Proteome-wide probing of the dual NMT-dependent myristoylation tradeoff unveils potent, mechanism-based suicide inhibitors

Abstract

N-myristoyltransferases (NMTs) catalyze protein myristoylation, a major and ubiquitous lipid modification. Originally thought to modify only N-terminal glycine -amino groups (G-myristoylation), NMTs are now known to modify lysine {varepsilon}-amino groups (K-myristoylation), the significance of which is uncertain. Here we exploited systematic structural proteomics analyses and a novel pipeline involving the Shigella IpaJ protease to discriminate K- and G-myristoylation with unprecedented accuracy and identify the specific features driving each modification. NMT-dependent K-myristoylation occurs post-translationally and only on lysines 1, 2, or 3 following G-myristoylation or caspase cleavage. Direct interactions between the substrates reactive amino group and the NMT catalytic base slow K-myristoylation catalysis. IpaJ unmasked novel K-myristoylation sites in a dozen human proteins. The unique properties of NMT-driven K-myristoylation allowed us to design potent, mechanism-based suicide NMT inhibitors. These analyses unravel the respective paths towards K-myristoylation, G-myristoylation, or NMT inhibition, which rely on a very subtle tradeoff embracing the chemical landscape around the reactive group. SIGNIFICANCE STATEMENTWe report the specific and unique elements guiding N-myristoyltransferase to either alpha or epsilon myristoylation, allowing us to establish the post-translational nature of N-myristoyltransferase-dependent lysine myristoylation and design novel, potent N-myristoyltransferase inhibitors.