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.