The X-linked lethal Ogden syndrome was the first reported human genetic disorder associated with a mutation in an N-terminal acetyltransferase (NAT) gene. The affected males harbor an Ser37Pro (S37P) mutation in the gene encoding Naa10, the catalytic subunit of NatA, the major human NAT involved in the co-translational acetylation of proteins. Structural models and molecular dynamics simulations of the human NatA and its S37P mutant highlight differences in regions involved in catalysis and at the interface between Naa10 and the auxiliary subunit hNaa15. Biochemical data further demonstrate a reduced catalytic capacity and an impaired interaction between hNaa10 S37P and Naa15 as well as Naa50 (NatE), another interactor of the NatA complex. N-Terminal acetylome analyses revealed a decreased acetylation of a subset of NatA and NatE substrates in Ogden syndrome cells, supporting the genetic findings and our hypothesis regarding reduced Nt-acetylation of a subset of NatA/NatE-type substrates as one etiology for Ogden syndrome. Furthermore, Ogden syndrome fibroblasts display abnormal cell migration and proliferation capacity, possibly linked to a perturbed retinoblastoma pathway. N-Terminal acetylation clearly plays a role in Ogden syndrome, thus revealing the in vivo importance of N-terminal acetylation in human physiology and disease.

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.

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23 N-terminal acetyltransferases (NATs) are enzymes catalysing the transfer of the acetyl from Ac-CoA to the 24 N-terminus of proteins, one of the most common protein modifications. Unlike NATs, lysine 25 acetyltransferases (KATs) transfer an acetyl onto the amine group of internal lysines. To date, not much is 26 known on the exclusive substrate specificity of NATs towards protein N-termini. All the NATs and some 27 KATs share a common fold called GNAT. The main difference between NATs and KATs is an extra 28 hairpin loop found only in NATs called β6β7 loop. It covers the active site as a lid. The hypothesized role of 29 the loop is that of a barrier restricting the access to the catalytic site and preventing acetylation of internal 30 lysines. We investigated the dynamics-function relationships of all available structures of NATs covering 31 the three domains of life. Using elastic network models and normal mode analysis, we found a common 32 dynamics pattern conserved through the GNAT fold; a rigid V-shaped groove, formed by the β4 and β5 33 strands and three relatively more dynamic loops α1α2, β3β4 and β6β7. We identified two independent 34 dynamical domains in the GNAT fold, which is split at the β5 strand. We characterized the β6β7 hairpin 35 loop slow dynamics and show that its movements are able to significantly widen the mouth of the ligand 36 binding site thereby influencing its size and shape. Taken together our results show that NATs may have 37 access to a broader ligand specificity range than anticipated. 38Author summary 39 N-terminal acetylation concerns 80% of eukaryotic proteins and is achieved by enzymes called the 40 N-terminal acetyltransferases (NATs). They belong to the large family of acetyltransferases and 41 adopt the GNAT fold. Interestingly most lysine acetyltransferases (KATs), which acetylate 42 specifically internal lysines, share the same fold. Rationale for the ligand recognition by the GNAT 43 enzymes remains unclear. Proteins are dynamic entities that utilize their structural flexibility to 44 carry out functions in living cells. By studying the dynamics throughout the entire NATs family, 45 we found that the slow dynamics of the fold is strongly conserved. We also revealed the mobility of 46 the active site lid, namely the -hairpin loop 67, which is one of the main structural differences between 47 the NATs and the KATs. The size and shape of the ligand binding site depend on movements of that -Dynamics-function relationship of NATs 3 48 hairpin loop. We suggest that in attempts of mapping NATs specificity or ligand design the fold flexibility 49 should be taken into consideration. 50 51 Acetyltransferases are enzymes catalysing the transfer of an acetyl group from the co-factor acetyl-52 coenzyme A (Ac-CoA) to a substrate. Among them, lysine acetyltransferases (KATs) and Nα-terminal 53 acetyltransferases (NATs) perform protein acetylation to either lysine side chains or N-termini of 54 polypeptide chains, respectively. NATs acetylate 80 to 90% of the proteins of the human ...