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

Abstract

N-terminal acetyltransferases (NATs) are enzymes catalysing the transfer of the acetyl from Ac-CoA to the N-terminus of proteins, one of the most common protein modifications. Unlike NATs, lysine acetyltransferases (KATs) transfer an acetyl onto the amine group of internal lysines. To date, not much is known on the exclusive substrate specificity of NATs towards protein N-termini. All the NATs and some KATs share a common fold called GNAT. The main difference between NATs and KATs is an extra hairpin loop found only in NATs called {beta}6{beta}7 loop. It covers the active site as a lid. The hypothesized role of the loop is that of a barrier restricting the access to the catalytic site and preventing acetylation of internal lysines. We investigated the dynamics-function relationships of all available structures of NATs covering the three domains of life. Using elastic network models and normal mode analysis, we found a common dynamics pattern conserved through the GNAT fold; a rigid V-shaped groove, formed by the {beta}4 and {beta}5 strands and three relatively more dynamic loops 12, {beta}3{beta}4 and {beta}6{beta}7. We identified two independent dynamical domains in the GNAT fold, which is split at the {beta}5 strand. We characterized the {beta}6{beta}7 hairpin loop slow dynamics and show that its movements are able to significantly widen the mouth of the ligand binding site thereby influencing its size and shape. Taken together our results show that NATs may have access to a broader ligand specificity range than anticipated.\n\nAuthor summaryN-terminal acetylation concerns 80% of eukaryotic proteins and is achieved by enzymes called the N-terminal acetyltransferases (NATs). They belong to the large family of acetyltransferases and adopt the GNAT fold. Interestingly most lysine acetyltransferases (KATs), which acetylate specifically internal lysines, share the same fold. Rationale for the ligand recognition by the GNAT enzymes remains unclear. Proteins are dynamic entities that utilize their structural flexibility to carry out functions in living cells. By studying the dynamics throughout the entire NATs family, we found that the slow dynamics of the fold is strongly conserved. We also revealed the mobility of the active site lid, namely the {beta}-hairpin loop {beta}6{beta}7, which is one of the main structural differences between the NATs and the KATs. The size and shape of the ligand binding site depend on movements of that {beta}-hairpin loop. We suggest that in attempts of mapping NATs specificity or ligand design the fold flexibility should be taken into consideration.