Molecular basis for N-terminal acetylation by human NatE and its modulation by HYPK

Abstract

The human N-terminal acetyltransferase E (NatE) contains NAA10 and NAA50 catalytic, and NAA15 auxiliary subunits and associates with HYPK, a protein with intrinsic NAA10 inhibitory activity. NatE co-translationally acetylates the N-terminus of half the proteome to mediate diverse biological processes, including protein half-life, localization, and interaction. The molecular basis for how NatE and HYPK cooperate is unknown. Here, we report the cryo-EM structures of human NatE and NatE/HYPK complexes and associated biochemistry. We reveal that NAA50 and HYPK exhibit negative cooperative binding to NAA15 in vitro and in human cells by inducing NAA15 shifts in opposing directions. NAA50 and HYPK each contribute to NAA10 activity inhibition through structural alteration of the NAA10 substrate-binding site. NAA50 activity is increased through NAA15 tethering, but is inhibited by HYPK through structural alteration of the NatE substrate-binding site. These studies reveal the molecular basis for coordinated N-terminal acetylation by NatE and HYPK.

Fig. 1. HYPK and hNatE form a tetrameric complex.

a Left—gel filtration elution profile of the hNatA/HYPK complex with excess hNAA50, using a Superdex S200 column. Right—Coomassie-stained SDS–PAGE of peak fractions. Red bars indicates the peak complex. b Fluorescence polarization assays with either hNatA or hNatA/MBP-HYPK titrated into fluorescein-5-maleimide-labeled hNAA50. The data is fit to calculate a dissociation constant (K_d). Replicates were shown in the curve with n = 3 independent experiments. Source data are provided as a Source Data file. c Representative ITC curve of MBP-HYPK titrated into hNatA. The calculated K_d is indicated. d Representative ITC curve of MBP-HYPK titrated into hNatE. e The ITC fitting information and calculated K_d is provided for curves c and d.

Fig. 2. HYPK binding negatively affects hNatE acetylation activity.

a Either MBP-HYPK (green), hNAA50 (red), or both (blue) are titrated into hNatA (100 nM) to evaluate their modulatory effect on hNatA activity against an SASE peptide substrate. A best line is drawn through the data points for ease of visualization. Source data are provided as a Source Data file. b Comparison of time course acetylation activity of hNAA50 (black), preformed hNatE (red), and preformed hNatE/HYPK (blue; all 500 nM) against an MLGP peptide substrate. Source data are provided as a Source Data file. c hNatA acetylation activity against an SASE peptide with addition of buffer, wild type (WT), or hNAA50 mutants. Errors were reported in SEM with n = 3 independent experiments. Source data are provided as a Source Data file. d Activity of hNAA50 WT and mutants against MLGP peptide was tested, either alone (black), in the context of hNatE (+hNatA; red), or in the context of preformed hNatE/HYPK (+hNatA/HYPK; blue). Data were normalized to WT hNatE activity as 100%, represented as mean ± SD, n = 3 independent experiments. Source data are provided as a Source Data file.

Fig. 3. hNatA forms competing complexes with hNAA50 and HYPK.

a Heat map showing the mean relative IBAQ intensity of NatA components from three independent MS analyses of immunoprecipitated NAA15 variants, NAA15-WT-V5, NAA15-T406Y-V5, and NAA15-L814P-V5. The IBAQ intensities of each component were normalized to the IBAQ intensity of NAA15 in the respective sample and to the corresponding NatA WT protein. Source data are provided as a Source Data file. b Western blot analysis and NatA Nt-acetylation assay of V5-immunoprecipiated NAA15 variants. The measured DPM signal for each reaction was normalized to the corresponding V5-band in the IP. The immunoprecipitation and activity assay were performed in n = 3 independent experiments, each with three technical replicates (indicated by dot plots) per assay. Source data are provided as a Source Data file.

Fig. 4. Cryo-EM structure of the hNatE complex.

a hNAA50(magenta), hNAA15(green), and hNAA10(orange) within the hNatE complex shown in cartoon. b Zoom-in view of the contacts between hNAA15 and hNAA50 with residues that participate in interaction labeled. c Zoom-in view of the contacts between hNAA10 and hNAA50 with residues that participate in interaction shown. Acetyl-CoA bound to hNAA10 is shown in ball and stick.

Fig. 5. Subunit crosstalk within the hNatE complex.

a hNAA50(magenta), hNAA15(green), and hNAA10(orange) within the hNatE complex overlay with ScNatE (gray, PDB: 6O07). The α3 helix and β7 strand of Naa50 is shown to shift toward Naa10 in the human structure (b) hNatE aligned with hNatA (light blue, PDB:6C9M) and hNAA50 (wheat, PDB: 3TFY). The top zoom-in area shows the alignment of free hNAA50 and hNatE. The below zoom-in area shows the hNAA10 conformational change induced by hNAA50 binding.

Fig. 6. Overall structure of the hNatE/HYPK complex.

a hNAA50 (pink), hNAA15 (green), hNAA10 (orange), and HYPK (red) within the hNatE complex is shown in cartoon. b Zoom-in view of the contacts between HYPK and hNAA15 with residues that participate in interaction shown. c Zoom-in view of the contacts between hNAA15 and hNAA50 with residues that participate in interaction shown. d Zoom-in view of the contacts between hNAA10 and hNAA50 with residues that participate in interaction shown.

Fig. 7. Molecular basis for hNAA50 and HYPK binding to hNatA.

a hNatE/HYPK overlayed onto hNatE (purple) with NAA50 aligned. b hNatE/HYPK overlayed onto hNatA/HYPK (PDB: 6C95, blue) with HYPK aligned. c Zoom-in view of HYPK C-terminal binding region as indicated in a. d Zoom-in view of HYPK α2 binding region as indicated in a. e Zoom-in view of HYPK N-terminal α1 domain binding region as indicated in a. f Zoom-in view shows the hNAA50 binding region on hNAA15 as indicated in b. g Zoom-in view shows the hNAA50 binding region on hNAA10 as indicated in b. h Zoom-in view shows the hNAA15 C-terminal helices conformal changes when HYPK bound as indicated in b.

Fig. 8. Molecular basis for the decrease of hNAA50 activity by HYPK.

hNatE/HYPK (green) overlayed onto hNatE (magenta) with hNatA aligned. Zoom-in view shows the local conformational change of hNAA50.