Reverse-direction movement of a molecular motor

German scientists mastermind a backwards-moving molecular motor

February 04, 2004

In a new study, which appears in the Feb. 5 issue of Nature, researchers based at Hannover Medical School and the Max Planck Institute for Medical Research in Germany 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 alpha-actinin repeats. Drs Tsiavaliaris, Fujita-Becker and Manstein’s results demonstrate that reverse-direction movement of myosins can be achieved simply by rotating the direction of the lever arm 180°.

Myosins are mechano-enzymes that contain a common motor domain, by which they convert the energy from the hydrolysis of adenosine triphosphate into movement exerted against polar actin filaments. On the basis of sequence comparisons the myosin superfamily can be divided into at least 18 classes. 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" end.

Molecular models of the artifical backwards moving myosin motor attached to F-actin. The recombinant protein consists of the myosin I motor domain (grey), the hGBP four-helix bundle (red) and the lever arm (orange). The motors are modelled in the ‘pre-power-stroke’ state attached to an actin protofilament consisting of five actin monomers (green and blue).

"The results lend support to a model that suggests that myosins and microtubule-based molecular motors of the kinesin family, which share a common fold consisting of seven beta-strands and six alpha-helices, are intrinsically plus-end directed motors. Conformational changes in the core motor domain are either amplified or amplified and redirected by the neck region in both protein classes," note the researchers.

"The work is based on a very simple idea namely that the translational movement of the tip of a lever depends on the angle of rotation and the direction in which the lever projects away from the axis of rotation. The difficult part was to rotate the direction of the lever arm in precisely the right orientation without creating sterical clashes between domains and compromising the stiffness of the domains and the joints between them," explains Dr. Manstein, who moved his laboratory from the MPI in Heidelberg to the Institute for Biophysical Chemistry in Hannover while the work was in progress.

"Our work shows beautifully how far protein design and engineering approaches have evolved. The generation of an artificial backwards moving myosin was not the result of an extensive trial and error approach but rather we had to reach only once into the toolbox of molecular building blocks to produce a protein with the predicted activity," Manstein notes.

The translational movement of the tip of a lever (black arrow) depends on the angle of rotation and the direction in which the lever projects away from the axis of rotation. Simply by attaching the lever to the opposite site of the axis of rotation, the same rotation leads to a reversal of the translational movement of the lever.

The researchers suggest that the engineering of proteins with new and well-defined properties from known building blocks derived from biologically unrelated proteins has a wide range of applications. "Currently we are combining fluorescent probe techniques with the engineered attachment of long amplifier elements to study the conformational dynamics of enzymes with high spatial and temporal resolution using near-field microscopy techniques," says Tsiavaliaris, who started working on the project during the final phase of his PhD project and is now one of the youngest professors in Germany.

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