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. 2010 Nov 12;40(3):433-43.
doi: 10.1016/j.molcel.2010.10.018.

WD40 repeat propellers define a ubiquitin-binding domain that regulates turnover of F box proteins

Natasha Pashkova  1 , Lokesh Gakhar, Stanley C Winistorfer, Liping Yu, S Ramaswamy, Robert C Piper
Affiliations

WD40 repeat propellers define a ubiquitin-binding domain that regulates turnover of F box proteins (V体育平台登录)

Natasha Pashkova et al. Mol Cell. .

Abstract

WD40-repeat β-propellers are found in a wide range of proteins involved in distinct biological activities VSports手机版. We define a large subset of WD40 β-propellers as a class of ubiquitin-binding domains. Using the β-propeller from Doa1/Ufd3 as a paradigm, we find the conserved top surface of the Doa1 β-propeller binds the hydrophobic patch of ubiquitin centered on residues I44, L8, and V70. Mutations that disrupt ubiquitin binding abrogate Doa1 function, demonstrating the importance of this interaction. We further demonstrate that WD40 β-propellers from a functionally diverse set of proteins bind ubiquitin in a similar fashion. This set includes members of the F box family of SCF ubiquitin E3 ligase adaptors. Using mutants defective in binding, we find that ubiquitin interaction by the F box protein Cdc4 promotes its autoubiquitination and turnover. Collectively, our results reveal a molecular mechanism that may account for how ubiquitin controls a broad spectrum of cellular activities. .

