Leading the Procession: New Insights into Kinesin Motors

Abstract

He view that proteins are, in essence, nanoscale macromolecular machines is helping to foster a new paradigm in biology, with an interdisciplinary em- phasis that incorporates strong elements of biophysics (1). Remarkable examples of cellular machinery abound, in- cluding ribosomes, polymerases, proteosomes, chapero- nins, and spliceosomes. But perhaps no more spectacular example of proteins-as-machines exists than the motor proteins, also called mechanoenzymes. These molecular assemblies drive both rotary and linear motions in virtu- ally all organisms. Rotary engines include the F 1 -F 0 ATP- ase (ATP synthase) and the bacterial flagellar motor, both powered by currents of protons. Linear motors, which in- clude members of the myosin, kinesin, and dynein super- families, are fueled by the hydrolysis of ATP. Among the linear motors, kinesin is the only mecha- noenzyme that is known to be processive—that is, it re- mains bound to its polymer substrate while undergoing multiple rounds of activity (23). Kinesin dimers, which consist of two identical heads attached to a common a -heli- cal coiled-coil stalk, are widely believed to proceed by a "hand-over-hand" mechanism, involving a strict alterna- tion of the two heads along the microtubule lattice. Three recent studies, two appearing in this issue (8, 19) and one in Biochemistry (31), shed new light on the mystery of kine- sin processivity and lend additional experimental support to the hand-over-hand model. Proceeding Processively There is overwhelming evidence for kinesin processivity. Single molecules of kinesin typically travel for distances of a micrometer along microtubules, with a constant proba- bility of dissociation per unit of distance traveled, corre- sponding to roughly 1% per molecular step (5, 28). This implies a processivity value of z 100 enzymatic turnovers catalyzed before dissociation or termination. This value compares favorably with that calculated for many nucleic acid enzymes, which move processively but are not nor- mally categorized as motor proteins, although it pales in comparison with DNA or RNA polymerase. Perhaps more impressively, single molecules of kinesin can pull tiny beads ( z 0.5 m m in diameter) against loads up to z 6 pN or more (24), all while taking steps measuring 8 nm (25). These numbers can be used to estimate an upper bound for the length of time any kinesin molecule might reason- ably spend apart from its microtubule substrate, assuming it were to unbind at all during the mechanical cycle. A load of z 3 pN (half of the stall force) can pull a dissociated bead rearwards through a distance equal to its forward stepsize, thereby precluding net progress, in z 20 m s. Any time spent unbound must be shorter than this (perhaps 0). This is one thousandth of the time required for molecules to complete their mechanical cycle, which is z 20 ms at z 400 nm/s (half the peak velocity). The effective "duty ra- tio" for a single kinesin molecule, defined as that fraction of the total cycle during which it remains tightly attached to the microtubule during the translocation process, is thus . 0.999, and possibly 1.0. Kinesin hangs on for dear life. But does kinesin move hand-over-hand? Implicit in that scenario is a kind of molecular legerdemain whereby the trailing head can't advance until the leading head is al- ready in position, and vice versa, so that strict alternation is accomplished. The first real indication for such coordi- nation came from elegant biochemical work by Hackney (7), who showed that there is a fundamental asymmetry between the two heads of a kinesin dimer molecule bound to a microtubule in the absence of ATP: one head domain was directly attached to the microtubule in a rigor-like manner and carried no bound nucleotide, while the other head appeared to be tethered and carried ADP in its cata- lytic site. Moreover, the binding of ATP to the rigor head induced ADP release by the partner head. Subsequent