Structural insights into human TM6SF2-mediated cholesterol coordination in apoB-100 interactions
摘要
Transmembrane 6 superfamily member 2 (TM6SF2) is a liver- and intestine-enriched ER membrane protein that promotes VLDL lipidation/secretion and protects against hepatic lipid accumulation. The common E167K mutation of TM6SF2 impairs protein stability and VLDL secretion while showing a paradoxical protective lipid/cardiovascular profile. However, it remains elusive how TM6SF2 contributes to lipoprotein maturation. Here, we first determined the cryo-EM structure of human TM6SF2 using HEK293F-expressed proteins. TM6SF2 forms a dimer, with each protomer comprising 10 transmembrane helices (TMs) that can be divided into an N-terminal domain (NTD, TM1-2) and two EXPERA domains—EXPERA-N (TM3-6) and EXPERA-C (TM7-10). Further analysis revealed that an endogenous cholesterol molecule was coordinated by the NTD and EXPEART-N domains. Disruption of cholesterol coordination compromised the TM6SF2-apoB-100 interaction in pull-down assays. When the structure was determined using proteins expressed under cholesterol-depleted conditions, the cholesterol densities disappeared, accompanied by more flexible conformations of the NTD and EXPEART-N domains. Molecular dynamics simulations suggested that the luminal loop between TM5 and TM6 of the EXPEART-N domain (TM5-6 linker) becomes more flexible under cholesterol-free conditions. The TM5-6 linker is essential for apoB-100 interaction, as supported by the AlphaFold3-predicted structure and pull-down assays. The common E167K mutation is located on the TM5-6 linker and weakens its direct interaction with apoB-100. Together, these observations suggest that cholesterol contributes to the TM6SF2-apoB-100 interaction by maintaining a stable conformation of the TM5-6 linker.
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