Mechanistic insights into the selective targeting of MLX to triacylglycerol-rich lipid droplets

The activation of transcription factor Max-like protein X (MLX) is modulated by competition between active dimerization and inactive association with cytosolic lipid droplets (LDs). However, LD association has been shown to depend on the neutral lipid composition. This work explores the mechanism by which MLX specifically targets LDs rich in triacylglycerol (TG) over those with abundant sterol esters (SEs). We compare the association ensembles for a potential minimal targeting sequence, an amphipathic helix-loop-helix hairpin, and the full dimerization and cytoplasmic localization domain, finding that the latter requires larger packing defects and quantifiably alters LD membrane properties. Surprisingly, direct interactions with TG neutral lipids are not observed for either sequence. Instead, targeting to SE-rich LDs is blocked for both sequences by insufficient packing defects. We additionally explore the full mechanism of hairpin association, aiming to understand sequence-specific features that enable strong membrane association. We find that there are multiple association pathways but that each involves a catch, dive, snorkeling, and embedding phase. The combination of multiple catch-and-dive residues placed on opposing ends of amphipathic helices lengthens the catch phase, greatly enhancing association in a manner that resembles kinetic selection. Once bound, locking interhelical interactions block dissociation. Collectively, our findings suggest that in addition to relative binding affinities, both kinetics and altered surface properties due to protein association could influence competition within the LD proteome. VIDEO ABSTRACT.