How Does The Dimeric Cytoplasmic Dynein Processively Walk on a Microtubule?

Abstract

Cytoplasmic dynein is a two-headed molecular motor, which can take hundreds steps along a microtubule (MT). Although the mechanism of this processive motion remains poorly understood, it is generally assumed that each of the two heads alternatively produces force on MT to move forward. To elucidate the mechanism of this processive motion, we expressed the hetero-dimeric construct of dynein motor domain, in which one domain completely lost its ATP-binding activity due to the K/T mutation in the Walker A motif in its AAA1 module (P1T mutation). Our single-molecule motility assays showed that the hetero-dimer of the wild type and the P1T mutant (Wild/P1T) moved processively on MT with its velocity approximately half of that of the wild-type homo-dimer. Because one head of the Wild/P1T hetero-dimer cannot bind ATP, its processive motion suggests that the “chemical gating” is not necessarily required for the processive stepping, but some type of “mechanical gating” may be responsible for it. We then examined if the intramolecular tension through the tail domain linking the two motor domains is responsible for this “mechanical gating”. We inserted a Gly-rich flexible linker with 20 or 40 residues between the tail domain and the hetero-dimerizer to reduce the tension. Unexpectedly, the Wild/P1T hetero-dimer with the flexible linker moved processively; their run length and velocity were similar to those of the hetero-dimer without the flexible linker. These results suggest that the tension through the tail domain does not play a critical role in the processive motion. The direct interaction of the two AAA rings in the motor domain may be responsible for the “mechanical gating” to sustain alternative steps of the two motor domains on MT.