Structural basis for ubiquitin recognition and autoubiquitination by Rabex-5

Abstract

Rabex-5 is an exchange factor for Rab5, a master regulator of endosomal trafficking. Rabex-5 binds monoubiquitin, undergoes covalent ubiquitination and contains an intrinsic ubiquitin ligase activity, all of which require an N-terminal A20 zinc finger followed immediately by a helix. The structure of the N-terminal portion of Rabex-5 bound to ubiquitin at 2.5-A resolution shows that Rabex-5-ubiquitin interactions occur at two sites. The first site is a new type of ubiquitin-binding domain, an inverted ubiquitin-interacting motif, which binds with approximately 29-microM affinity to the canonical Ile44 hydrophobic patch on ubiquitin. The second is a diaromatic patch on the A20 zinc finger, which binds with approximately 22-microM affinity to a polar region centered on Asp58 of ubiquitin. The A20 zinc-finger diaromatic patch mediates ubiquitin-ligase activity by directly recruiting a ubiquitin-loaded ubiquitin-conjugating enzyme.

Fig. 1. Structure of the Rabex-5 A20 ZnF domain and IUIM

(a) The domain structure of Rabex-5 and the construct used in this study. The ZnF domain and the IUIM are colored magneta and green, respectively. (b) Density-modified Fourier synthesis from prime and switch phasing of the complex structure in space group P6₁. Rabex-5 and ubiquitin models are colored green and yellow, respectively. (c) Rabex-5 (ribbon model) contacts ubiquitin (surface models in orange and marine) at two sites in the crystal lattice. (d) Superposition of the four crystallographically independent molecules of the A20 ZnF domain and IUIM. The P6₁ molecule is colored green, and the three independent molecules in the asymmetric unit of the C2 lattice are colored pink, purple, and red. (e) The zinc binding site in the A20 ZnF domain. The Zn ion is colored red. (f) Ribbon and ball-and-stick representation showing exposed hydrophobic side-chains. (g) Surface of the A20 ZnF domain and IUIM colored by residue type: green (hydrophobic), red (acidic), blue (basic), and white (uncharged polar).

Fig. 2. Ubiquitin recognition by Rabex-5

(a) Ribbon and ball-and-stick representation of ubiquitin (orange) shown bound to a surface model of Rabex-5 IUIM. (b) Ribbon and ball-and-stick representation of Rabex-5 IUIM bound to a surface model of ubiquitin. (c) Hydrogen bonds near the N-terminus of the IUIM. (d) Hydrogen bonds near the C-terminus of the IUIM. (e) Ribbon and ball-and-stick representation of ubiquitin (marine) shown bound to a surface model of Rabex-5 A20 ZnF domain (magenta). (f) Ribbon and ball-and-stick representation of Rabex-5 A20 ZnF domain (magenta) bound to a surface model of ubiquitin (marine). (g) The ubiquitin Asp58 hydrogen bonds with Rabex-5 A20 ZnF Ser36. (h) Ubiquitin binds to Rabex-5 through two different non-overlapping surfaces on ubiquitin. Surfaces contacting IUIM are colored orange, and those contacting the A20 ZnF are colored marine.

Fig. 3. Conservation of IUIMs and relationship to UIMs

(a) Superposition of Rabex-5 IUIM and Vps27 UIM (PDB ID 1Q0W) based on overlay of ubiquitin molecules. (b) Sequence of Rabex-5 IUIM aligned with secondary structure elements and annotated by function. Sequences of UIMs of yeast Vps27, human Hrs (Vps27 ortholog), and human S5a are shown below ordered from C terminal to N-terminal. Residues contacting ubquitin are marked with orange (IUIM) and marine (Vps27 UIM-1) triangles, respectively. (c) Vps27 UIM (ribbon) bound to ubiquitin (translucent surface with underlying ribbon). (d) Rabex-5 IUIM (ribbon) in the same orientation as panel (b) for comparison.

Fig. 4. Conservation of A20 ZnF domains

(a) Structure-based sequence alignment of A20 zinc fingers, labeled b, bovine; m, murine; h, human. Conserved Cys residues are indicated as black triangles at bottom, and ubiquitin-binding residues are indicated with blue triangles at bottom. Sequence numbering is for bovine Rabex-5. (b) The surface of the Rabex-5 A20 ZnF colored by conservation. Ubiquitin-binding residues are labeled marine.

Fig. 5. The ubiquitin:Rabex-5 interaction in vitro

(a) ITC analysis of the binding of ubiquitin to the Rabex-5 (9–73) constructs in vitro. The inset shows the raw heat change elicited by successive injections of ubiquitin into a solution of Rabex-5 (9–73) wild type. The main Fig. depicts the normalized integration data (kcal/mol of ubiquitin) as a function of the molar ratio of ubiquitin to the various Rabex-5 (9–73) constructs. The results shown are representative of four independent experiments carried out with the wild-type construct and two independent experiments carried out with each individual mutant. (b) Surface plasmon resonance sensorgrams for selected Rabex-5(9–73) mutants with GST-ubiquitin wild-type immobilized. (c-e) Histograms showing relative affinities of Rabex-5(9–73) mutants with GST-ubiquitin wild-type (c), I44D (d) and D58A (e). K_A values were obtained by taking inverse value of K_D values from Table 2 and normalized so that K_A for Rabex-5(9–73) wild-type was set to 100. A two-site model was used to fit the data for GST-ubiquitin wild-type (c) and one-site model for GST-ubiquitin I44D (d) and D58A (e). Values greater than 100 in panels c-e probably reflect the limitations of the simple one and two site models used to fit the data, rather than real increases in affinity.

Fig. 6. E2 recruitment and E3 ligase activity of Rabex-5 and its mutants

(a) Detection of [³⁵S]-labeled E2 enzymes shows that ubiquitin-loaded UbcH5 selectively binds to Rabex-5 with an intact A20 ZnF domain, but not to the Y25A/Y26A Rabex-5 mutant. The E2 enzyme Ubc7 binds to the Ubc-binding domain of gp78 independent of its ubiquitin loading, and does not bind to Rabex-5. (b) Wild-type Rabex-5 has ubiquitin E3 ligase activity as indicated by detection of Flag-ubiquitin. Tyr mutations in the A20 ZnF domain block this activity, while the A58D IUIM mutant enhances activity. The arrow marks the position corresponding to the molecular weight of unmodified GST-Rabex-5 (9–73).

Supplementary Figure 1

A speculative model for ubiquitin-loaded E2 binding to Rabex-5(9–73). Ubiquitin bound to ZnF of Rabex-5(9–73) was superimposed to SUMO-1 of the SUMO-RanGAP1-Ubc9-Nup358 complex structure (PDB ID: 1Z5S). Green, IUIM; magenta, ZnF; red, zinc ion; marine, ubiquitin bound to ZnF; cyan, SUMO-1; yellow, Ubc9. Some key residues including Y25 and Y26 on ZnF, I44 on ubiquitin and the active site residue C93 on Ubc9 are labeled. Note that the C-terminus of SUMO-1 is in proximity to C93 of Ubc9.