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

Abstract

SUMMARY N-terminal acetylation is a common and important protein modification catalysed by N-terminal acetyltransferases (NATs). Six human NATs (NatA-NatF) contain one catalytic subunit each, Naa10-Naa60, respectively. In contrast to the ribosome associated NatA-NatE, NatF/Naa60 specifically associates with Golgi membranes and acetylates transmembrane proteins. To gain insight into the molecular basis for function of Naa60, we developed a 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 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, this data reveals the molecular basis for Naa60-specific acetyltransferase activity with implications for its Golgi-specific functions.

Molecular Basis for N-terminal Alpha-Synuclein Acetylation by Human NatB

“…NatA accounts for 38% of the human N-terminal acetylome, acetylating small N-terminal residues after removal of the initial methionine residue [23], while NatB modifies N-termini with sequences of MD-, ME-, MNand MQ- [24,25]. NatC/E/F have overlapping substrates, acting on N-terminal methionine when it is followed by several residues excluding D, E, N and Q [23,[26][27][28][29][30][31]. When another catalytic subunit (NAA50) binds to NatA, a dual enzyme complex NatE is formed [32,33], with catalytic crosstalk between NAA10 and NAA50 [33,34].…”

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

“…The α1 and α2 helices are also part of the binding interface with Naa15, the auxiliary subunit of the NatA complex [17] (S2 Table). The loop β3β4 is 10 to 15 residues longer in Naa60 than in the other NATs and mutations on key residues disrupt interactions with the β5, β6, and β7 strands, leading to altered catalytic efficiency and protein stability [70]. Finally the β6β7 loop, one of the most flexible loops, contains two tyrosines conserved through most of the NATs, which make hydrogen bonds with the backbone of the first and second amino acids of the substrate [19].…”

“…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]. The X-ray structure of the human NatF bound to bisubstrate CoA-Ac-MKAV 7 shows that the inhibitor is placed in the Ac-CoA and substrate binding site with the β6β7 hairpin loop hanging over the top of it [28] (Fig.…”

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

“…Importantly, the C-terminal tail of Naa60 (amino acids 182-242), which constitutes an extra segment, not present in the other catalytic Naas, was defined as the part providing organellar localization. A recent crystal structure of Naa60 exposed the molecular determinants for substrate-specific acetylation and revealed that Naa60 employs a catalytic mechanism close to that of Naa50 (29). 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).…”

“…A recent crystal structure of Naa60 exposed the molecular determinants for substrate-specific acetylation and revealed that Naa60 employs a catalytic mechanism close to that of Naa50 (29). 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).…”