Specificity and Versatility of Substrate Binding Sites in Four Catalytic Domains of Human N-Terminal Acetyltransferases

Abstract

Nt-acetylation is among the most common protein modifications in eukaryotes. Although thought for a long time to protect proteins from degradation, the role of Nt-acetylation is still debated. It is catalyzed by enzymes called N-terminal acetyltransferases (NATs). In eukaryotes, several NATs, composed of at least one catalytic domain, target different substrates based on their N-terminal sequences. In order to better understand the substrate specificity of human NATs, we investigated in silico the enzyme-substrate interactions in four catalytic subunits of human NATs (Naa10p, Naa20p, Naa30p and Naa50p). To date hNaa50p is the only human subunit for which X-ray structures are available. We used the structure of the ternary hNaa50p/AcCoA/MLG complex and a structural model of hNaa10p as a starting point for multiple molecular dynamics simulations of hNaa50p/AcCoA/substrate (substrate = MLG, EEE, MKG), hNaa10p/AcCoA/substrate (substrate = MLG, EEE). Nine alanine point-mutants of the hNaa50p/AcCoA/MLG complex were also simulated. Homology models of hNaa20p and hNaa30p were built and compared to hNaa50p and hNaa10p. The simulations of hNaa50p/AcCoA/MLG reproduce the interactions revealed by the X-ray data. We observed strong hydrogen bonds between MLG and tyrosines 31, 138 and 139. Yet the tyrosines interacting with the substrate’s backbone suggest that their role in specificity is limited. This is confirmed by the simulations of hNaa50p/AcCoA/EEE and hNaa10p/AcCoA/MLG, where these hydrogen bonds are still observed. Moreover these tyrosines are all conserved in hNaa20p and hNaa30p. Other amino acids tune the specificity of the S1’ sites that is different for hNaa10p (acidic), hNaa20p (hydrophobic/basic), hNaa30p (basic) and hNaa50p (hydrophobic). We also observe dynamic correlation between the ligand binding site and helix that tightens under substrate binding. Finally, by comparing the four structures we propose maps of the peptide-enzyme interactions that should help rationalizing substrate-specificity and lay the ground for inhibitor design.

A Saccharomyces cerevisiae Model Reveals In Vivo Functional Impairment of the Ogden Syndrome N-Terminal Acetyltransferase NAA10 Ser37Pro Mutant

“…Recently, the first structures of NATs and a NAT-complex were solved, providing a molecular understanding of the sequence specific Nt-acetylation of protein N termini (27)(28)(29)(30). Structural analyses of noncomplexed Naa10 and NatA from Schizosaccharomyces pombe reveal an allosteric modulator function of Naa15 in steering Naa10 specificity and provide a rational for the distinctive substrate specificity profiles observed when assaying non-complexed versus complexed Naa10 (10,27), with both forms co-existing in cells (10).…”

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

“…Using relatively short 10 nanoseconds-long molecular dynamics simulations of the catalytic domains of two human NATs, we earlier showed for hNaa50 that (i) helix α2 undergoes flexibility changes upon ligand binding and (ii) the β6β7 hairpin loop was the most flexible region of the protein [19]. Likewise, we observed the β6β7 loop of the human Naa10 to be flexible [30] in 100 ns-long molecular simulations.…”

“…In the region of the helices α1 and α2, we notice a similar pattern of flexibility between all the structures where the loop α1α2 and the helix α2 fluctuate more. Molecular dynamics simulations of the human Naa50 and Naa10 have shown that the flexibility of helix α2 is decreased in the presence of a substrate [19]. This region is also involved in the complex formation with the subunit Naa15 [30].…”

“…Two tyrosines located at positions 239 and 240 of the alignment (Y138 and Y139 in Naa50), and conserved across several groups (not in RimL, Naa40 and Naa80), are located on loop β6β7. They are known to be involved in substrate binding and form hydrogen bonds with the substrate [19].…”

“…NAT substrates usually position two to three residues in the peptide binding site [15–18]. Two conserved tyrosines located on the β6β7 loop and another one on the α1α2 loop have been shown to interact with the substrate backbone via hydrogen bonds [19]. Both loops cover the groove containing the catalytic site (Fig.…”