Structural Basis for Regulation of ESCRT-III Complexes by Lgd

Abstract

The ESCRT-III complex induces outward membrane budding and fission through homotypic polymerization of its core component Shrub/CHMP4B. Shrub activity is regulated by its direct interaction with a protein called Lgd in flies, or CC2D1A or B in humans. Here, we report the structural basis for this interaction and propose a mechanism for regulation of polymer assembly. The isolated third DM14 repeat of Lgd binds Shrub, and an Lgd fragment containing only this DM14 repeat and its C-terminal C2 domain is sufficient for in vivo function. The DM14 domain forms a helical hairpin with a conserved, positively charged tip, that, in the structure of a DM14 domain-Shrub complex, occupies a negatively charged surface of Shrub that is otherwise used for homopolymerization. Lgd mutations at this interface disrupt its function in flies, confirming functional importance. Together, these data argue that Lgd regulates ESCRT activity by controlling access to the Shrub self-assembly surface.

Keywords: CC2D1A; CC2D1B; CHMP4B; DM14 domain; Shrub; Snf7; lethal giant discs; membrane fission; multivesicular body.

Figure 1

A, Domain organization of Lgd, highlighting the four DM14 modules and C-terminal C2 domain. B-N, Wing imaginal discs of Drosophila late third instar larvae. B, a wildtype disc, stained for Wg. C-N, In each panel, the indicated Lgd construct was transgenically expressed at the same insertion site in lgd null (lgd^d7/lgd^d7), and lgd null/shrub heterozygous (lgd^d7/lgd^d7, shrub^4-1/+) backgrounds. Each imaginal disc was stained for the Notch target gene, wingless (wg), as a surrogate for MVB dysfunction. Wingless expression is restricted to the dorsal/ventral (D/V) boundary line during normal development (arrowhead in B). Loss of ESCRT function results in ectopic broadening of the Wg D/V stripe, as seen in C. Lethality in early L3 stage of development was observed in parts D and J. Scale bar: 200 µm. O, Interaction of Shrub (6–106) with Lgd (DM14-3, orange; DM14-4, red; DM14-3+4, grey) detected using SPR. Response units are normalized to the molecular mass of each Lgd construct. See also Figures S1, S2, and Table S1.

Figure 2

Structure of the third DM14 domain (residues 359–423) of Lgd. A, Cartoon representation, colored on a sliding scale from N-terminus (blue) to C-terminus (red). B, Surface representation, colored by conservation on a sliding scale from cyan (least conserved) to maroon (most conserved). C, Surface representation colored by electrostatic potential from negative (red) to positive (blue). See also Figure S2.

Figure 3

Crystal structure of the Lgd-Shrub fusion protein. A, Left panel: Lgd-Shrub complex. DM14-3 is shown as a cartoon (green), and Shrub as an electrostatic surface on a sliding scale from negative (red) to positive (blue). The polyglycine linker connecting Lgd and Shrub, illustrated here with a dotted line, is not visible in the structure. Right panel: crystallographic model of a Shrub homopolymer (based on PDB entry 5J45). B, Key residues at the Lgd:Shrub binding interface. Lgd is shown as a cartoon in green. Shrub is shown as a cartoon in cyan. Charged residues at the interface are shown as sticks with salt bridges illustrated by dotted lines. See also Figure S3.

Figure 4

Analysis of the Lgd-Shrub interface. A, Interaction between DM14 domain three of Lgd (residues 359–423) and Shrub (6–106) measured using SPR. Interaction between wildtype molecules is shown in orange. Interaction between wildtype Shrub and Lgd R393A is shown in blue. Interaction between Shrub E86R and wildtype Lgd is shown in green. B-G, Truncated Lgd (3-4-C2 construct) molecules with the listed mutations were transgenically expressed in the indicated genetic backgrounds. Fly wing discs were stained for Wg expression as a surrogate for MVB dysfunction. H-M, Full-length Lgd molecules (1-2-3-4-C2) with the listed mutations were transgenically expressed in the indicated genetic backgrounds. Fly wing discs were stained for Wg expression as a surrogate for MVB dysfunction. Scale bar: 200 µm. See also Table S1.