1. Volume 13, Issue 5, Pages 665-676 (March 2004)
Cdc42 Regulates the Par-6 PDZ Domain through an Allosteric CRIB-PDZ Transition 
Francis C. Peterson, Rhiannon R. Penkert, Brian F. Volkman, Kenneth E. Prehoda 
Molecular Cell 
Volume 13, Issue 5, Pages (March 2004)
DOI: /S (04)
Copyright © 2004 Cell Press Terms and Conditions

2. Figure 1 Par-6 Domain Structure and PDZ Interactions
(A) Domain structure of Par-6 and known CRIB-PDZ ligands (PB1: Phox and Bem1; CRIB: Cdc42/Rac interactive binding; PDZ: PSD-95, Discs Large, and ZO-1). The Par-6 CRIB has also been termed a “semi-CRIB” because it lacks several histidine residues found in canonical CRIB domains. Coupling between the CRIB and PDZ domains (dashed line) may lead to regulation of PDZ ligands by Cdc42.
(B) Par-6 binding to Par-3 is not regulated by Cdc42. The amount of Par-3 (Bazooka) pulled down by a GST fusion of Drosophila Par-6, visualized by Coomassie blue staining, is not affected by saturating concentrations of Cdc42•GNPPNP (50 μM).
(C) The Par-6-PALS1 interaction is not regulated by Cdc42. The amount of the Par-6 CRIB-PDZ domain pulled down by a GST fusion of Pals is not affected by the presence of saturating concentrations of Cdc42.
(D) Cdc42 regulation of a Par-6 carboxy-terminal ligand. Through screening of a carboxy-terminal peptide library, Par-6 was found to bind to the sequence VKESLV in a Cdc42-dependent manner. Binding to a GST fusion of the VKESLV sequence is shown in the absence and presence of saturating concentrations of Cdc42.
Molecular Cell  , DOI: ( /S (04) )
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3. Figure 2
Coupling of Cdc42 and Carboxy-Terminal Ligand Binding in Par-6
(A) Cdc42 dependence of Par-6 binding to rhodamine-labeled VKESLV. Fluorescence anisotropy was used to measure the affinity of Drosophila Par-6 in the absence and presence of Cdc42. The affinity of Par-6 CRIB-PDZ for VKESLV is significantly enhanced by the presence of Cdc42 (Kd,Cdc42-bound, 6 μM; Kd,free, 80 μM).
(B) VKESLV is a carboxy-terminal ligand. Addition of a C-terminal alanine to the VKESLV sequence disrupts binding to Par-6 as measured by GST pull-down.
(C) Competition of Par-3 and Pals1 binding with carboxy-terminal peptide. Soluble synthetic peptide was added from 0 to 2 mM in GST pull-down reactions.
(D) Identification of residues critical for Par-6 binding in the carboxy-terminal peptide. Residues at each position of the ligand were substituted with alanine. GST fusions of these peptide sequences were tested for their ability to pull down the Par-6 CRIB-PDZ domain.
Molecular Cell  , DOI: ( /S (04) )
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4. Figure 3
Coupling of Cdc42 and Peptide Binding Is Required for Par-6 Function in MDCK Cells
(A) The Par-6 P171G mutation decouples Cdc42 and carboxy-terminal ligand binding. The P171G mutant no longer binds to the VKESLV PDZ ligand in the presence of Cdc42. However, the mutant protein retains the capability to bind Pals1, Par-3, and Cdc42.
(B) The decoupling mutant fails to regulate tight junction formation in MDCK cells. Transient transfection of mammalian Par-6B in MDCK cells leads to disruption of tight junctions as is shown by the lack of ZO-1 localization (white arrows). Cells transfected with the decoupling P171G (Drosophila residue numbering) mutant protein are indistinguishable from untransfected cells.
Molecular Cell  , DOI: ( /S (04) )
Copyright © 2004 Cell Press Terms and Conditions

5. Figure 4 Structure and Dynamics of the Par-6 PDZ Domain
(A) Solution structure of the Drosophila Par-6 PDZ domain. The ensemble of structures from the NMR structure refinement is shown. The CRIB domain, which is highly dynamic in free Par-6, is not shown.
(B) Heteronuclear NOE measurements of the Par-6 CRIB-PDZ in the free and Cdc42-bound forms. Heteronuclear NOEs are a measure of dynamics on the picosecond to nanosecond timescales, with smaller values indicating more relative motion. Secondary structure elements for the PDZ domain are shown below the plot. Heteronuclear NOE values of zero in the plot were not measured. For free Par-6, the NOE values were truncated at −1.0, as values for residues at the beginning of the CRIB are less than −4.0.
Molecular Cell  , DOI: ( /S (04) )
Copyright © 2004 Cell Press Terms and Conditions

6. Figure 5
Comparison of Par-6 PDZ Domain in the Free and Cdc42-Bound Forms
(A) Ribbon overlay of the free (green) and Cdc42 bound (orange) Par-6 PDZ domain. The root mean square deviation (rmsd) between the free PDZ ensemble is shown compared to the rmsd between the free ensemble and the Cdc42-bound structure. The sequence of human Par-6B used for the crystal structure is shown for comparison.
(B) Comparison of the free and Cdc42-bound Par-6 PDZ domains to other PDZ structures. Known PDZ structures (black; PDB codes: 1BE9, 1G9O, 1GM1, 1I92, 1IHJ, 1KEF, 1KWA, 1PDR, 1QAU; only residues from the PDZ domains of these structures are shown) have a tightly clustered conformation that closely resembles the Cdc42-bound Par-6 PDZ domain. The free Par-6 structure deviates from the canonical PDZ fold, however. A statistical analysis of the conformational differences between the free and Cdc42-bound Par-6 PDZ domains and PSD-95 PDZ3 (PDB code 1BE9) is shown below the overlay.
Molecular Cell  , DOI: ( /S (04) )
Copyright © 2004 Cell Press Terms and Conditions

7. Figure 6
Par-6 PDZ Peptide Binding Induces Conversion to the High-Affinity Conformation
(A) The free Par-6 PDZ domain exists in a low-affinity conformation (green) that deviates from the canonical PDZ conformation. Binding of Cdc42 or peptide induces conversion to the high-affinity form (orange). Once one ligand has bound, the other ligand binds with an enhanced affinity (by a cooperativity factor, c).
(B) The Drosophila Par-6 PDZ-VKESLV peptide complex structure. Electron density for the peptide from a 2Fo − Fc map in which the peptide was omitted from the calculation of the phases is shown.
(C) Comparison of the crystal structure of Par-6 in complex with VKESLV peptide (orange) and Cdc42-bound Par-6 and PSD-95 PDZ3 (both black).
(D) Structure of the peptide binding pocket. The Par-6 PDZ domain (orange) is shown with bound peptide (violet).
(E) Spatial and temporal Par-6 regulation model. Cdc42 is lipid modified and becomes associated with the membrane when activated, which may play a role in Par-6 localization thereby coupling membrane translocation with activity modulation.
Molecular Cell  , DOI: ( /S (04) )
Copyright © 2004 Cell Press Terms and Conditions