Figure 4.
Figure 4: Crystallization and structural modelling of PA-Rac1.
a, Dark state crystal structure of PA-Rac1. Blue, LOV domain;
red, J helix;
green, Rac1. b, Interacting residues at the LOV–Rac interface
(arrow in panel a), including Trp 56. c, Mutating Cdc42 to
include the Trp involved in stabilizing the LOV2–Rac1
interaction substantially improved LOV inhibition of Cdc42. Lane
1, PA-Cdc42; linking LOV to Cdc42 using the same truncations
that produced good inhibition for Rac does not inhibit
Cdc42–PAK binding. Lane 2, PA-Cdc42–CRIB; covalently linking
the CRIB domain of PAK to PA-Cdc42 blocks PAK binding. Lane 3,
PA-Cdc42(F56W) (PA-Cdc42W); introduction of the tryptophan
substantially improves LOV inhibition of Cdc42 binding to PAK.
Lane 4, lit state mutant of PA-Cdc42(F56W) (PA-Cdc42W(I539E),
showing that Cdc42 inhibition is sensitive to the lit/dark state
of the LOV domain. Supplementary Movie 16 and Supplementary Fig.
14 demonstrate the ability of PA-Cdc42(F56W) to produce
filopodia and protrusions in living cells.
The above figure is reprinted
by permission from Macmillan Publishers Ltd:
Nature
(2009,
461,
104-108)
copyright 2009.
helix;
green, Rac1. b, Interacting residues at the LOV–Rac interface
(arrow in panel a), including Trp 56. c, Mutating Cdc42 to
include the Trp involved in stabilizing the LOV2–Rac1
interaction substantially improved LOV inhibition of Cdc42. Lane
1, PA-Cdc42; linking LOV to Cdc42 using the same truncations
that produced good inhibition for Rac does not inhibit
Cdc42–PAK binding. Lane 2, PA-Cdc42–CRIB; covalently linking
the CRIB domain of PAK to PA-Cdc42 blocks PAK binding. Lane 3,
PA-Cdc42(F56W) (PA-Cdc42W); introduction of the tryptophan
substantially improves LOV inhibition of Cdc42 binding to PAK.
Lane 4, lit state mutant of PA-Cdc42(F56W) (PA-Cdc42W(I539E),
showing that Cdc42 inhibition is sensitive to the lit/dark state
of the LOV domain. Supplementary Movie 16 and Supplementary Fig.
14 demonstrate the ability of PA-Cdc42(F56W) to produce
filopodia and protrusions in living cells.