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Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002.

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Biochemistry. 5th edition.

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Section 34.3Kinesin and Dynein Move Along Microtubules

In addition to actin, the cytoskeleton includes other components, notably intermediate filaments and microtubules. Microtubules serve as tracks for two classes of motor proteins — namely, kinesins and dyneins. Kinesins moving along microtubules usually carry cargo such as organelles and vesicles from the center of a cell to its periphery. Dyneins are important in sliding microtubules relative to one other during the beating of cilia and flagella on the surfaces of some eukaryotic cells.

Image caduceus.jpg Some members of the kinesin family are crucial to the transport of organelles and other cargo to nerve endings at the periphery of neurons. It is not surprising, then, that mutations in these kinesins can lead to nervous system disorders. For example, mutations in a kinesin called KIF1Bβ can lead to the most common peripheral neuropathy (weakness and pain in the hands and feet), Charcot-Marie-Tooth disease, which affects 1 in 2500 people. A glutamine-to-leucine mutation in the P-loop of the motor domain of this kinesin has been found in some affected persons. Knockout mice with a disruption of the orthologous gene have been generated. Mice heterozygous for the disruption show symptoms similar to those observed in human beings; homozygotes die shortly after birth. Mutations in other kinesin genes have been tentatively linked to a predisposition to schizophrenia. In these disorders, defects in kinesin-linked transport may impair nerve function directly, and the decrease in the activity of specific neurons may lead to other degenerative processes.

34.3.1. Microtubules Are Hollow Cylindrical Polymers

Microtubules are built from two kinds of homologous 50-kd subunits, α- and β-tubulin, which assemble in an helical array of alternating tubulin types to form the wall of a hollow cylinder (Figure 34.21). Alternatively, a microtubule can be regarded as 13 protofilaments that run parallel to its long axis. The outer diameter of a microtubule is 30 nm, much larger than that of actin (5 nm). Like actin, microtubules are polar structures. One end, termed the minus end, is anchored near the center of a cell, whereas the plus end extends toward the cell surface.

Figure 34.21. Microtubule Structure.

Figure 34.21

Microtubule Structure. Schematic views of the helical structure of a microtubule. α-tubulin is shown in dark red and β-tubulin in light red. (A) Top view. (B) Side view.

Microtubules are also key components of cilia and flagella present on some eukaryotic cells. For example, sperm propel themselves through the motion of flagella containing microtubules. The microtubules present in these structures adopt a common architecture (Figure 34.22). A bundle of microtubules called an axoneme is surrounded by a membrane contiguous with the plasma membrane. The axoneme is composed of a peripheral group of nine microtubule pairs surrounding two singlet microtubules. This recurring motif is often called a 9 + 2 array. Dynein drives the motion of one member of each outer pair relative to the other, causing the overall structure to bend.

Figure 34.22. Microtubule Arrangement.

Figure 34.22

Microtubule Arrangement. Electron micrograph of a cross section of a flagellar axoneme shows nine microtubule doublets surrounding two singlets. [Courtesy of Dr. Joel Rosenbaum.]

Microtubules are important in determining the shapes of cells and in separating daughter chromosomes in mitosis. They are highly dynamic structures that grow through the addition of α- and β-tubulin to the ends of existing structures. Like actin, tubulins also bind and hydrolyze nucleoside triphosphates, although for tubulin the nucleotide is GTP rather than ATP. The critical concentration for the polymerization of the GTP forms of tubulin is lower than that for the GDP forms. Thus, a newly formed microtubule consists primarily of GTP-tubulins. Through time, the GTP is hydrolyzed to GDP. The GDP-tubulin subunits in the interior length of a microtubule remain stably polymerized, whereas GDP subunits exposed at an end have a strong tendency to dissociate. Marc Kirschner and Tim Mitchison found that some microtubules in a population lengthen while others simultaneously shorten. This property, called dynamic instability, arises from random fluctuations in the number of GTP- or GDP-tubulin subunits at the plus end of the polymer. The dynamic character of microtubules is crucial for processes such as mitosis, which require the assembly and disassembly of elaborate microtubule-based structures.

Image tree.jpg The structure of tubulin was determined at high resolution by electron crystallographic methods (Figure 34.23). As expected from their 40% sequence identity, α- and β-tubulin have very similar three-dimensional structures. Further analysis revealed that the tubulins are members of the P-loop NTPase family and contain a nucleotide-binding site adjacent to the P-loop. Tubulins are present only in eukaryotes, although a prokaryotic homolog has been found. Sequence analysis identified a prokaryotic protein called FtsZ (for filamentous temperature-sensitive mutant Z) that is quite similar to the tubulins. The homology was confirmed when the structure was determined by x-ray crystallography. Interestingly, this protein participates in bacterial cell division, forming ring-shaped structures at the constriction that arises when a cell divides. These observations suggest that tubulins may have evolved from an ancient cell-division protein.

Figure 34.23. Tubulin.

Figure 34.23

Tubulin. Image mouse.jpg Microtubules can be viewed as an assembly of α-tubulin - β-tubulin dimers. The structures of α-tubulin and β-tubulin are quite similar; each includes a P-loop NTPase domain (purple shading) and a bound guanine (more...)

Image caduceus.jpg The continual lengthening and shortening of microtubules is essential to their role in cell division. Taxol, a compound isolated from the bark of the Pacific yew tree, was discovered through its ability to interfere with cell proliferation. Taxol binds to microtubules and stabilizes the polymerized form.

Image ch34fu3.jpg

Taxol and its derivatives have been developed as anticancer agents because they preferentially affect rapidly dividing cells, such as those in tumors.