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Figures

Figure 1
Figure 1. The Doa1 WD40 β-Propeller Binds Ub
(A) Schematic of S. cerevisiae Doa1 domains including WD40 repeats forming the β-propeller, a central (P)LAA (F)amily (U)b-binding (PFU) domain, and a C-terminal (P)LAA, (U)fd3p and (L)ub1p (PUL) domain that binds to p97/Cdc48. (B) V5-epitope-tagged fragments of Doa1 encompassing the N-terminal β-Prp, either alone or with the PFU domain, were expressed in yeast or bacteria and subjected to pull-down assays using GST alone (ø) or GST-ubiquitin (Ub). Samples were immunoblotted with anti-V5 antibodies. Input lysate sample represents a 10% equivalent. (C) NMR HSQC data of 15N-Ub (25 μM) in the presence of Doa1 β-Prp. Peaks of the indicated backbone amides are shown in the absence (blue) or presence of increasing amounts of Doa1 β-Prp (Doa1:Ub molar ratios of 1:1, 2:1, 4:1, 6:1, 8:1,10:1, and 12:1). Scale bars indicate 0.5 ppm for the 15N dimension and 0.05 ppm for 1H dimension. (D) Chemical shift perturbations in the titration series were quantified using the formula (0.2*[ΔN]2 + [ΔH]2)1/2 and plotted as a function of Doa1 β-Prp concentration yielding a Kd of 224 μM (±27 SD). (E) Residues undergoing selective chemical perturbation (+1 SD above the mean ΔChemical Shift) data from HSQC data of 15N-Ub and Doa1 at a 1:1 ratio were mapped onto the structure of Ub (PDB ID code 1UBQ). Side chains corresponding to the residues with the highest perturbations are red. Below is the corresponding molecular surface of Ub indicating a large contiguous patch of residues including L8, R42, I44, V70, and R72 that may form the Doa1 β-Prp-binding interface. (F) Bacterially produced V5-Doa1 β-Prp was subjected to GST pull-down assays with GST alone (ø); or GST fused to wild-type ubiquitin (Ub); or ubiquitin with L8A, R42E, and I44A mutations (ub*). Input lysate represents a 10% equivalent.
Figure 2
Figure 2. Structure and Function of the Ub-Binding Doa1 β-Propeller
(A) The 1.35Å structure of the Doa1 β-Prp (residues 2–295) as viewed from the top, side, and bottom perspectives. Left column shows a cartoon of Doa1 where color spectrum of blue to red depicts N terminus to C terminus. Middle column shows the molecular surface of Doa1 β-Prp where residues conserved among Doa1 homologs are indicated in dark blue. Right columns depict summary of mutagenesis data mapped onto the Doa1 β-Prp structure. Mutations with little to no impact on Ub binding are colored light blue, and those that blocked Ub binding are colored red on the molecular surface of Doa1. (B) Binding assays with GST alone (ø) and GST-Ub using bacterially produced wild-type and mutant Doa1 β-Prps. β-Prps were made as V5-epitope-tagged proteins and immunoblotted with anti-V5 antibodies together with a 3% equivalent of input. (C) Effect of Doa1 mutations on in vivo function. Mutant doa1Δ null cells were transformed with low-copy plasmids encoding C-terminally HA-tagged Doa1 with either no mutations (WT), a mutation that disrupts Ub binding by the central PFU domain (PFUΔUb), mutations that disrupt Ub binding by the β-Prp (residues D15, F222, W265), or a combination of UBD mutations in both the PFU and β-Prp domains. Left shows an anti-V5 immunoblot of whole-cell lysates from the corresponding transformants showing comparable expression of each Doa1-V5 mutant. Right shows growth assays of serially diluted cells grown on minimal media at 30°C, 37°C, or 30°C in the presence of 0.2% caffeine or 75 μg/ml L-azetidine-2-carboxylic acid (ADCB).
Figure 3
Figure 3. WD40 β-Propellers from Functionally Diverse Proteins Bind Ub
β-Prps (defined in Table S1) were expressed as N-terminally V5-epitope-tagged proteins in bacteria and subjected to binding assays with GST alone (ø), Ub-GST, or a mutant Ub-GST protein (ub*). A 3% equivalent of input lysate was also immunoblotted. The mutant Ub (ub*) used contained L8A, R42E, and I44A mutations. Also shown is the molecular surface of Ub with those residues involved in Doa1 binding in blue. Shown below is the same view but with the positions of L8, R42, and I44 in red.
Figure 4
Figure 4. Shared Binding Mode of Duf1 and Cdc4 β-Propellers to Ub
(A) Mapping of chemical shift perturbation data onto Ub from HSQC data of 15N-labeled Ub in the presence and absence of unlabelled β-Prp from Duf1 (1:15). Left shows side chains of affected residues, and right shows corresponding molecular surface. (B) Similar analysis as in (A) for Cdc4:Skp1 complex at a 6:1 ratio with Ub. (C) The Ub binding surfaces, as defined by NMR chemical shift perturbation, used by Duf1 (left) and Cdc4 (right) compared with that used by Doa1 β-Prps. Residues involved in Duf1 and Cdc4 binding are in green, those involved in Doa1 binding are in red, and those in common (merge) are in yellow.
Figure 5
Figure 5. Prediction and Validation of the Ub-Binding Surface of Cdc4 β-Propeller
(A) Molecular surface of Cdc4 β-Prp (residues 367–744 from PDB ID code 1NEX). Conserved surface residues are red. A subset of conserved residues involved in binding phosphodegrons is shown in pink. (B) HIT map of Cdc4 β-Prp. Shown is the Cα molecular surface color coded as a function of how often a given Cα is within an interface with Ub throughout a series of rigid body docks calculated by ZDOCK. The spectrum describing the probability of being near the Ub docking sites is arranged from dark blue (low probability) to red (high probability). Details on HIT map calculations are provided in the Supplemental Information and Supplemental Experimental Procedures. (C) Mapping of mutagenesis data onto the molecular surface of Cdc4 β-Prp. Residues in blue are ones that when mutated do not affect Ub binding. Residues in red are those that disrupt binding when altered. The positions of R485 and R534 that are involved in phosphodegron binding are also indicated. (D) Ub-binding data used for mutagenesis mapping experiments. V5-epitope-tagged Cdc4 β-Prp proteins were made in bacteria and subjected to GST pull-down experiments with GST alone (ø) or GST fused to Ub. Fractions were immunoblotted with anti-V5 antibodies. Input represents a 3% equivalent.
Figure 6
Figure 6. Ub Binding Promotes Ubiquitination and Degradation of F Box Proteins
(A) Temperature-sensitive cdc4-1 cells were transformed with low-copy-expressing wild-type HA-epitope-tagged Cdc4 (CDC4), no Cdc4 (vector), or Cdc4ΔUb mutants with the indicated residue changes that block Ub binding by the β-Prp. Expression was driven by the CDC4 promoter. Cell dilutions were plated onto SC minimal media plates or YPD-rich media plates and grown at 25°C or 36°C, which inactivates the endogenous Cdc4-1 protein. (B) Cells expressing wild-type HA-Cdc4 and the indicated HA-Cdc4ΔUb mutant cells were subjected cycloheximide chase experiments to estimate protein half-life. At the indicated times after cycloheximide addition, aliquots were processed and analyzed by immunoblotting with anti-HA antibodies. As internal loading controls, blots were subsequently immunoblotted for Pgk1. Our anti-HA blotting conditions also revealed a constant background band (*) that provided an additional loading standard. (C) In vitro ubiquitination assays with WT Skp1:Fbw7 and mutant Fbw7ΔUbD560S,D600S. Skp1:Fbw7 complexes (1 μM) were incubated with 50 μM Ub, 0.2 μM E1, 5 mM ATP, 2 μM each UbcH5 and hCdc34, 1 μM Rbx1:Cul1Nedd8 complex, and 0.5 μM cyclin-E/CDK2 at 30°C. Aliquots were taken at the indicated time points, diluted with SDS-containing sample buffer, and immunoblotted for cyclin-E and Fbw7 using anti-cyclin E and anti-Fbw7 antibodies. (D) WT Skp1:Fbw7, mutant Skp1:Fbw7ΔUbY545A, L583S, or the Duf1 β-Prp was covalently attached to Sepharose beads and incubated with cyclin-E/CDK complex in PBS containing 5 mM MgCl2, 1 mM ATP, 10 nM cyclin-E/CDK, and 0.05 mg/ml BSA in a volume of 200 μl in the presence or absence of 120 μM diUb. (E) Model for how Ub binding could promote ubiquitination and degradation of F box proteins. Above shows a model (based on Hoeller et al., 2007; Sorkin, 2007) for E3-independent ubiquitination of proteins containing a ubiquitin-binding domain (UBD). The UBD would bind Ub~E2, facilitating attack from an acceptor lysine residue within the UBD-containing protein. Below is the same scheme, except that this would happen within the context of a fully assembled SCF complex. The close proximity of the E2 to the F box protein afforded by the Cullin/Skp1/Rbx1 complex would facilitate E2~Ub:β-Prp interaction and compensate for the modest affinity that F box β-Prps show independently. Binding of substrate might occlude Ub interaction, thereby prolonging the life of the F box protein until available substrate is depleted.
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