Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase

Abstract

N-terminal acetylation (Nt-acetylation), carried out by N-terminal acetyltransferases (NATs), is a conserved and primary modification of nascent peptide chains. Naa60 (also named NatF) is a recently identified NAT found only in multicellular eukaryotes. This protein was shown to locate on the Golgi apparatus and mainly catalyze the Nt-acetylation of transmembrane proteins, and it also harbors lysine Nε-acetyltransferase (KAT) activity to catalyze the acetylation of lysine ε-amine. Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA). The hNaa60 protein contains an amphipathic helix following its GNAT domain that may contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures. Remarkably, we found that the side-chain of Phe 34 can influence the position of the coenzyme, indicating a new regulatory mechanism involving enzyme, co-factor and substrates interactions. Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved β3-β4 long loop participates in the regulation of hNaa60 activity.

Molecular determinants of the N-terminal acetyltransferase Naa60 anchoring to the Golgi membrane

“…The predictions and modeling indicated the presence of two ␣-helices ( Fig. 1), of which the first one matched with previous crystallization data (30). Our CD also confirmed the presence of helical structure, although incapable of providing information on the number of helices.…”

“…We here expanded this work by further investigating how this C-terminal segment might interact with membranes and initially identified two ␣-helices that were predicted by PSIPRED (31), herein referred to as Pred-␣1 and Pred-␣2 ( Fig. 1A), of which the former is in agreement with the Naa60-(1-211) crystal structure (30). Plotting helical wheels for both these predicted helices revealed their amphipathic character (Fig.…”

“…However, this structure does not contain the localization-determining C-terminal tail because it was necessary to omit this part to obtain purified Naa60 amenable to structural analysis (29). Another crystallization study of Naa60 met similar challenges and the resulting structure only shows amino acids 1-211, although the full-length protein (242 amino acids) was used (30). This structure shows an amphipathic ␣-helix (aa 190 -202) that could conceivably be involved in membrane binding.…”

Dynamics-function relationship of N-terminal acetyltransferases: the β6β7 loop modulates substrate accessibility to the catalytic site

“…The extra β-hairpin β6β7, the α1α2 loop and the helix α2 contain amino acids forming the boundary of the binding pocket, which we hereafter refer to as the mouth of the binding pocket. NAT substrates usually position two to three residues in the peptide binding site [15–18]. Two conserved tyrosines located on the β6β7 loop and another one on the α1α2 loop have been shown to interact with the substrate backbone via hydrogen bonds [19].…”

“…The loops have been proposed to prevent the access of internal lysine to the catalytic site and as a consequence prevent their acetylation by NATs [15,20]. However, there have been reports of lysine acetylations by NATs [18,21–27]. Moreover, NATs can be inhibited by so-called bisubstrate inhibitors consisting of a short polypeptide covalently bound to the Ac-CoA [6,17,28,29].…”