34.3.2. Kinesin Motion Is Highly Processive

Kinesins are motor proteins that move along microtubules. We have seen that myosin moves along actin filaments by a process in which actin is released in each cycle; a myosin head group acting independently dissociates from actin after every power stroke. In contrast, when a kinesin molecule moves along a microtubule, the two head groups of the kinesin molecule operate in tandem — one binds, and then the next one does. A kinesin molecule may take many steps before both heads groups are dissociated at the same time. In other words, the motion of kinesin is highly processive. Single-molecule measurements allow processive motion to be observed (Figure 34.24). A single kinesin molecule will typically take 100 or more steps toward the plus end of a microtubule in a period of seconds before the molecule becomes detached from the microtubule. These measurements also revealed that the average step size is approximately 80 Å, a value that corresponds to the distance between consecutive α- or β-tubulin subunits along each protofilament.

Figure 34.24. Monitoring Movements Mediated by Kinesin.

Figure 34.24

Monitoring Movements Mediated by Kinesin. (A) The movement of beads or vesicles, carried by individual kinesin dimers along a microtubule, can be directly observed. (B) A trace shows the displacement of a bead carried by a kinesin molecule. Multiple steps (more...)

An additional fact is crucial to the development of a mechanism for kinesin motion — namely, that the addition of ATP strongly increases the affinity of kinesin for microtubules. This behavior stands in contrast with the behavior of myosin; ATP binding to myosin promotes its dissociation from actin. Do these differences imply that kinesin and myosin operate by completely different mechanisms? Indeed not. Kinesin-generated movement appears to proceed by a mechanism that is quite similar to that used by myosin (Figure 34.25). Let us begin with a two-headed kinesin molecule in its ADP form, dissociated from a microtubule. Recall that the neck linker binds the head domain when ATP is bound and is released when ADP is bound. The initial interaction of one of the head domains with a tubulin dimer on a microtubule stimulates the release of ADP from this head domain and the subsequent binding of ATP. The binding of ATP triggers a conformational change in the head domain that leads to two important events. First, the affinity of the head domain for the microtubule increases, essentially locking this head domain in place. Second, the neck linker binds to the head domain. This change repositions the other head domain acting through the coiled-coil domain that connects the two kinesin monomers. In its new position, the second head domain is close to a second tubulin dimer, 80 Å along the microtubule in the direction of the plus end. Meanwhile, the intrinsic ATPase activity of the first head domain hydrolyzes the ATP to ADP and Pi. When the second head domain binds to the microtubule, the first head releases ADP and binds ATP. Again, ATP binding favors a conformational change that pulls the first domain forward. This process can continue for many cycles until, by chance, both head domains are in the ADP form simultaneously and kinesin dissociates from the microtubule. Because of the relative rates of the component reactions, a simultaneous dissociation occurs approximately every 100 cycles.

Figure 34.25. Kinesin Moving Along a Microtubule.

Figure 34.25

Kinesin Moving Along a Microtubule. (1) One head of a two-headed kinesin molecule, initially with both heads in the ADP form, binds to a microtubule. (2) Release of ADP and binding of ATP results in a conformational change that locks the head to the microtubule (more...)

Kinesin hydrolyzes ATP at a rate of approximately 80 molecules per second. Thus, given the step size of 80 Å per molecule of ATP, kinesin moves along a microtubule at a speed of 6400 Å per second. This rate is considerably slower than the maximum rate for myosin, which moves relative to actin at 80,000 Å per second. Recall, however, that myosin movement depends on the independent action of hundreds of different head domains working along the same actin filament, whereas the movement of kinesin is driven by the processive action of kinesin head groups working in pairs. Muscle myosin evolved to maximize the speed of the motion, whereas kinesin functions to achieve steady, but slower, transport in one direction along a filament.

34.3.3. Small Structural Changes Can Reverse Motor Polarity

Most members of the kinesin family move toward the plus end of microtubules. However, a small number, including the protein ncd (for nonclaret disjunctional, first identified in Drosophila), move toward the minus end. From an engineering perspective, there are many ways to change the polarity of a motor. How is polarity changed in this case?

Determination of the core structure of ncd revealed great similarity to other kinesins in the mechanical parts of the motor domain, including the structures of the switch regions, the relay helix, and the parts that bind microtubules. Significantly, however, the motor domain of ncd lies near the carboxyl terminus of the protein, whereas it lies near the amino terminus of conventional kinesin. Furthermore, when a larger fragment of ncd bound to ADP was analyzed, a short region just before the motor domain was seen to form an α helix that docks against the motor domain in a position similar to that occupied by the neck linker of conventional kinesin in the ATP form (Figure 34.26). This finding suggests that ncd moves toward the minus end of a microtubule by a mechanism only slightly different from that used by conventional kinesin to move in the opposite direction. Whereas ATP binding by conventional kinesin leads to the binding of the neck linker, ATP binding by ncd releases the helical region. Its release allows the second motor domain of the ncd dimer to bind to a site on the microtubule farther toward the minus end (Figure 34.27). We see once again the economical refinement of a protein by evolution — in this case, subtle adjustments have produced an opposite mechanical result in the activity of the protein assembly.

Figure 34.26. Structure of Ncd.

Figure 34.26

Structure of Ncd. Image mouse.jpg The head domain of ncd is quite similar to that of conventional kinesin, including the presence of a P-loop NTPase domain (shaded in purple). In the ADP form of ncd (shown), the amino-terminal part of this fragment forms an α-helix (more...)

Figure 34.27. Motion in Ncd.

Figure 34.27

Motion in Ncd. The replacement of ATP for ADP releases the α-helical region from the head domain, moving the other head domain toward the minus end of the microtubule.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2002, W. H. Freeman and Company.
Bookshelf ID: NBK22572


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