Biochemical and structural analysis of N-myristoyltransferase mediated protein tagging.

N-terminal myristoylation is an essential eukaryotic modification crucial for cellular homeostasis in the context of many physiological processes. Myristoylation is a lipid modification resulting in a C14 saturated fatty acid addition. This modification is challenging to capture due to its hydrophobicity, low abundance of target substrates, and the recent discovery of unexpected NMT reactivity including myristoylation of lysine side chains and N-acetylation in addition to classical N-terminal Gly-myristoylation. This chapter details the high-end approaches developed to characterize the different features of N-myristoylation and its targets through in vitro and in vivo labeling.

Kinetic and catalytic features of N-myristoyltransferases.

N-myristoyltransferases (NMTs) are members of the large family of GCN5-related N-acetyltransferases (GNATs). NMTs mainly catalyze eukaryotic protein myristoylation, an essential modification tagging protein N-termini and allowing successive subcellular membrane targeting. NMTs use myristoyl-CoA (C14:0) as major acyl donor. NMTs were recently found to react with unexpected substrates including lysine side-chains and acetyl-CoA. This chapter details the kinetic approaches that have allowed the characterization of the unique catalytic features of NMTs in vitro.

Structural and Large-scale Analysis Unveil the Intertwined Paths Promoting NMT-catalyzed Lysine and Glycine Myristoylation.

N-myristoyltransferases (NMTs) catalyze protein myristoylation, a lipid modification crucial for cell survival and a range of pathophysiological processes. Originally thought to modify only N-terminal glycine α-amino groups (G-myristoylation), NMTs were recently shown to also modify lysine ε-amino groups (K-myristoylation). However, the clues ruling NMT-dependent K-myristoylation and the full range of targets are currently unknown. Here we combine mass spectrometry, kinetic studies, in silico analysis, and crystallography to identify the specific features driving each modification. We show that direct interactions between the substrate's reactive amino group and the NMT catalytic base promote K-myristoylation but with poor efficiency compared to G-myristoylation, which instead uses a water-mediated interaction. We provide evidence of depletion of proteins with NMT-dependent K-myristoylation motifs in humans, suggesting evolutionary pressure to prevent this modification in favor of G-myristoylation. In turn, we reveal that K-myristoylation may only result from post-translational events. Our studies finally unravel the respective paths towards K-myristoylation or G-myristoylation, which rely on a very subtle tradeoff embracing the chemical landscape around the reactive group.