Kinesin and myosin are motor proteins that share a common structural core and bind to microtubules and actin filaments, respectively. While the actomyosin interface has been well studied, the location of the microtubule-binding site on kinesin has not been identified. Using alanine-scanning mutagenesis, we have found that microtubule-interacting kinesin residues are located in three loops that cluster in a patch on the motor surface. The critical residues are primarily positively charged, which is consistent with a primarily electrostatic interaction with the negatively charged tubulin molecule. The core of the microtubule-binding interface resides in a highly conserved loop and helix (L12/alpha5) that corresponds topologically to the major actin-binding domain of myosin. Thus, kinesin and myosin have developed distinct polymer-binding domains in a similar region with respect to their common catalytic cores.

Members of the kinesin superfamily share a similar motor catalytic domain yet move either toward the plus end (e.g., conventional kinesin) or the minus end (e.g., Ncd) of microtubules. The structural features that determine the polarity of movement have remained enigmatic. Here, we show that kinesin's catalytic domain (316 residues) in a dimeric construct (560 residues) can be replaced with the catalytic domain of Ncd and that the resultant motor moves in the kinesin direction. We also demonstrate that this chimera does not move processively over many tubulin subunits, which is similar to Ncd but differs from the highly processive motion of conventional kinesin. These findings reveal that the catalytic domain contributes to motor processivity but does not control the polarity of movement. We propose that a region adjacent to the catalytic domain serves as a mechanical transducer that determines directionality.

Abstract. To understand the interactions between the microtubule-based motor protein kinesin and intracellular components, we have expressed the kinesin heavy chain and its different domains in CV-1 monkey kidney epithelial cells and examined their distributions by immunofluorescence microscopy. For this study, we cloned and sequenced cDNAs encoding a kinesin heavy chain from a human placental library. The human kinesin heavy chain exhibits a high level of sequence identity to the previously cloned invertebrate kinesin heavy chains; homologies between the COOHterminal domain of human and invertebrate kinesins and the nonmotor domain of the Aspergillus kinesinlike protein bimC were also found. The gene encoding the human kinesin heavy chain also contains a small upstream open reading frame in a G-C rich 5' untranslated region, features that are associated with transla-