Kinesin tail domains are intrinsically disordered

Kinesin motor proteins transport a wide variety of molecular cargoes in a spatially and temporally regulated manner. Kinesin motor domains, which hydrolyze ATP to produce a directed mechanical force along a microtubule, are well conserved throughout the entire superfamily. Outside of the motor domains, kinesin sequences diverge along with their transport functions. The nonmotor regions, particularly the tails, respond to a wide variety of structural and molecular cues that enable kinesins to carry specific cargoes in response to particular cellular signals. Here, we demonstrate that intrinsic disorder is a common structural feature of kinesins. A bioinformatics survey of the full‐length sequences of all 43 human kinesins predicts that significant regions of intrinsically disordered residues are present in all kinesins. These regions are concentrated in the nonmotor domains, particularly in the tails and near sites for ligand binding or post‐translational modifications. In order to experimentally verify these predictions, we expressed and purified the tail domains of kinesins representing three different families (Kif5B, Kif10, and KifC3). Circular dichroism and NMR spectroscopy experiments demonstrate that the isolated tails are disordered in vitro, yet they retain their functional microtubule‐binding activity. On the basis of these results, we propose that intrinsic disorder is a common structural feature that confers functional specificity to kinesins. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

Drivers and passengers wanted! the role of kinesin-associated proteins

Members of the kinesin superfamily of proteins participate in a wide variety of cellular processes. Although much attention has been devoted to the structural and biophysical properties of the force-generating motor domain of kinesins, the factors controlling the functional specificity of each kinesin have only recently been examined. Genetic and biochemical approaches have identified two classes of proteins that associate physically with the diverse non-motor domains of kinesins. These proteins can be divided into two general classes: first, those that form tight complexes with the kinesin and are instrumental in directing the distinct function of the motor (i.e. drivers) and, second, those proteins that might transiently interact with the motor or be an integral part of the motor's cargo (i.e. passengers). Here, we discuss known kinesin-binding proteins, and how they might participate in the activity of their motor partners.     

Structural links to kinesin directionality and movement

The kinesin motor proteins generate directional movement along microtubules and are involved in many vital processes, including cell division, in eukaryotes. The kinesin superfamily is characterized by a conserved motor domain of approximately 320 residues. Dimeric constructs of N and C class kinesins, with the motor domains at opposite ends of the heavy chain, move towards microtubule plus and minus ends, respectively. Their crystal structures differ mainly in the region linking the motor domain core to the alpha-helical coiled coil dimerization domain. Chimeric kinesins show that regions outside of the motor domain core determine the direction of movement and mutations in the linker region have a strong effect on motility. Recent work on chimeras and mutants is discussed in a structural context giving insights to possible molecular mechanisms of kinesin directionality and motility.     

A novel split kinesin assay identifies motor proteins that interact with distinct vesicle populations

Identifying the kinesin motors that interact with different vesicle populations is a longstanding and challenging problem with implications for many aspects of cell biology. Here we introduce a new live-cell assay to assess kinesin-vesicle interactions and use it to identify kinesins that bind to vesicles undergoing dendrite-selective transport in cultured hippocampal neurons. We prepared a library of "split kinesins," comprising an axon-selective kinesin motor domain and a series of kinesin tail domains that can attach to their native vesicles; when the split kinesins were assembled by chemical dimerization, bound vesicles were misdirected into the axon. This method provided highly specific results, showing that three Kinesin-3 family members-KIF1A, KIF13A, and KIF13B-interacted with dendritic vesicle populations. This experimental paradigm allows a systematic approach to evaluate motor-vesicle interactions in living cells.     

Identification and phylogenetic analysis ofDictyostelium discoideumkinesin proteins

Background

Kinesins constitute a large superfamily of motor proteins in eukaryotic cells. They perform diverse tasks such as vesicle and organelle transport and chromosomal segregation in a microtubule- and ATP-dependent manner. In recent years, the genomes of a number of eukaryotic organisms have been completely sequenced. Subsequent studies revealed and classified the full set of members of the kinesin superfamily expressed by these organisms. ForDictyostelium discoideum, only five kinesin superfamily proteins (Kif's) have already been reported.

