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The Wiskott-Aldrich syndrome protein (WASP) and neural WASP (N-WASP) are key players in regulating actin cytoskeleton via the Arp2/3 complex. It has been widely reported that the WASP proteins are activated by Rho family small GTPase Cdc42 and that Rac1 acts through SCAR/WAVE proteins. However, a systematic study of the specificity of different GTPases for different Arp2/3 activators has not been conducted. In this study, we have expressed, purified, and characterized completely soluble, highly active, and autoinhibited full-length human WASP and N-WASP from mammalian cells. We show a novel N-WASP activation by Rho family small GTPase Rac1. This GTPase exclusively stimulates N-WASP and has no effects on WASP. Rac1 is a significantly more potent N-WASP activator than Cdc42. In contrast, Cdc42 is a more effective activator of WASP than N-WASP. Lipid vesicles containing PIP2 significantly improve actin nucleation by the Arp2/3 complex and N-WASP in the presence of Rac1 or Cdc42. PIP2 vesicles have no effect on WASP activity alone. Moreover, the inhibition of WASP-stimulated actin nucleation in the presence of Cdc42 and PIP2 vesicles has been observed. We found that adaptor proteins Nck1 or Nck2 are the most potent WASP and N-WASP activators with distinct effects on the WASP family members. Our in vitro data demonstrates differential regulation of full-length WASP and N-WASP by cellular activators that highlights fundamental differences of response at the protein-protein level.

Direct inhibition of smooth muscle myosin (SMM) is a potential means to treat hypercontractile smooth muscle diseases. The selective inhibitor CK-2018571 prevents strong binding to actin and promotes muscle relaxation in vitro and in vivo. The crystal structure of the SMM/drug complex reveals that CK-2018571 binds to a novel allosteric pocket that opens up during the "recovery stroke" transition necessary to reprime the motor. Trapped in an intermediate of this fast transition, SMM is inhibited with high selectivity compared with skeletal muscle myosin (IC 50 = 9 nM and 11,300 nM, respectively), although all of the binding site residues are identical in these motors. This structure provides a starting point from which to design highly specific myosin modulators to treat several human diseases. It further illustrates the potential of targeting transition intermediates of molecular machines to develop exquisitely selective pharmacological agents.M yosins comprise a family of ATP-dependent motor proteins capable of producing directed force via interaction with their track, the F-actin filament. Force production by these motors powers numerous cellular processes such as muscle contraction, intracellular transport, and cell migration and division (1). Several myosins have also been linked to genetic disorders where either gain or loss of motor function can lead to disease. These motor proteins represent promising targets for the development of drugs modulating force production in cells, tissues, and muscle (2-4). Here we report a selective, smallmolecule inhibitor of smooth muscle myosin (SMM) able to induce muscle relaxation. This mechanism of action has potential relevance for many diseases where smooth muscle contractility is central to the pathophysiology, such as asthma (5, 6) and chronic obstructive pulmonary disease (7).Smooth muscle contractility can be activated through different pathways. Existing airway smooth muscle relaxants, such as β-adrenergic agonists and muscarinic antagonists, ultimately inhibit the activity of SMM. However, they do so via specific upstream signaling pathways. Direct inhibition of SMM contractility has the advantage of relaxing contracted smooth muscle regardless of the molecular stimulus driving it. Moreover, application of SMM inhibitors to the airway provides a means of selectively modulating contractility of these tissues by delivering a high local concentration of drug. We thus set about identifying selective inhibitors of SMM that can effectively relax muscle in vivo, leading to the discovery of a highly selective, small-molecule inhibitor, CK-2018571 (CK-571).The detailed inhibitory mechanism of CK-571 was elucidated by a combination of in vitro characterization of the step in which the drug traps the motor and determination of the high-resolution structure of SMM cocrystallized with CK-571. The drug targets an intermediate state that occurs during the recovery stroke, the large conformational rearrangement that enables repriming of the motor. Blocking this critical transiti...