Electrostatic Interactions between Elongated Monomers Drive Filamentation of Drosophila Shrub, a Metazoan ESCRT-III Protein

Abstract

The endosomal sorting complex required for transport (ESCRT) is a conserved protein complex that facilitates budding and fission of membranes. It executes a key step in many cellular events, including cytokinesis and multi-vesicular body formation. The ESCRT-III protein Shrub in flies, or its homologs in yeast (Snf7) or humans (CHMP4B), is a critical polymerizing component of ESCRT-III needed to effect membrane fission. We report the structural basis for polymerization of Shrub and define a minimal region required for filament formation. The X-ray structure of the Shrub core shows that individual monomers in the lattice interact in a staggered arrangement using complementary electrostatic surfaces. Mutations that disrupt interface salt bridges interfere with Shrub polymerization and function. Despite substantial sequence divergence and differences in packing interactions, the arrangement of Shrub subunits in the polymer resembles that of Snf7 and other family homologs, suggesting that this intermolecular packing mechanism is shared among ESCRT-III proteins.

Figure 1. Structural Comparison of Shrub with CHMP3 and Snf7

(A) Snf7 domain architecture aligned by primary sequence. Helices are indicated by colored boxes and prolines by purple triangles. For Shrub (light blue) and Snf7 (beige), helices are denoted based on the crystal structures, and the fragments used for crystallization of Shrub (10–143; this work) and Snf7 (12–150; Tang et al., 2015) are represented by bars above the structural schematic colored based on alignment to the canonical α1–α4 helices of CHMP3 shown above. (B) Cartoon representations of CHMP3 (1–222, 3FRT; 1–150, 3FRV), Shrub (10–143, 5J45), Snf7 (12–150, 5FD9), and CHMP1B (4–163, 3JC1), colored using the scheme in (A). See also Figures S1 and S3.

Figure 2. Key Lattice Interactions in the Shrub Crystal Structure

(A) Three molecules from adjacent unit cells are shown and labeled as “−1,” “0,” and “+1.” The “0” subunit is represented as a surface, colored by charge (blue, positive to red, negative). Extensive crystal contacts formed by electrostatic interactions occur between molecules in adjacent unit cells. (B) The electrostatic interface between adjacent molecules represented in “open book” form. Dashed lines indicate salt bridge interactions. (C) Superposition of Shrub (cyan) onto the two conformations of the yeast ortholog Snf7 (tan, 5FD9; gray, 5FD7). Two subunits, and their observed crystal contacts, are shown for each of the independent crystal forms. (D) Electrostatic surface representations (blue, positive to red, negative) for the indicated region of subunit “1.”

Figure 3. Filamentation Is Driven by Electrostatic Interactions

(A) The 6-106 fragment of Shrub at a concentration of 250 μM, was polymerized by dialysis into low salt buffer. Filaments were visualized by negative stain electron microscopy at a magnification of 68,000. Scale bars, 100 nM. (B) Circular dichroism spectra of wild-type, R59E, and E86R variants of Shrub (6–106), acquired at 20°C. (C) Wild-type Shrub and the indicated mutants were dialyzed to low salt and visualized by negative stain electron microscopy at a magnification of 30,000. (D) Summary of the results from experiments attempting to rescue shrb^4-1 (null) lethality using wild-type or mutant forms of Shrub delivered to the identical genomic landing site. (E) Overlay of the observed crystal contact interfaces of Shrub (cyan) or Snf7 (light orange). The Shrub side-chains involved in direct salt bridge interactions (dotted lines) and the analogous yeast residues, are represented by sticks. (F) Primary sequence alignment of the fly, human, and yeast orthologs. Residues at the crystal contact interfaces (within 5 Å of the neighboring protomer) are boxed and colored by class (acidic, red; basic, blue; polar, green; non-polar, gray). Residues involved in salt bridge interactions (dotted lines) are indicated by residue number and with a colored circle above (Shrub) or below (Snf7) the aligned sequences. See also Figures S2 and S3.