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

Abstract

NatB is one of three major N-terminal acetyltransferase (NAT) complexes (NatA-NatC), which co-translationally acetylate the N-termini of eukaryotic proteins. Its substrates account for about 21% of the human proteome, including well known proteins such as actin, tropomyosin, CDK2, and αsynuclein (aSyn). Human NatB (hNatB) mediated N-terminal acetylation of αSyn has been demonstrated to play key roles in Parkinson's disease pathogenesis and as a potential therapeutic target for hepatocellular carcinoma.Here we report the cryo-EM structure of hNatB bound to a CoA-aSyn conjugate, together with structure-guided analysis of mutational effects on catalysis. This analysis reveals functionally important differences with human NatA and Candida albicans NatB, resolves key hNatB protein determinants for aSyn Nterminal acetylation, and identifies important residues for substrate-specific recognition and acetylation by NatB enzymes. These studies have implications for developing small molecule NatB probes and for understanding the mode of substrate selection by NAT enzymes.

Molecular mechanism of N-terminal acetylation by the ternary NatC complex

“…Inositol hexaphosphate (IP6) binding contributes to yeast NatC complex stability. Earlier reports demonstrated that IP6 interacts with and stabilizes both the yeast and human NatA and NatE complexes (Cheng et al, 2019;Deng et al, 2019;Deng et al, 2020a;Gottlieb and Marmorstein, 2018), but does not appear to interact with human NatB (Deng et al, 2020b). To determine if IP6 could bind to the recombinant NatC complex, we employed isothermal titration calorimetry (ITC).…”

“…While Naa30 displays a canonical NAT fold with mixed α/β secondary structure, the Naa35/38 complex forms a clamp that pinches Naa30 on opposite sides. This clamp-like structure is distinct from the architecture of the large auxiliary subunits of NatA and NatB, which surround the base of their respective catalytic subunits (Deng et al, 2020b;Gottlieb and Marmorstein, 2018;Hong et al, 2017;. The Naa35 architecture is mostly helical, with 25 helices, three short β-strands in its N-terminal region and a single β-hairpin at its C-terminal region that are unique to the NatC complex (Figure 2b and Supplementary Figure 3).…”

“…Analogous segments in the NatA and NatB complexes are used for N-terminal substrate recognition. In these complexes, however, the β6-β7 segment is exposed to solvent (Deng et al, 2020b;Gottlieb and Marmorstein, 2018;Hong et al, 2017;. By contrast, this interaction between Naa30 and Naa35 is facilitated by hydrogen bonds between the Naa30 α1-α2 loop and Naa35 α3-β3 segment: the sidechain of Naa30-Ser28 and the backbone carbonyl of Naa35-Ala70; between the sidechains of Naa30-Gln20 and Naa35-Glu75; and the backbone carbonyl of Naa30-Gln20 and the sidechain of Naa35-Lys77.…”

“…Overall, there are three notable differences between NatC and the other multi-subunit NATs -NatA, NatE and NatB. First, the auxiliary subunits of NatA, NatE and NatB contain only helical secondary structure, forming 10-13 conserved tetratricopeptide repeat (TPR) motifs (Deng et al, 2019;Deng et al, 2020a;Deng et al, 2020b;Gottlieb and Marmorstein, 2018;Hong et al, 2017;. In contrast, the NatC auxiliary subunits (Naa35 and Naa38) do not contain TPR repeats and but do contain several beta strands that make key interactions in the complex (Figure 2b).…”

“…Substrate recognition by eukaryotic NATs is usually dictated by the first few residues of the substrate via the active site structure (Deng et al, 2019;Deng and Marmorstein, 2021;Deng et al, 2020b;Goris et al, 2018;Hong et al, 2017;Magin et al, 2015;Stove et al, 2016). The active site of NatA contains a relatively small binding pocket to accommodate a small, uncharged residue at position 1 such as alanine, valine or threonine residues that remains following initiator methionine (iMet) -cleavage .…”