Insights into the specificity of lysine acetyltransferases

Abstract

Reversible lysine acetylation by protein acetyltransferases is a conserved regulatory mechanism that controls diverse cellular pathways. Gcn5-related N-acetyltransferases (GNATs), named after their founding member, are found in all domains of life. GNATs are known for their role as histone acetyltransferases, but non-histone bacterial protein acetytransferases have been identified. Only structures of GNAT complexes with short histone peptide substrates are available in databases. Given the biological importance of this modification and the abundance of lysine in polypeptides, how specificity is attained for larger protein substrates is central to understanding acetyl-lysine-regulated networks. Here we report the structure of a GNAT in complex with a globular protein substrate solved to 1.9 Å. GNAT binds the protein substrate with extensive surface interactions distinct from those reported for GNAT-peptide complexes. Our data reveal determinants needed for the recognition of a protein substrate and provide insight into the specificity of GNATs.

Keywords: Acetyl Coenzyme A (Acetyl-CoA); Bacterial Metabolism; Enzyme Inactivation; Histone Acetylase; Post-translational Modification (PTM); Substrate Specificity.

FIGURE 1.

SlPatA^GNAT domain and in vivo function. A, regulation of SeAcs^WT activity by SePat^WT or SlPatA^GNAT in S. enterica. B, domain arrangements of characterized wild-type protein acetyltransferases are listed for S. lividans (SlPatA), S. enterica (SePat), and Mycobacterium tuberculosis (MtPatA). cNMP, cyclic mononucleotide monophosphate-binding domain. C, ribbon representation of SlPatA^GNAT with its catalytic residue Glu¹²³ shown in purple (PBD code 4NXY). Ac-CoA was modeled into the SlPatA^GNAT structure using the closely related MtPatA^WT structure as a guide (44) (PDB code 4AVB). D, alignment of SlPatA^GNAT (beige) with MtPatA (teal) shows the location of SlPatA^GNAT residues Phe¹²⁶ and Met¹⁶⁸, which overlap with the modeled Ac-CoA. Homologous residues of MtPatA (Phe²³⁸ and Met²⁸⁰) are shown and accommodate the bound Ac-CoA. SlPatA^GNAT active site residue Glu¹²³ is colored purple, and putative active site MtPatA Glu²³⁵ is shown in dark teal. E, growth of S. enterica Δpat cobB⁺ (open symbols) and Δpat ΔcobB strains (filled symbols), producing SlPatA^GNAT (squares), SlPatA^GNAT active site variant E123Q (triangles), or an empty vector (circles) on acetate. F, ClusPro interaction model of SlPatA^GNAT (beige) with SeAcs (N-terminal domain in green, C-terminal domain in blue) with active site residue Glu¹²³ (red) and SeAcs^WT target residue Lys⁶⁰⁹ (blue) shown as spheres.

FIGURE 2.

Cross-linking of variants SlPatA^GNAT S73C and SeAcs^CTD H567C with a disulfide bond results in a productive complex. A, ClusPro model of the SlPatA^GNAT-SeAcs interface. SlPatA^GNAT active site residue Glu¹²³ (red) and SeAcs target residue Lys⁶⁰⁹ (blue) are shown as spheres. Cross-linked residues are highlighted. B, schematic of SeAcs^CTD (light blue) and SlPatA^GNAT (beige) with relative location of the cross-linked residues (spheres). Residues Glu¹²³ of SlPatA^GNAT and Lys⁶⁰⁹ of SeAcs^CTD are red and blue spheres, respectively. C, chemical structures of 2.0–19.0 Å cross-link, where atoms and bonds in red are directly inserted between the cysteine residues (AT-2, aldriothiol-2; BMOE, bis(maleimido)ethane; BMB, 1,4-bis(maleimido)butane; BMH, bis(maleimido)hexane; DPDPB, 1,4-di-(3′-(2′-pyridyldithio)-propionamido). D, SlPatA^GNAT was incubated with SeAcs^CTD at molar ratios of 1:3, 1:1, and 10:1 (SlPatA^GNAT/SeAcs^CTD) in the presence of [1-¹⁴C]acetyl-CoA to visualize acetyl transfer to SeAcs^CTD (phosphor images labeled [¹⁴C] Acetylation). Samples were quenched after 60 min, separated by SDS-PAGE, and stained with Coomassie Blue to visualize proteins (labeled SDS-PAGE). Full-length SlPatA was incubated with SeAcs^CTD at a molar ratio of 1:3 (SlPatA^WT/SeAcs^CTD) for reference. Images of Coomassie Blue-stained gels and phosphor images were cropped to bands corresponding to the SeAcs^CTD. Acetylation was quantified relative to the signal obtained with SlPatA plus SeAcs^CTD and is reported as the mean (n = 3). S.D. was ≤18% of the mean value. E, transfer of the acetyl moiety from [1-¹⁴C]acetyl-CoA to the SeAcs^CTD was tested (phosphor images) for each of the SlPatA^GNAT-SeAcs^CTD complexes. Images of Coomassie Blue-stained gels (labeled SDS-PAGE) and phosphor images (labeled [¹⁴C] Acetylation) were cropped to bands corresponding to the SlPatA^GNAT-SeAcs^CTD heterodimers.