Results

Here, we report the identification of thirteen kinesin genes exploiting the information from the raw shotgun reads of theDictyostelium discoideumgenome project. A phylogenetic tree of 390 kinesin motor domain sequences was built, grouping theDictyosteliumkinesins into nine subfamilies. According to known cellular functions or strong homologies to kinesins of other organisms, four of theDictyosteliumkinesins are involved in organelle transport, six are implicated in cell division processes, two are predicted to perform multiple functions, and one kinesin may be the founder of a new subclass.

Conclusion

This analysis of theDictyosteliumgenome led to the identification of eight new kinesin motor proteins. According to an exhaustive phylogenetic comparison,Dictyosteliumcontains the same subset of kinesins that higher eukaryotes need to perform mitosis. Some of the kinesins are implicated in intracellular traffic and a small number have unpredictable functions.     

One axon,many kinesins: What's the logic?

A large number of membrane-bounded organelles, protein complexes, and mRNAs are transported along microtubules to different locations within the neuronal axon. Axonal transport in the anterograde direction is carried out by members of a superfamily of specialized motor proteins, the kinesins. All kinesins contain a conserved motor domain that hydrolyses ATP to generate movement along microtubules. Regions outside the motor domain are responsible for cargo binding and regulation of motor activity. Present in a soluble, inactive form in the cytoplasm, kinesins are activated upon cargo binding. Selective targeting of different types of kinesin motors to specific cargoes is directed by amino acid sequences situated in their variable tails. Cargo proteins with specific function at their destination, bind directly to specific kinesins for transport. Whereas most kinesins move to microtubule plus-ends, a small number of them move to microtubule minus-ends, and may participate in retrograde axonal transport. Axonal transport by kinesins has a logic: Fully assembled, multisubunit, functional complexes (e.g., ion channel complexes, signaling complexes, RNA-protein complexes) are transported to their destination by kinesin motors that interact transiently (i.e., during transport only) with one of the complexes' subunits.     

Comprehensive comparative analysis of kinesins in photosynthetic eukaryotes

Background

Kinesins, a superfamily of molecular motors, use microtubules as tracks and transport diverse cellular cargoes. All kinesins contain a highly conserved ~350 amino acid motor domain. Previous analysis of the completed genome sequence of one flowering plant (Arabidopsis) has resulted in identification of 61 kinesins. The recent completion of genome sequencing of several photosynthetic and non-photosynthetic eukaryotes that belong to divergent lineages offers a unique opportunity to conduct a comprehensive comparative analysis of kinesins in plant and non-plant systems and infer their evolutionary relationships.

Results

We used the kinesin motor domain to identify kinesins in the completed genome sequences of 19 species, including 13 newly sequenced genomes. Among the newly analyzed genomes, six represent photosynthetic eukaryotes. A total of 529 kinesins was used to perform comprehensive analysis of kinesins and to construct gene trees using the Bayesian and parsimony approaches. The previously recognized 14 families of kinesins are resolved as distinct lineages in our inferred gene tree. At least three of the 14 kinesin families are not represented in flowering plants. Chlamydomonas, a green alga that is part of the lineage that includes land plants, has at least nine of the 14 known kinesin families. Seven of ten families present in flowering plants are represented in Chlamydomonas, indicating that these families were retained in both the flowering-plant and green algae lineages.

Conclusion

The increase in the number of kinesins in flowering plants is due to vast expansion of the Kinesin-14 and Kinesin-7 families. The Kinesin-14 family, which typically contains a C-terminal motor, has many plant kinesins that have the motor domain at the N terminus, in the middle, or the C terminus. Several domains in kinesins are present exclusively either in plant or animal lineages. Addition of novel domains to kinesins in lineage-specific groups contributed to the functional diversification of kinesins. Results from our gene-tree analyses indicate that there was tremendous lineage-specific duplication and diversification of kinesins in eukaryotes. Since the functions of only a few plant kinesins are reported in the literature, this comprehensive comparative analysis will be useful in designing functional studies with photosynthetic eukaryotes.     

