SUMMARY Fundamental to cellular processes are directional movements driven by molecular motors. A common theme for these and other molecular machines driven by ATP is that controlled release of hydrolysis products is essential to use the chemical energy efficiently. Mechanochemical transduction by myosin motors on actin is coupled to unknown structural changes that result in the sequential release of inorganic phosphate (Pi) and MgADP. We present here a myosin structure possessing an actin-binding interface and a tunnel (back door) that creates an escape route for Pi with a minimal rotation of the myosin lever arm that drives movements. We propose that this state represents the beginning of the powerstroke on actin, and that Pi translocation from the nucleotide pocket triggered by actin binding initiates myosin force generation. This elucidates how actin initiates force generation and movement, and may represent a strategy common to many molecular machines.

Microvillus inclusion disease (MVID) is one of the most severe congenital intestinal disorders and is characterized by neonatal secretory diarrhea and the inability to absorb nutrients from the intestinal lumen. MVID is associated with patient-, family-, and ancestry-unique mutations in the MYO5B gene, encoding the actin-based motor protein myosin Vb. Here, we review the MYO5B gene and all currently known MYO5B mutations and for the first time methodologically categorize these with regard to functional protein domains and recurrence in MYO7A associated with Usher syndrome and other myosins. We also review animal models for MVID and the latest data on functional studies related to the myosin Vb protein. To congregate existing and future information on MVID geno-/phenotypes and facilitate its quick and easy sharing among clinicians and researchers, we have constructed an online MOLGENIS-based international patient registry (www.MVID-central.org). This easily accessible database currently contains detailed information of 137 MVID patients together with reported clinical/phenotypic details and 41 unique MYO5B mutations, of which several unpublished. The future expansion and prospective nature of this registry is expected to improve disease diagnosis, prognosis, and genetic counseling.

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...