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. 2016 Jul 6;24(7):1044-56.
doi: 10.1016/j.str.2016.04.020. Epub 2016 Jun 16.

Crystal Structure of the Golgi-Associated Human Nα-Acetyltransferase 60 Reveals the Molecular Determinants for Substrate-Specific Acetylation

Svein Isungset Støve  1 , Robert S Magin  2 , Håvard Foyn  3 , Bengt Erik Haug  4 , Ronen Marmorstein  5 , Thomas Arnesen  6
Affiliations

Crystal Structure of the Golgi-Associated Human Nα-Acetyltransferase 60 Reveals the Molecular Determinants for Substrate-Specific Acetylation

Svein Isungset Støve et al. Structure. .

Abstract

N-Terminal acetylation is a common and important protein modification catalyzed by N-terminal acetyltransferases (NATs). Six human NATs (NatA-NatF) contain one catalytic subunit each, Naa10 to Naa60, respectively. In contrast to the ribosome-associated NatA to NatE, NatF/Naa60 specifically associates with Golgi membranes and acetylates transmembrane proteins. To gain insight into the molecular basis for the function of Naa60, we developed an Naa60 bisubstrate CoA-peptide conjugate inhibitor, determined its X-ray structure when bound to CoA and inhibitor, and carried out biochemical experiments. We show that Naa60 adapts an overall fold similar to that of the catalytic subunits of ribosome-associated NATs, but with the addition of two novel elongated loops that play important roles in substrate-specific binding. One of these loops mediates a dimer to monomer transition upon substrate-specific binding. Naa60 employs a catalytic mechanism most similar to Naa50. Collectively, these data reveal the molecular basis for Naa60-specific acetyltransferase activity with implications for its Golgi-specific functions.

Keywords: N-terminal acetylation; NAT; Naa60; NatF; acetyltransferase; crystal structure.

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Figures

Figure 1
Figure 1. Sequence alignment of selected NATs and overall structure of the binary hNaa60/CoA-Ac-MKAV7 complex
A) Sequence alignment of hNaa60 (1–184) and NAT homologues hNaa50, hNaa40 (50–237), SsNAT (50–216) and SpNaa10 for which crystal structures already have been determined, and the human homologues hNaa20 and hNaa30 (200–362). Sequence identity is shown with white letters in red boxes and sequence similarity with red letters in blue frames. The secondary structure elements of hNaa60 are shown on top of the alignment. The β3–β4 loop is marked in pink and the β6–β7 loop in light blue. B) IC50 values of the two bisubstrate analogues CoA-Ac-MAPL7 and CoA-Ac-MKAV7 in addition to the control compound desulfoCoA. C) Overall structure of the binary hNaa60/CoA-Ac-MKAV7 complex. The β3–β4 loop is shown in pink (on the back side of the structure), the β6–β7 loop in light blue, and the CoA-Ac-MKAV7 inhibitor in green sticks. See also Figure S1–S3.
Figure 2
Figure 2. Structural alignment highlighting hNaa60 specific features
A) Superimposition of hNaa60 (yellow) with hNaa50 (cyan), SpNaa10 (orange) and ssNAT (white). CoA-Ac-MKAV7 bound to hNaa60 is shown in green sticks. B) Superimposition of hNaa60 (yellow), hNaa50 (cyan), SpNaa10 (orange) and SsNAT (white) β3–β4 loop. C) Superimposition of hNaa60 (yellow), hNaa50 (cyan), SpNaa10 (orange) and SsNAT (white) β6–β7 loop containing a hairpin structure with two shorts strands (β6′ and β7′).
Figure 3
Figure 3. Catalytic mechanism and peptide binding site of hNaa60
A) Superimposition of the hNaa60 and hNaa50 active site (shown in yellow and cyan respectively). Residues associated with catalysis (Y97, H138 of hNaa60 and Y73, H112 of Naa50) are shown in sticks. An ordered water molecule involved in catalysis is shown in yellow and cyan for the hNaa60 and hNaa50 structure respectively. The bisubstrate analogue CoA-Ac-MKAV7 is shown in green sticks in all figures (A–F). B) Residues F34, P35 I36, L140, Y165 and I167 (yellow sticks) are forming a hydrophobic pocket surrounding the substrate peptide M1p side chain. C) Structural alignment of hNaa60 and hNaa50 active sites. Residues L99, Y38, and Y165 of hNaa60 that plays important roles in peptide anchoring and which are structurally conserved between hNaa60 and hNaa50 are shown as yellow sticks. D) Superimposition of the cavity surrounding the hNaa60 and hNaa50 peptide binding site. E) Active site surface representation of hNaa60. F) Active site surface representation of hNaa50.
Figure 4
Figure 4. Catalytic parameters of hNaa60 WT and mutants
A) kcat values (black bars) and Km values (yellow bars) of wild-type hNaa60 and selected hNaa60 mutants toward the substrate polypeptide MKAV24. B) Catalytic efficiency of hNaa60 (black bars) and hNaa50 (blue bars) towards different substrate polypeptides representing previously identified hNaa60 or hNaa50 substrates in vivo. See also Tables S1 and S2.
Figure 5
Figure 5. The hNaa60-specific β3–β4 loop
A) Residues D81 and D83 (pink sticks) of the extended β3–β4 loop of hNaa60 (shown in pink) that form salt bridges with residues H138 and H159 (yellow sticks) of β5 and β7 are shown. E80 and D81 (pink sticks) that form hydrogen bonds with Y164 and T176 (yellow sticks) of β6 and the carbonyl backbone of I84 (pink sticks) that form a hydrogen bond with Y180 (yellow sticks) are also shown. The CoA-Ac-MKAV is shown in green sticks. B) Residues I77, I84 and L85 (pink sticks) that form a hydrophobic interior of the loop and participate in van der Waals interactions with residues V95, A134, Y136 and V178 (yellow sticks) of β5, β6 and β7 strands is shown. See also Figure S4.
Figure 6
Figure 6. hNaa60 dimerization
A) Crystal structure of the hNaa60-CoA homodimer complex. CoA is shown in green sticks. B) Superimposition of the β6–β7 loop in a monomeric state (shown in yellow) and in a dimeric state (shown in red). C) Superimposition of the hNaa60/CoA-Ac-MKAV7 structure (only showing CoA-Ac-MKAV7) with the hNaa60/CoA dimer. D) Contacts between the two hNaa60 protomers. The side-chain of K172b was omitted for clarity. E) Size exclusion chromatography of hNaa60 1–184 mixed with either 2× or 10× excess of Ac-CoA or 0.5×, 2× or 10× excess of the bisubstrate analogue CoA-Ac-MAPL7. The first peak corresponds to a hNaa60 homodimer and the second peak corresponds to a hNaa60 monomer. F) Sedimentation equilibrium analytical ultracentrifugation experiments of hNaa60 mixed with the bisubstrate analogue CoA-Ac-MAPL7, which eluted as a dimer. G) Sedimentation equilibrium analytical ultracentrifugation experiments of hNaa60 mixed with the bisubstrate analogue CoA-Ac-MAPL7 which eluted as a dimer. See also Figure S5.
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