Extended Data Fig. 4: Release of Lgl from aPKC-Par6 is driven by Cdc42, Crb and ADP-ATP exchange. | Nature Structural & Molecular Biology

Extended Data Fig. 4: Release of Lgl from aPKC-Par6 is driven by Cdc42, Crb and ADP-ATP exchange.

From: Capture, mutual inhibition and release mechanism for aPKC–Par6 and its multisite polarity substrate Lgl

Extended Data Fig. 4: Release of Lgl from aPKC-Par6 is driven by Cdc42, Crb and ADP-ATP exchange.

(a) Steric clash between K162 (human Par6α numbering) of the Par6 PDZ domain in the Cdc42-induced conformation (5I7Z) shown in purple and P712 from a structural superposition of the Llgl1 internal PBM (this study) shown in blue. The clash indicates that the Cdc42-induced conformation is incompatible with a bound internal PBM. Cdc42 is coloured in green and the Par6α PDZ domain of the reported structure in salmon. (b) aPKCι release kinetics quantified from the aPKCι-Par6α-Llgl2 complex western blots as shown in Fig. 5c. Quantified from n = 4 biological replicates represented as mean ± SEM (c) Par6 release kinetics shown in Fig. 5c. Quantified from n = 4 biological replicates represented as mean ± SEM. Points fitted to a one phase exponential decay dissociation curve using GraphPad Prism (d) Schematic representation of the predicted proportions of tripartite complex with aPKCι in the apo or ADP-bound state when preloaded with ADP.Mg2+ and their respective trajectories for release. Proportions inferred from residually bound protein levels in panels b and c. (e) Time course of aPKCι-Par6 release from Llgl2 and the influence of the indicated factors without preincubation with ADP.Mg2+. Representative western blot of n = 2 biological replicates. (f) aPKCι release kinetics quantified from western blots and shown in e. Quantified from n = 2 biological replicates represented as mean ± SEM (g) Par6 release kinetics as in e. Quantified from n = 2 biological replicates represented as mean ± SEM. Points fitted to a one-phase exponential decay dissociation curve using GraphPad Prism (h) Schematic representation of the predicted proportions of complex obtained as apo or ADP-bound and their respective trajectories for release. Proportions inferred from residually bound protein levels in panels f and g. (i) Time course of complex disassembly induced by ATP.Mg2+ versus AMP-PNP.Mg2+ when pre-incubated with ADP-Mg2+. Representative western blot of n = 3 biological replicates.

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