FIGURE 3.

Charged surface residues mediate interactions between SlPatA^GNAT and SeAcs^CTD. A, ribbon representation of the SlPatA^GNAT S73C-SeAcs^CTD H567C crystal structure with residues Glu¹²³ of SlPatA^GNAT and Lys⁶⁰⁹ of SeAcs^CTD shown as sticks in red and blue, respectively. B and C, electrostatic potential distribution at the interface for SeAcs^CTD and SlPatA^GNAT colored red for negatively charged, white for neutral, and blue for positively charged areas. D, interaction of the SlPatA^GNAT active site cleft with SeAcs^CTD (Arg⁶⁰⁶–Arg⁶¹⁶ shown in blue). The region shown in the black box is enlarged (E) to show specific interaction of SeAcs^CTD Arg⁶⁰⁶–Arg⁶¹⁶ with SlPatA^GNAT. F, electrostatic potential distribution at the interface for Gcn5 interaction with substrate H3 peptide (PDB code 1QSN) colored as described above. The H3 peptide is colored green with the target Lys¹⁴ shown in blue.

FIGURE 4.

SlPatA^GNAT and SeAcs^CTD residues at the interaction interface are conserved. A, alignment of sequences in and around the Acs^CTD PX₄GK motif (black box) from S. enterica (SeAcs, accession number NP_463140), Saccharomyces cerevisiae (Acs2p, accession number NP_013254), Halobacterium salinarum (HsAcs, accession number WP_0109027), and S. lividans (SlAcs, accession number EFD68454). Blue shaded boxes indicate conserved positively charged residues. *, fully conserved residue; :, residues with high similarity; ., residues with low similarity. B, alignment of GNAT domain from homologues of SlPatA (accession number EFD66247) from S. enterica (SePat, accession number XNP_461586), R. palustris (RpPat, accession number NP_494576), and M. tuberculosis (MtPat, accession number WP_003906490). Notation is described as above. Red and green shaded boxes indicate negatively charged and hydrophobic residues, respectively, observed at the SlPatA^GNAT-SeAcs^CTD interaction interface. Sequence alignment generated in ClustalW2 (45).

FIGURE 5.

Reversing charges at the interaction surface disrupts interactions between SlPatA^GNAT and SeAcs^CTD. A, effect of different Zif-SlPatA^GNAT variants and empty vector on transcription in vivo from promoter PlacZif 1–61 with ω-SeAcs^CTD variants or empty vector. B, data from the reciprocal bait-prey experiment (Zif-SeAcs^CTD and ω-SlPatA^GNAT). “ω only” refers to empty prey plasmid expressing only the ω subunit of the RNA polymerase α subunit. Zif only, empty bait plasmid expressing only the zinc finger protein. *, p < 0.0001; ∧, interactions were detected in bait-prey reciprocal experiments. Error bars, S.D.

FIGURE 6.

Full-length variants SeAcs^R606E and SeAcs^R613D are poorly acetylated by protein acetyltransferases. A, SeAcs^WT, SeAcs^R606E, and SeAcs^R613D were incubated with SlPatA or SePat in a 3:1 molar ratio (SeAcs/SlPatA or SeAcs/SePat) in the presence of [1-¹⁴C]Ac-CoA. Samples were quenched at 0, 15, 30, 60, and 90 min and separated by SDS-PAGE and stained with Coomassie Blue to visualize proteins (labeled SDS-PAGE). Acetylation was visualized by phosphor imaging (labeled [¹⁴C] Acetylation). B, reaction controls lacking SePat were incubated for 90 min and imaged as described above. Gels and phosphor images were cropped to the SeAcs bands and labeled as described in A. C, phosphor signal associated with each band in A and B was quantified as described under “Experimental Procedures.” D, SeAcs, SeAcs^R606E, and SeAcs^R613D were incubated with SlPatA (white bars) or SePat (gray bars) at the ratio described above in the presence or absence of Ac-CoA. After 90 min, SeAcs activity was measured in an NADH consumption assay. All data points are mean ± S.D. (error bars) (n = 6). DLU, digital light units.

FIGURE 7.

SlPatA^GNAT D185R interacts poorly with SeAcs in vivo. S. enterica Δpat cobB⁺ (open shapes) and S. enterica Δpat ΔcobB (filled shapes) strains producing wild-type SlPatA^GNAT (pGNAT^WT, circles), and variants SlPatA^GNAT E121R (pGNAT^E121R, squares) and SlPatA^GNAT D185R (pGNAT^D185R, triangles) in the absence of inducer during growth on no-carbon essential minimal medium supplemented with acetate (10 mm). B, growth of S. enterica Δpat cobB⁺ (squares) and S. enterica Δpat ΔcobB (circles) strains producing variant SlPatA^GNAT D185R (pGNAT^D185R) in the presence of inducer (no inducer (white), 5 μm inducer (light gray), 10 μm inducer (dark gray), 25 μm inducer (black)) during growth on no-carbon essential minimal medium supplemented with acetate (10 mm). Growth experiments were performed at 37 °C using a microtiter plate and a microtiter plate reader (Bio-Tek Instruments). All data points represent the mean value. All S.D. values are <0.015 absorbance units (n = 4).