Dynamics-function relationship in the catalytic domains of N-terminal acetyltransferases

Abboud, Angèle; Bedoucha, Pierre; Arnesen, Thomas; Reuter, Nathalie

doi:10.1016/j.csbj.2020.02.017

“…The same tyrosine is present in the KAT14 β6-β7 loop ( S5B Text ). This tyrosine has been shown to be essential for substrate binding [ 49 , 54 , 78 ] and it has been suggested that the size and flexibility of the β6-β7 loop plays an important role in substrate recognition [ 2 , 83 ]. Based on similarity between the β6-β7 loop of KAT14 and the NATs from Groups 1 and 2 and given the fact that the β6-β7 loop differs in size and primary sequence in other acetyltransferases, it is not excluded that KAT14 might be able to accommodate the same type of substrate as NATs and acetylate N-termini of proteins.…”

Section: Resultsmentioning

confidence: 99%

Classification and phylogeny for the annotation of novel eukaryotic GNAT acetyltransferases

Krtenic

¹

,

Drazic

²

,

Arnesen

³

et al. 2020

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The enzymes of the GCN5-related N-acetyltransferase (GNAT) superfamily count more than 870 000 members through all kingdoms of life and share the same structural fold. GNAT enzymes transfer an acyl moiety from acyl coenzyme A to a wide range of substrates including aminoglycosides, serotonin, glucosamine-6-phosphate, protein N-termini and lysine residues of histones and other proteins. The GNAT subtype of protein N-terminal acetyltransferases (NATs) alone targets a majority of all eukaryotic proteins stressing the omnipresence of the GNAT enzymes. Despite the highly conserved GNAT fold, sequence similarity is quite low between members of this superfamily even when substrates are similar. Furthermore, this superfamily is phylogenetically not well characterized. Thus functional annotation based on sequence similarity is unreliable and strongly hampered for thousands of GNAT members that remain biochemically uncharacterized. Here we used sequence similarity networks to map the sequence space and propose a new classification for eukaryotic GNAT acetyltransferases. Using the new classification, we built a phylogenetic tree, representing the entire GNAT acetyltransferase superfamily. Our results show that protein NATs have evolved more than once on the GNAT acetylation scaffold. We use our classification to predict the function of uncharacterized sequences and verify by in vitro protein assays that two fungal genes encode NAT enzymes targeting specific protein N-terminal sequences, showing that even slight changes on the GNAT fold can lead to change in substrate specificity. In addition to providing a new map of the relationship between eukaryotic acetyltransferases the classification proposed constitutes a tool to improve functional annotation of GNAT acetyltransferases.

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“…The flexible β6–β7 loop is common to all NATs, forming the peptide substrate-binding pocket along with the α1–α2 loop and residues of the α2 helix. The conformation of the β6–β7 loop is a major determinant of how open is the substrate channel ( 52 ). The extended β6–β7 loop is able to exhibit a multitude of conformations, some of which may hinder substrate or Ac-CoA binding ( Fig.…”

Section: Discussionmentioning

confidence: 99%

PFN2 and NAA80 cooperate to efficiently acetylate the N-terminus of actin

Ree

¹

,

Kind

²

,

Kaziales

³

et al. 2020

Journal of Biological Chemistry

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The actin cytoskeleton is of profound importance to cell shape, division, and intracellular force generation. Profilins bind to globular (G-)actin and regulate actin filament formation. Although profilins are well-established actin regulators, the distinct roles of the dominant profilin, profilin 1 (PFN1), versus the less abundant profilin 2 (PFN2) remain enigmatic. In this study, we use interaction proteomics to discover that PFN2 is an interaction partner of the actin N-terminal acetyltransferase NAA80, and further confirmed this by analytical ultracentrifugation. Enzyme assays with NAA80 and different profilins demonstrate that PFN2 binding specifically increases the intrinsic catalytic activity of NAA80. NAA80 binds PFN2 through a proline-rich loop, deletion of which abrogates PFN2 binding. Small-angle X-ray scattering shows that NAA80, actin and PFN2 form a ternary complex and that NAA80 has partly disordered regions in the N-terminus and the proline-rich loop, the latter of which is partly ordered upon PFN2 binding. Furthermore, binding of PFN2 to NAA80 via the proline-rich loop promotes binding between the globular domains of actin and NAA80, and thus acetylation of actin. However, the majority of cellular NAA80 is stably bound to PFN2 and not to actin, and we propose that this complex acetylates G-actin before it is incorporated into filaments. In conclusion, we reveal a functionally specific role of PFN2 as a stable interactor and regulator of the actin N-terminal acetyltransferase NAA80, and establish the modus operandi for NAA80-mediated actin N-terminal acetylation, a modification with a major impact on cytoskeletal dynamics. Data are available via ProteomeXchange with identifier PXD021408.

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“…The flexible β6-β7 loop is common to all NATs, forming the peptide substrate-binding pocket along with the α1-α2 loop and residues of the α2 helix. The conformation of the β6-β7 loop is a major determinant of how open the substrate channel is 47 . The extended β6-β7 loop is able to exhibit a multitude of conformations, some of which may hinder substrate or Ac-CoA binding (Fig.…”

Section: Discussionmentioning

confidence: 99%

PFN2 and NAA80 cooperate to efficiently acetylate the N-terminus of actin

Ree

¹

,

Kind

²

,

Kaziales

³

et al. 2020

Preprint

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The actin cytoskeleton is of profound importance to cell shape, division, and intracellular force generation. Profilins bind to globular (G-)actin and regulate actin filament formation. Although profilins are well-established actin regulators, the distinct roles of the dominant profilin, profilin 1 (PFN1), versus the less abundant profilin 2 (PFN2) remain enigmatic. Here, we define a specific role for PFN2 as a stable interactor and regulator of the actin N-terminal acetyltransferase NAA80. PFN2 binding increases the intrinsic catalytic activity of NAA80. Furthermore, binding of PFN2 to NAA80 via its proline-rich loop promotes binding between the globular domains of actin and NAA80, and thus acetylation of actin. The majority of NAA80 is stably bound to PFN2, and we propose that this complex acetylates G-actin before it is incorporated into filaments. In conclusion, we reveal a functionally specific role of PFN2, and establish the modus operandi for NAA80-mediated actin N-terminal acetylation. Data are available via ProteomeXchange with identifier PXD020188.

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Dynamics-function relationship in the catalytic domains of N-terminal acetyltransferases

Cited by 11 publications

References 87 publications

Classification and phylogeny for the annotation of novel eukaryotic GNAT acetyltransferases

Classification and phylogeny for the annotation of novel eukaryotic GNAT acetyltransferases

PFN2 and NAA80 cooperate to efficiently acetylate the N-terminus of actin

PFN2 and NAA80 cooperate to efficiently acetylate the N-terminus of actin

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