Molecular engineering of a backwards−moving myosin motor

Abstract

All members of the diverse myosin superfamily have a highly conserved globular motor domain that contains the actin− and nucleotide−binding sites and produces force and movement1, 2. The light−chain−binding domain connects the motor domain to a variety of functionally specialized tail domains and amplifies small structural changes in the motor domain through rotation of a lever arm3, 4. Myosins move on polarized actin filaments either forwards to the barbed (+ ) or backwards to the pointed (− ) end5, 6. Here, we describe the engineering of an artificial backwards−moving myosin from three pre−existing molecular building blocks. These blocks are: a forward−moving class I myosin motor domain, a directional inverter formed by a four−helix bundle segment of human guanylate−binding protein−1 and an artificial lever arm formed by two −actinin repeats. Our results prove that reverse−direction movement of myosins can be achieved simply by rotating the direction of the lever arm 180°. Most myosins move towards the barbed (+ ) end of actin filaments, but recent studies have established that at least one member of the family, myosin VI, moves towards the pointed (− ) end5. The structural basis for reverse−direction movement has not been established. Two mechanisms for achieving reversal of myosin motility on the inherently polar actin filament have been suggested. On the basis of direct functional assays, electron microscopy and sequence analysis, Sweeney and co−workers proposed a model whereby reversal is achieved by rotating the lever arm in the opposite direction to conventional myosin lever arm movement5 (Fig. 1). Ikebe and co−workers, however, measured the motile properties of chimaeric constructs and proposed that the core of the motor domain is the sole determinant of directionality6