VHS domains of ESCRT-0 cooperate in high-avidity binding to polyubiquitinated cargo

Abstract

VHS (Vps27, Hrs, and STAM) domains occur in ESCRT-0 subunits Hrs and STAM, GGA adapters, and other trafficking proteins. The structure of the STAM VHS domain-ubiquitin complex was solved at 2.6 A resolution, revealing that determinants for ubiquitin recognition are conserved in nearly all VHS domains. VHS domains from all classes of VHS-domain containing proteins in yeast and humans, including both subunits of ESCRT-0, bound ubiquitin in vitro. ESCRTs have been implicated in the sorting of Lys63-linked polyubiquitinated cargo. Intact human ESCRT-0 binds Lys63-linked tetraubiquitin 50-fold more tightly than monoubiquitin, though only 2-fold more tightly than Lys48-linked tetraubiquitin. The gain in affinity is attributed to the cooperation of flexibly connected VHS and UIM motifs of ESCRT-0 in avid binding to the polyubiquitin chain. Mutational analysis of all the five ubiquitin-binding sites in yeast ESCRT-0 shows that cooperation between them is required for the sorting of the Lys63-linked polyubiquitinated cargo Cps1 to the vacuole.

Figure 1

The STAM VHS (Vps27, Hrs, and STAM) domain–ubiquitin (Ub) complex. (A) Overall structure of the complex with the STAM1 VHS domain shown in yellow and Ub in light blue. The side-chains of the key Ub residues Ile44, Lys48, and Lys63, are shown. (B) The STAM1 VHS domain binds to the hydrophobic patch (colored green) consisting of Ub Ile44, Gly47, and Val70. Basic residues Arg42, Lys48, and His68 involved in binding are colored dark blue, whereas Gln49 is colored white. The remainder of the non-interacting residues on the Ub surface are colored light blue. (C) Interactions in the complex. Hydrogen bonds are shown with dashed lines. (D) Binding of wild-type and mutant VHS domains to Ub. Binding curves for wild-type and mutant VHS domains are identified in the inset.

Figure 2

The structural ubiquitin (Ub)-binding motif is conserved in nearly all VHS (Vps27, Hrs, and STAM) domains. (A) Aligned sequences of VHS domain-containing proteins in the region corresponding to the N-terminal half of the domain. The secondary structure of the human STAM1 VHS domain is shown with solid bars to indicate α-helices. Residues shown to be critical for VHS domain–Ub binding are shaded in red, and other residues involved in, but not essential for, the interaction are shaded in blue. Identically conserved residues that are not directly involved in binding are shaded gray. Species abbreviations: Hs, Homo sapiens; Dm, Drosophila melanogaster; and Sc, Saccharomyces cerevisiae. (B) Schematic of the domain organization of selected VHS domain-containing proteins. Of the sequences shown in (A), the domain structure of STAM2_Hs is like that of STAM1_Hs; Hrs_Dm like that of Hrs_Hs, GGA1_Hs, and GGA2_Hs like GGA3_Hs; and GGA2_Sc like GGA1_Sc. SH3, Src homology-3 domain; core, ESCRT-0 core GAT and coiled coil region responsible for heterodimerization; PSAP, PTAP, PSDP are ESCRT-I binding motifs; CB, clathrin binding; and GAE, γ-adaptin ear domain.

Figure 3

Ubiquitin binds to nearly all VHS (Vps27, Hrs, and STAM) domains. Surface plasmon resonance (SPR)-binding curves between ubiquitin (Ub )and the indicated VHS domains are identified by the key in the inset.

Figure 4

ESCRT-0 and VHS (Vps27, Hrs, and STAM) domain-containing fragments of STAM1 bind polyubiquitin chains avidly. (A) Surface plasmon resonance (SPR) curves for full length ESCRT-0. (B) SPR curves for STAM VHS domain and VHS–UIM construct. (C) SPR curves for Hrs DUIM and VHS–FYVE–DUIM construct. SPR binding curves between the constructs noted at top and the ubiquitin (Ub) chains identified by the key in the inset. Binding curves are normalized to 100% for the saturable component. The total binding includes a non-saturable, non-specific component in some cases, leading to values higher than 100%. (D) Model for mono and polyubiquitinated cargo recognition by human and yeast ESCRT-0.

Figure 5

Cps1 sorting in yeast depends on the cooperative action of ESCRT-0 Ub binding domains. The indicated plasmids were transformed into vps27Δhse1Δ yeast cells and imaged for GFP-Cps1 (cargo) and FM4-64 (membrane) fluorescence as shown individually in panels (A–J).