Actin's N-terminal acetyltransferase uncovered

Abstract

Humans express six highly conserved actin isoforms, which differ the most at their N-termini. Actin's N-terminus undergoes co- and post-translational processing unique among eukaryotic proteins. During translation, the initiator methionine of the two cytoplasmic isoforms is N-terminally acetylated (Nt-acetylated) and that of the four muscle isoforms is removed and the exposed cysteine is Nt-acetylated. Then, an unidentified acetylaminopeptidase post-translationally removes the Ac-Met (or Ac-Cys), and all six isoforms are re-acetylated at the N-terminus. Despite the vital importance of actin for cellular processes ranging from cell motility to organelle trafficking and cell division, the mechanism and functional consequences of Nt-acetylation remained unresolved. Two recent studies significantly advance our understanding of actin Nt-acetylation. Drazic et al. (2018, Proc Natl Acad Sci U S A, 115, 4399-4404) identify actin's dedicated N-terminal acetyltransferase (NAA80/NatH), and demonstrate that Nt-acetylation critically impacts actin assembly in vitro and in cells. NAA80 knockout cells display increased filopodia and lamellipodia formation and accelerated cell motility. In vitro, the absence of Nt-acetylation leads to a decrease in the rates of filament depolymerization and elongation, including formin-induced elongation. Goris et al. (2018, Proc Natl Acad Sci U S A, 115, 4405-4410] describe the structure of Drosophila NAA80 in complex with a peptide-CoA bi-substrate analog mimicking the N-terminus of β-actin. The structure reveals the source of NAA80's specificity for actin's negatively-charged N-terminus. Nt-acetylation neutralizes a positive charge, thus enhancing the overall negative charge of actin's unique N-terminus. Actin's N-terminus is exposed in the filament and influences the interactions of many actin-binding proteins. These advances open the way to understanding the many likely consequences and functional roles of actin Nt-acetylation.

Keywords: N-terminal acetylation; actin assembly; cell motility.

Figure 1. Nt-acetylation of actin isoforms

(a) Humans express six actin isoforms, sharing 94–99% sequence identity. Most of the differences among isoforms concentrate near the N-terminus, which is additionally acetylated in all the isoforms. Dark blue to white background coloring indicates amino acids conservation scores of 100%, 67%, 50% and <50%. The UniProt accession codes are (in the order of the alignment): P60709, P63261, P68133, P68032, P62736, P63267. (b) The acetylation of actin’s N-terminus proceeds through a multi-step process, unique among all eukaryotic proteins, which involved both co- and post-translational modification. Muscle (class II) actins contain Met-Cys at the N-terminus after synthesis, and the initial step of co-translational acetylation (likely catalyzed by NatA) is preceded by the removal of the initiator methionine. Cytoplasmic (class I) actins are co-translationally Nt-acetylated directly on the initiator methionine, a reaction catalyzed by NatB (Van Damme et al. 2012; Arnesen et al. 2009; Polevoda and Sherman 2003). A still non-identified acetylaminopeptidase removes the acetylated N-terminal residues, which is followed by the last step of post-translational Nt-acetylation catalyzed by NatH (NAA80) (Goris et al. 2018; Drazic et al. 2018) (c) Actin’s N-terminus is prominently exposed in the actin filament, such that most F-actin-binding protein likely “see” the difference between acetylated and non-acetylated actin, as well as differences among actin isoforms. Actin subunits along the short-pitch helix of the actin filament are named A1–5 (from the filament pointed to barbed ends). Of particular interest is myosin, which binds in a cleft between two actin subunits along the long-pitch helix of the actin filament, within contact distance to the N-termini of both actin subunits. The figure shows the recently determined cryo-EM structure of ADP-myosin-IB bound to the actin filament (Mentes et al. 2018).

Figure 2. Source of NAA80’s specificity for actin

(a) Alignment of the sequences of Drosophila and human NAA80 (UniProt accession codes Q59DX8 and Q93015-2, respectively). The two proteins share only ~29% sequence identity (blue background color indicates identical amino acids). Different from Drosophila NAA80, the human protein has a long extension at the N-terminus and a large, proline-rich insertion near the C-terminus. The N-terminal extension is unique to mammalian NAA80, and precedes the catalytic core that is most highly conserved among all NATs. The N-terminal extension could possible recruit still to be identified auxiliary subunits that might regulate NAA80’s activity in cells. The proline-rich insertion contains a classical profilin-binding site, as observed in many cytoskeletal proteins (Dominguez 2016). (b) Electrostatic surface representation of the structure of Drosophila NAA80 in complex with an actin peptide-CoA bi-substrate analog, mimicking the N-terminus of β-actin (Goris et al. 2018). The positively-charged substrate-binding groove makes direct and water-mediated contacts with the side chains of Asp-3 and Asp-4 of β-actin and appears uniquely adapted for binding actin’s negatively-charged N-terminus, which is unique among all eukaryotic proteins.