There are currently at least forty-six unique protein kinase crystal structures, twenty-four of which are available in an active state. Here we examine these structures using a structural bioinformatics approach to understand how the conformation of the activation segment controls kinase activity.
Polymerization of actin filaments directed by the Arp2/3 complex supports many types of cellular movements1. However, questions remain regarding the relative contributions of Arp2/3 complex versus other mechanisms of actin filament nucleation to processes such as path finding by neuronal growth cones owing to the lack of simple methods to inhibit Arp2/3 complex reversibly in living cells. Here we describe two classes of small molecules that bind to different sites on Arp2/3 complex and inhibit its ability to nucleate actin filaments. CK-636 binds between Arp2 and Arp3 where it appears to block movement of Arp2 and Arp3 into their active conformation. CK-548 inserts into the hydrophobic core of Arp3 and alters its conformation. Both classes of compounds inhibit formation of actin filament comet tails by Listeria and podosomes by monocytes. Two inhibitors with different mechanisms of action provide a powerful approach for studying Arp2/3 complex in living cells.
Summary
Arp2/3 complex is a seven-subunit assembly that nucleates branched actin filaments. Small molecule inhibitors CK-666 and CK-869 bind to Arp2/3 complex and inhibit nucleation, but their modes of action are unknown. Here we use biochemical and structural methods to determine the mechanism of each inhibitor. Our data indicate that CK-666 stabilizes the inactive state of the complex, blocking movement of the Arp2 and Arp3 subunits into the activated filament-like (short pitch) conformation, while CK-869 binds to a serendipitous pocket on Arp3 and allosterically destabilizes the short pitch Arp3-Arp2 interface. These results provide key insights into the relationship between conformation and activity in Arp2/3 complex, and will be critical for interpreting the influence of the inhibitors on actin filament networks in vivo.
The arginine-serine (RS)-rich domain of the SR protein ASF/SF2 is phosphorylated by SR protein kinases (SRPKs) and Clk/Sty kinases. However, the mode of phosphorylation by these kinases and their coordination in the biological regulation of ASF/SF2 is unknown. Here, we report the crystal structure of an active fragment of human SRPK1 bound to a peptide derived from an SR protein. This structure led us to identify a docking motif in ASF/SF2. We find that this docking motif restricts phosphorylation of ASF/SF2 by SRPK1 to the N-terminal part of the RS domain - a property essential for its assembly into nuclear speckles. We further show that Clk/Sty causes release of ASF/SF2 from speckles by phosphorylating the C-terminal part of its RS domain. These results suggest that the docking motif of ASF/SF2 is a key regulatory element for sequential phosphorylation by SRPK1 and Clk/Sty and, thus, is essential for its subcellular localization.
Actin-related protein (Arp) 2/3 complex mediates the formation of actin filament branches during endocytosis and at the leading edge of motile cells. The pathway of branch formation is ambiguous owing to uncertainty regarding the stoichiometry and location of VCA binding sites on Arp2/3 complex. Isothermal titration calorimetry showed that the CA motif from the C terminus of fission yeast WASP (Wsp1p) bound to fission yeast and bovine Arp2/3 complex with a stoichiometry of 2 to 1 and very different affinities for the two sites (
K
d
s of 0.13 and 1.6 μM for fission yeast Arp2/3 complex). Equilibrium binding, kinetic, and cross-linking experiments showed that (
i
) CA at high-affinity site 1 inhibited Arp2/3 complex binding to actin filaments, (
ii
) low-affinity site 2 had a higher affinity for CA when Arp2/3 complex was bound to actin filaments, and (
iii
) Arp2/3 complex had a much higher affinity for free CA than VCA cross-linked to an actin monomer. Crystal structures showed the C terminus of CA bound to the low-affinity site 2 on Arp3 of bovine Arp2/3 complex. The C helix is likely to bind to the barbed end groove of Arp3 in a position for VCA to deliver the first actin subunit to the daughter filament.
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