SUMMARYAmino-terminal acetylation is ubiquitous among eukaryotic proteins and controls a myriad of biological processes. Of the N-terminal acetyltransferases (NATs) that facilitate this co-translational modification, the heterodimeric NatA complex harbors the most diversity for substrate selection and modifies the majority of all amino-terminally acetylated proteins. Here, we report the X-ray crystal structure of the 100 kDa holo-NatA complex from Schizosaccharomyces pombe in the absence and presence of a bisubstrate peptide-CoA conjugate inhibitor, as well as the structure of the uncomplexed Naa10p catalytic subunit. The NatA-Naa15p auxiliary subunit contains 13 TPR motifs and adopts a ring-like topology that wraps around the NatA-Naa10p subunit, an interaction that alters the Naa10p active site for substrate-specific acetylation. These studies have implications for understanding the mechanistic details of other NAT complexes and how regulatory subunits modulate the activity of the broader family of protein acetyltransferases.

The impact of N ␣ -terminal acetylation on protein stability and protein function in general recently acquired renewed and increasing attention. Although the substrate specificity profile of the conserved enzymes responsible for N ␣ -terminal acetylation in yeast has been well documented, the lack of higher eukaryotic models has hampered the specificity profile determination of N ␣ -acetyltransferases (NATs) of higher eukaryotes. The fact that several types of protein N termini are acetylated by so far unknown NATs stresses the importance of developing tools for analyzing NAT specificities. Here, we report on a method that implies the use of natural, proteome-derived modified peptide libraries, which, when used in combination with two strong cation exchange separation steps, allows for the delineation of the in vitro specificity profiles of NATs. The human NatA complex, composed of the auxiliary hNaa15p (NATH/hNat1) subunit and the catalytic hNaa10p (hArd1) and hNaa50p (hNat5) subunits, cotranslationally acetylates protein N termini initiating with Ser, Ala, Thr, Val, and Gly following the removal of the initial Met. In our studies, purified hNaa50p preferred Met-Xaa starting N termini (Xaa mainly being a hydrophobic amino acid) in agreement with previous data. Surprisingly, purified hNaa10p preferred acidic N termini, representing a group of in vivo acetylated proteins for which there are currently no NAT(s) identified. The most prominent representatives of the group of acidic N termini are ␥-and ␤-actin. Indeed, by using an independent quantitative assay, hNaa10p strongly acetylated peptides representing the N termini of both ␥-and ␤-actin, and only to a lesser extent, its previously characterized substrate motifs. The immunoprecipitated NatA complex also acetylated the actin N termini efficiently, though displaying a strong shift in specificity toward its known Ser-starting type of substrates. Thus, complex formation of NatA might alter the substrate specificity profile as compared with its isolated catalytic subunits, and, furthermore,

SignificanceMore than 80% of human proteins are N-terminal (Nt)–acetylated during translation. In contrast, actin, the most abundant protein in the cytoplasm of animal cells, is Nt-acetylated posttranslationally and following a unique multistep mechanism that has remained poorly characterized. Here, we describe the discovery of actin’s N-terminal acetyltransferase (NAT), NAA80. We further demonstrate that actin Nt-acetylation plays essential roles in filament assembly, cytoskeleton organization, and cell motility, resulting in a net increase in the ratio of monomeric to filamentous actin and fewer lamellipodia and filopodia. These effects converge to reduce cell hypermotility. This work establishes the role of Nt-acetylation for the most abundant cytoskeletal protein in animals and reveals a NAT acting posttranslationally and on a single dedicated substrate.

No abstract

SignificanceN-terminal acetylation performed by N-terminal acetyltransferases (NATs) is a common protein modification in human cells. A unique NAT, NAA80, was recently found to control actin N-terminal acetylation and cytoskeletal dynamics. In this study, we developed potent and specific bisubstrate inhibitors against NAA80 and determined the crystal structure of NAA80 in complex with an inhibitor mimicking the β-actin N terminus, thus revealing molecular determinants for the substrate specificity and selective inhibition of NAA80. A yeast model uncovered how a cellular determinant, the NatB enzyme, acts to restrict the number of in vivo NAA80 substrates relative to the broader intrinsic capacity of NAA80. Our data provide a starting point for further development of inhibitors for the regulation of actin and cytoskeletal functions.

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.

The N-termini of 80-90% of human proteins are acetylated by the N-terminal acetyltransferases (NATs), NatA-NatF. The major NAT complex, NatA, and particularly the catalytic subunit hNaa10 (ARD1) has been implicated in cancer development. For example, knockdown of hNaa10 results in cancer cell death and the arrest of cell proliferation. It also sensitized cancer cells to drug-induced cytotoxicity. Human NatE has a distinct substrate specificity and is essential for normal chromosome segregation. Thus, NAT inhibitors may potentially be valuable anticancer therapeutics, either directly or as adjuvants. Herein, we report the design and synthesis of the first inhibitors targeting these enzymes. Using the substrate specificity of the enzymes as a guide, we synthesized three bisubstrate analogues that potently and selectively inhibit the NatA complex (CoA-Ac-SES4; IC50 = 15.1 μM), hNaa10, the catalytic subunit of NatA (CoA-Ac-EEE4; Ki = 1.6 μM), and NatE/hNaa50 (CoA-Ac-MLG7; Ki* = 8 nM); CoA-Ac-EEE4 is a reversible competitive inhibitor of hNaa10, and CoA-Ac-MLG7 is a slow tight binding inhibitor of hNaa50. Our demonstration that it is possible to develop NAT selective inhibitors should assist future efforts to develop NAT inhibitors with more drug-like properties that can be used to chemically interrogate in vivo NAT function.

SynopsisN-terminal acetylation, catalysed by N-terminal acetyltransferases (NATs), is among the most common protein modifications in eukaryotes and involves the transfer of an acetyl group from acetyl-CoA to the α-amino group of the first amino acid. Functions of N-terminal acetylation include protein degradation and sub-cellular targeting. Recent findings in humans indicate that a dysfunctional Nα-acetyltransferase (Naa) 10, the catalytic subunit of NatA, the major NAT, is associated with lethality during infancy. In the present study, we identified the Danio rerio orthologue zebrafish Naa 10 (zNaa10). In vitro N-terminal acetylation assays revealed that zNaa10 has NAT activity with substrate specificity highly similar to that of human Naa10. Spatiotemporal expression pattern was determined by in situ hybridization, showing ubiquitous expression with especially strong staining in brain and eye. By morpholino-mediated knockdown, we demonstrated that naa10 morphants displayed increased lethality, growth retardation and developmental abnormalities like bent axis, abnormal eyes and bent tails. In conclusion, we identified the zebrafish Naa10 orthologue and revealed that it is essential for normal development and viability of zebrafish.