Importance of a flexible hinge near the motor domain in kinesin-driven motility.

Conventional kinesin is a molecular motor consisting of an N-terminal catalytic motor domain, an extended stalk and a small globular C-terminus. Whereas the structure and function of the catalytic motor domain has been investigated, little is known about the function of domains outside the globular head. A short coiled-coil region adjacent to the motor domain, termed the neck, is known to be important for dimerization and may be required for kinesin processivity. We now provide evidence that a helix-disrupting hinge region (hinge 1) that separates the neck from the first extended coiled-coil of the stalk plays an essential role in basic motor activity. A fast fungal kinesin from Syncephalastrum racemosum was used for these studies. Deletion, substitution by a coiled-coil and truncation of the hinge 1 region all reduce motor speed and uncouple ATP turnover from gliding velocity. Insertion of hinge 1 regions from two conventional kinesins, Nkin and DmKHC, fully restores motor activity, whereas insertion of putative flexible linkers of other proteins does not, suggesting that hinge 1 regions of conventional kinesins can functionally replace each other. We suggest that this region is essential for kinesin movement in its promotion of chemo-mechanical coupling of the two heads and therefore the functional motor domain should be redefined to include not only the catalytic head but also the adjacent neck and hinge 1 domains.     

The crystal structure of the minus-end-directed microtubule motor protein ncd reveals variable dimer conformations

BACKGROUND: The kinesin superfamily of microtubule-associated motor proteins are important for intracellular transport and for cell division in eukaryotes. Conventional kinesins have the motor domain at the N terminus of the heavy chain and move towards the plus end of microtubules. The ncd protein is necessary for chromosome segregation in meiosis. It belongs to a subfamily of kinesins that have the motor domain at the C terminus and move towards the minus end of microtubules. RESULTS: The crystal structure of dimeric ncd has been obtained at 2.9 A resolution from crystals with the C222(1) space group, with two independent dimers per asymmetric unit. The motor domains in these dimers are not related by crystallographic symmetry and the two ncd dimers have significantly different conformations. An alpha-helical coiled coil connects, and interacts with, the motor domains. CONCLUSIONS: The ncd protein has a very compact structure, largely due to extended interactions of the coiled coil with the head domains. Despite this, we find that the overall conformation of the ncd dimer can be rotated by as much as 10 degrees away from that of the twofold-symmetric archetypal ncd. The crystal structures of conventional kinesin and of ncd suggest a structural rationale for the reversal of the direction of movement in chimeric kinesins.     

The family-specific K-loop influences the microtubule on-rate but not the superprocessivity of kinesin-3 motors

The kinesin-3 family (KIF) is one of the largest among the kinesin superfamily and an important driver of a variety of cellular transport events. Whereas all kinesins contain the highly conserved kinesin motor domain, different families have evolved unique motor features that enable different mechanical and functional outputs. A defining feature of kinesin-3 motors is the presence of a positively charged insert, the K-loop, in loop 12 of their motor domains. However, the mechanical and functional output of the K-loop with respect to processive motility of dimeric kinesin-3 motors is unknown. We find that, surprisingly, the K-loop plays no role in generating the superprocessive motion of dimeric kinesin-3 motors (KIF1, KIF13, and KIF16). Instead, we find that the K-loop provides kinesin-3 motors with a high microtubule affinity in the motor''s ADP-bound state, a state that for other kinesins binds only weakly to the microtubule surface. A high microtubule affinity results in a high landing rate of processive kinesin-3 motors on the microtubule surface. We propose that the family-specific K-loop contributes to efficient kinesin-3 cargo transport by enhancing the initial interaction of dimeric motors with the microtubule track.     

Motoring down the highways of the cell

All eukaryotic cells contain large numbers of motor proteins (kinesins, dyneins and myosins), each of which appears to carry out a specialized force-generating function within the cell. They are known to have roles in muscle contraction, ciliary movement, organelle and vesicle transport, mitosis and cytokinesis. These motor proteins operate on different cytoskeletal filaments; myosins move along actin filaments, and kinesins and dyneins along microtubules. Recently published crystal structures of the motor domains of two members of the kinesin superfamily reveal that they share the same overall fold that is also found at the core of the larger myosin motor. This suggests that they may share a common mechanism as well as a common ancestry.     

