Structural basis for membrane targeting by the MVB12-associated β-prism domain of the human ESCRT-I MVB12 subunit

Abstract

MVB12-associated β-prism (MABP) domains are predicted to occur in a diverse set of membrane-associated bacterial and eukaryotic proteins, but their existence, structure, and biochemical properties have not been characterized experimentally. Here, we find that the MABP domains of the MVB12A and B subunits of ESCRT-I are functional modules that bind in vitro to liposomes containing acidic lipids depending on negative charge density. The MABP domain is capable of autonomously localizing to subcellular puncta and to the plasma membrane. The 1.3-Å atomic resolution crystal structure of the MVB12B MABP domain reveals a β-prism fold, a hydrophobic membrane-anchoring loop, and an electropositive phosphoinositide-binding patch. The basic patch is open, which explains how it senses negative charge density but lacks stereoselectivity. These observations show how ESCRT-I could act as a coincidence detector for acidic phospholipids and protein ligands, enabling it to function both in protein transport at endosomes and in cytokinesis and viral budding at the plasma membrane.

Fig. 1.

The MABP domain is necessary and sufficient for liposome binding by ESCRT-I. (A) The MABP domain binds to Folch fraction liposomes. (B) The MABP domain is sufficient to confer lipid binding on ESCRT-I. (C) The MABP is necessary for lipid binding by ESCRT-I. “S” indicates supernatant, and “P” indicates pellet in vesicle sedimentation assays.

Fig. 2.

The MABP domain binds to highly acidic liposomes with little head group specificity. (A) Binding of MVB12B-MABP to liposomes composed of the indicated mole fraction of PS or to the charge-equivalent amount of PI(4,5)P₂, assuming charges of -1 and -4, respectively, per molecule. (B and C) Binding of MVB12B-MABP to the indicated mixtures of PS and phosphoinositides. All phosphoinositide:PS mixtures are equivalent in net charge to 70% PS. In A and B, PC is used as the background lipid in all cases. “S” indicates supernatant, “P” indicates pellet, and “blank” indicates a control containing no liposomes.

Fig. 3.

Subcellular targeting by the MABP domain. (A) mCherry-MVB12B-MABP is predominantly cytosolic with some punctate localization (arrows). (B) The tandem construct mCherry-(MVB12B-MABP)₂ localizes predominantly to the plasma membrane and punctate structures (arrows).

Fig. 4.

Structure and membrane-binding mechanism of the MABP domain. (A) Electron density (2F_o - F_c) synthesis highlighting the tip of the β2–β3 loop of subdomain I. (B) Overall fold of the MABP domain colored by subdomain. (C) Model for membrane binding colored according to electrostatic potential, with blue electropositive and red electronegative. Hydrophobic residues at the tip of the β2–β3 loop are highlighted and their potential role in membrane insertion shown.

Fig. 5.

Mechanism of membrane binding by the MABP domain. (A and B) Binding of MVB12B-MABP mutants to Folch liposomes. (C) Subcellular localization of mCherry-MVB12B-MABP hydrophobic loop mutant is diffuse with no detectable punctate localization.