Journal of Molecular Biology
Structure and Substrate Binding Properties of cobB, a Sir2 Homolog Protein Deacetylase from Escherichia coli
Introduction
Sirtuins are NAD+-dependent protein deacetylase enzymes that are broadly conserved within the three domains of bacteria, archaea and eukaryotes.1 The founding member of this family of proteins is the yeast Sir2 protein that silences gene expression through the deacetylation of specific acetyl-lysine residues in histones H3 and H4, and whose activity is correlated with longevity in yeast and worms.2., 3., 4. However, analysis of sirtuins from various species indicates that they have much broader biological functions and substrate specificities than yeast Sir2. For example, of the seven known mammalian sirtuins, the nuclear SIRT1 has been shown to target the p53 tumor suppressor protein for deacetylation to suppress the apoptotic program in response to DNA damage,5., 6. and the cytoplasmic SIRT2 homolog has been shown to target α-tubulin for deacetylation for the maintenance of cell integrity.7 In addition, the Sir2 homolog from the archaea Sulfolobus solftaricus deacetylates the non-specific DNA protein Alba to mediate transcription repression.8
cobB is a particularly interesting bacterial Sir2 homologue. This protein was originally found to be involved in cabalamin synthesis in the bacterium Salmonella typhimurium LT2 and to partially compensate for the inactivation of cobT, a phosphoribosyltransferase enzyme. Subsequent studies revealed that cobB and yeast Sir2 harbored weak NAD+-dependent ADP-ribosyltransferase activity,9., 10. and later studies revealed that these enzymes had robust NAD+-dependent histone/protein deacetylase activity.11., 12., 13. In Salmonella enterica, cobB was shown to activate acetyl-CoA synthetase (Acs) by specifically hydrolyzing the acetyl group from an active site acetyl-lysine of Acs to promote its activity.14 The finding that yeast Sir2 homologues can also stimulate the activity of Acs from S. enterica15 further suggests that some eukaryotic sirtuin proteins may play similar metabolic roles.
Insights into the mechanism of sirtuin activity have been provided by the structure of eukaryotic and archaeal proteins in various liganded forms. Specifically, the structures of human SIRT2 and yeast Hst216., 17. have been determined in nascent form, and the structures of archaeal Sir2 proteins have been determined in binary complex with either NAD+18 or an acetyl-lysine-containing peptide substrate from the human p53 tumor suppressor protein.19 Finally, the structure of yeast Hst2 has been determined in binary complex with NAD+ and in ternary complex with an acetyl-lysine-containing histone H4 peptide and the NAD+ product, 2′-O-acetyl-ADP-ribose.20 Together, these structures reveal a structurally similar catalytic core containing a large Rossmann fold domain that shows high-level structural conservation among the sirtuin proteins and a smaller zinc-binding domain that shows more variability. In each of the structures, these two domains are held together by a series of loops that traverse four times between the two domains, and contribute to the formation of a cleft between the large and small domains. The liganded structures reveal that the NAD+ and acetyl-lysine substrates bind to adjacent sides of the cleft. These structures, together with biochemical and enzymatic studies reveal that sirtuin conserved residues mediate catalysis with formation of the products lysine, nicotinamide and 2′-O-acetyl-ADP-ribose that equilibrates to a roughly 50/50 mixture with a 3′-O-acetyl-ADP-ribose product.21., 22., 23. Each of the sirtuin complexes with acetyl-lysine bearing substrates also show a highly conserved mode of acetyl-lysine recognition involving highly conserved residues. The mode of substrate selectivity by the Sir2 proteins has been more difficult to resolve. This is contributed by the fact that many Sir2 proteins show poor discrimination for substrates in vitro and there is no available structure of a Sir2 protein bound to its true cognate substrate. Indeed, the Sir2 proteins that have been crystallized with acetyl-lysine-bearing substrates, Af2-Sir2 and yeast Hst2, do not yet have identified substrates.
In order to obtain further insight into the evolutionary conservation of the sirtuin proteins, we determined the crystal structure of the first bacterial Sir2 homolog, cobB, from Escherichia coli in complex with an acetyl-lysine-bearing peptide form the histone H4 protein. To also investigate the discrimination of cobB for cognate and non-cognate acetyl-lysine-bearing substrates, we used isothermal calorimetry experiment to quantitatively compare the binding properties of cobB for cognate acetylated Acs protein and peptide as well as peptides from other known sirtuin substrates. The results of these studies provide new mechanistic insights into the evolutionary relationship of sirtuin proteins and their mode of substrate selectivity.
Section snippets
Overall structure of the cobB/histone H4 complex
The crystal structure of the E. coli cobB core domain (residues 40–274) in complex with an 11-residue peptide containing residues 12–19 of histone H4 and acetylated at lysine 16 was determined by a combination of Zn2+ and Se multiwavelength anomalous diffraction (MAD) to 1.96 Å resolution (Table 1). The final model contains 230 ordered protein residues, with residues 38–39, 175–181, 246–248 and 275–279 omitted from the model due to poor electron density in the corresponding protein region, and
CobB expression and purification
The cobB gene coding for the conserved Sir2 catalytic core (residues 38–279) was PCR amplified from genomic E. coli DNA and inserted into the pRSET-A expression vector. The protein was overexpressed in the E. coli BL21 (DE3) strain by initially growing cells at 37 °C to an absorbance of 0.5–0.7 at A590, and inducing by addition of 0.5 mM IPTG and overnight growth at 15 °C. Cells were disrupted by sonication in buffer A (20 mM Hepes (pH 7.5), 50 mM NaCl, 10 mM DTT, and 1 mM PMSF) and the
Acknowledgments
We thank the staff at the Brookhaven National Laboratory for assistance at beamline X25; the staff at Advanced Photon Source for help at beamline 19ID; and K. D. Speicher, D. F. Reim & T. Beer of the Proteomics Facility at The Wistar Institute for help with the MALDI mass spectrometry. This work was supported by NIH grants to R.M. and by a grant from the Commonwealth Universal Research Enhancement Program, Pennsylvania Department of Health awarded to the Wistar Institute.
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2021, European Journal of Medicinal ChemistryCitation Excerpt :In particular, amino acid residues corresponding to positions 8–19 of H4 and having the KRHR sequence (residues 16–19) are important for substrate recognition by deacetylase enzymes [24]. CobB is a Sirt2 homolog protein deacetylase found in E. coli and studies have shown interaction of CobB with the histone H4 peptide (12-KGGAKAcRHRKIL-22), pointing to the importance of KRHR sequence in recognition by prokaryotic and eukaryotic deacetylase enzymes [26]. Similar enzymes (SAV0325) are found in S. aureus [17] and also in Candida called as Sirt2 and Hst proteins [27].