Mechanisms of motor protein reversal

Members of the kinesin superfamily of microtubule-based motors and the myosin superfamily of actin-based motors that move 'backwards' have been identified. As the core catalytic domains of myosins and kinesins are similar in structure, this raises the intriguing questions of how direction reversal is accomplished and whether kinesins and myosins share mechanisms for switching their motors into reverse.     

Identification and characterization of a gene encoding a kinesin-like protein in Drosophila

We identified and sequenced a cDNA clone encoding a kinesin-like protein from Drosophila. The predicted product of this cDNA has a carboxy-terminal domain that is substantially similar to the motor domain of kinesin heavy chain. The amino-terminal domain is unlike that found in previously identified kinesins or kinesin-like proteins. Analyses of this new sequence suggest that the maximal motor unit in the kinesin superfamily may be as little as 350 amino acids, and that the existence of both kinesin and kinesin-like molecules must be an evolutionarily ancient feature of eukaryotes. We also tested some of the biochemical properties of the protein encoded by this cDNA and found them to be similar to those of kinesin. Finally, the clone we isolated appears to correspond to the non-claret disjunctional (ncd) gene, which when mutant causes defects in meiotic and early embryonic mitotic chromosome segregation, and whose recently determined sequence predicts a kinesin-like domain.     

Universal and unique features of kinesin motors: insights from a comparison of fungal and animal conventional kinesins.

Kinesins are microtubule motors that use the energy derived from the hydrolysis of ATP to move unidirectionally along microtubules. The founding member of this still growing superfamily is conventional kinesin, a dimeric motor that moves processively towards the plus end of microtubules. Within the family of conventional kinesins, two groups can be distinguished to date, one derived from animal species, and one originating from filamentous fungi. So far no conventional kinesin has been reported from plant cells. Fungal and animal conventional kinesins differ in several respects, both in terms of their primary sequence and their physiological properties. Thus all fungal conventional kinesins move at velocities that are 4-5 times higher than those of animal conventional kinesins, and all of them appear to lack associated light chains. Both groups of motors are characterized by a number of group-specific sequence features which are considered here with respect to their functional importance. Animal and fungal conventional kinesins also share a number of sequence characteristics which point to common principles of motor function. The overall domain organization is remarkably similar. A C-terminal sequence motif common to all kinesins, which constitutes the only region of high homology outside the motor domain, suggests common principles of cargo association in both groups of motors. Consideration of the differences of, and similarities between, fungal and animal kinesins offers novel possibilities for experimentation (e. g., by constructing chimeras) that can be expected to contribute to our understanding of motor function.     

Characterization of a novel rice kinesin O12 with a calponin homology domain

Genomic analysis predicted that the rice (Oryza sativa var. japonica) genome encodes at least 41 kinesin-like proteins including the novel kinesin O12, which is classified as a kinesin-14 family member. O12 has a calponin homology (CH) domain that is known as an actin-binding domain. In this study, we expressed the functional domains of O12 in Escherichia coli and determined its enzymatic characteristics compared with other kinesins. The microtubule-dependent ATPase activity of recombinant O12 containing the motor and CH domains was significantly reduced in the presence of actin. Interestingly, microtubule-dependent ATPase activity of the motor domain was also affected by actin in the absence of the CH domain. Our findings suggest that the motor activity of the rice plant-specific kinesin O12 may be regulated by actin.     

