A225 K207 Zinc rRNA E119


Distance (Å) 5.3 4.5 2.6 3.6 3.0 5.7 3.2 4.2 3.5 3.8 4.3 4.4 4.4 5.4 2.3 3.4 3.4 5.3 2.7 2.9 3.0 2.7 2.6 2.9 4.7 4.4 5.9 2.8 3.9 2.6 2.8 5.4 4.1 5.8 2.6 2.8 5.9 3.1 1 2.8


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

TTHA1186

1UB3

TTHA1343

1IXE

TTHA1465 TTHA1483

3D5B 1UF3

TTHA1487

1QVR

TTHA1524 TTHA1552 TTHA1613 TTHA1622 TTHA1625 TTHA1634 TTHA1664 TTHA1664 TTHA1675 TTHA1684 TTHA1686 TTHA1688 TTHA1689

2DKJ 2YWC 1VCH 2EG4 1UKK 2D5W 2O5J 1DOQ 3OHY 2Y15 3OHY 3OHY 2Y15

TTHA1695

3IZP

TTHA1755 TTHA1799 TTHA1812 TTHA1813 TTHA1838

1VEF 2ZXV 1IW7 2O5I 2D2F

TTHA1965

2PRD

TTHB059

2EHW

K151 K120 K273 K292 K300 K7 K147 K347 K448 K558 K630 K326 K359 K117 K146 K120 K395 K194 K270 K145 K23 K45 K18 K102 K4 K10 K423 K320 K110 K1490 K1004 K237 K30 K41 K48

Schiff-base Electrostatic interaction Electrostatic interaction Electrostatic interaction Electrostatic interaction Nucleic acid binding Intersubunit interaction Electrostatic interaction Electrostatic interaction Electrostatic interaction Electrostatic interaction Electrostatic interaction Electrostatic interaction Intersubunit interaction Hydrogen bond Hydrogen bond Electrostatic interaction Electrostatic interaction Electrostatic interaction Electrostatic interaction Electrostatic interaction Hydrogen bond Nucleic acid binding Nucleic acid binding Electrostatic interaction Electrostatic interaction Hydrogen bond Electrostatic interaction Hydrogen bond Intersubunit interaction Electrostatic interaction Electrostatic interaction Ligand binding Intersubunit interaction Electrostatic interaction

HPD 2 D106 E272 E295 E296 rRNA E145 D471 E445 E554 E629 E330 D221 D34 Q144 V135 E390 E26 E271 E149 E20 G41 rRNA rRNA E368 D285 K471 E319 T94 T38 D1003 E233 Sulfate E87 E15

0.0 2.6 4.7 2.7 4.5 4.8 2.6 3.1 3.4 4.3 4.2 4.3 5.1 5.8 2.6 2.3 2.7 3.8 5.1 3.9 5.4 2.7 5.2 3.2 2.6 2.7 2.7 4.0 2.8 3.0 3.2 2.6 3.2 3.3 4.1

This distance, 3.1 Å, didn't satisfy our criteria about a hydrogen bond. However, previous study suggested that

this lysine residue participates in protein-RNA interaction by hydrogen bond. 2

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1-hydroxy-pentane-3, 4-diol-5-phosphate


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Abstract graphic


Acetylome with Structural Mapping Reveals the Significance of Lysine

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