Origin and evolution of Kinesin-like calmodulin-binding protein

Kinesin-like calmodulin-binding protein (KCBP), a member of the Kinesin-14 family, is a C-terminal microtubule motor with three unique domains including a myosin tail homology region 4 (MyTH4), a talin-like domain, and a calmodulin-binding domain (CBD). The MyTH4 and talin-like domains (found in some myosins) are not found in other reported kinesins. A calmodulin-binding kinesin called kinesin-C (SpKinC) isolated from sea urchin (Strongylocentrotus purpuratus) is the only reported kinesin with a CBD. Analysis of the completed genomes of Homo sapiens, Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and a red alga (Cyanidioschyzon merolae 10D) did not reveal the presence of a KCBP. This prompted us to look at the origin of KCBP and its relationship to SpKinC. To address this, we isolated KCBP from a gymnosperm, Picea abies, and a green alga, Stichococcus bacillaris. In addition, database searches resulted in identification of KCBP in another green alga, Chlamydomonas reinhardtii, and several flowering plants. Gene tree analysis revealed that the motor domain of KCBPs belongs to a clade within the Kinesin-14 (C-terminal motors) family. Only land plants and green algae have a kinesin with the MyTH4 and talin-like domains of KCBP. Further, our analysis indicates that KCBP is highly conserved in green algae and land plants. SpKinC from sea urchin, which has the motor domain similar to KCBP and contains a CBD, lacks the MyTH4 and talin-like regions. Our analysis indicates that the KCBPs, SpKinC, and a subset of the kinesin-like proteins are all more closely related to one another than they are to any other kinesins, but that either KCBP gained the MyTH4 and talin-like domains or SpKinC lost them.     

Analysis of the weak interactions of ADP-Unc104 and ADP-kinesin with microtubules and their inhibition by MAP2c

Microtubule based motors like conventional kinesin (Kinesin-1) and Unc104 (Kinesin-3), and classical microtubule associated proteins (MAPs), including MAP2, are intimately involved in neurite formation and organelle transport. The processive motility of both these kinesins involves weak microtubule interactions in the ADP-bound states. Using cosedimentation assays, we have investigated these weak interactions and characterized their inhibition by MAP2c. We show that Unc104 binds microtubules with five-fold weaker affinity and two-fold higher stoichiometry compared with conventional kinesin. Unc104 and conventional kinesin binding affinities are primarily dependent on positively charged residues in the Unc104 K-loop and conventional kinesin neck coiled-coil and removal of these residues affects Unc104 and conventional kinesin differently. We observed that MAP2c acts primarily as a competitive inhibitor of Unc104 but a mixed inhibitor of conventional kinesin. Our data suggest a specific model in which MAP2c differentially interferes with each kinesin motor by inhibiting its weak attachment to the tubulin C-termini. This is reminiscent of the defects we have observed in Unc104 and kinesin mutants in which the positively charged residues in K-loop and neck coiled-coil domains were removed.     

Dissection of Kinesin's Processivity

The protein family of kinesins contains processive motor proteins that move stepwise along microtubules. This mechanism requires the precise coupling of the catalytic steps in the two heads, and their precise mechanical coordination. Here we show that these functionalities can be uncoupled in chimera of processive and non-processive kinesins. A chimera with the motor domain of Kinesin-1 and the dimerization domain of a non-processive Kinesin-3 motor behaves qualitatively as conventional kinesin and moves processively in TIRF and bead motility assays, suggesting that spatial proximity of two Kinein-1 motor domains is sufficient for processive behavior. In the reverse chimera, the non-processive motor domains are unable to step along microtubules, despite the presence of the Kinesin-1 neck coiled coil. Still, ATP-binding to one head of these chimera induces ADP-release from the partner head, a characteristic feature of alternating site catalysis. These results show that processive movement of kinesin dimers requires elements in the motor head that respond to ADP-release and induce stepping, in addition to a proper spacing of the motor heads via the neck coiled coil.     

The Translocation Selectivity of the Kinesins that Mediate Neuronal Organelle Transport

Polarized kinesin‐driven transport is crucial for development and maintenance of neuronal polarity. Kinesins are thought to recognize biochemical differences between axonal and dendritic microtubules in order to deliver their cargoes to the appropriate domain. To identify kinesins that mediate polarized transport, we prepared constitutively active versions of all the kinesins implicated in vesicle transport and expressed them in cultured hippocampal neurons. Seven kinesins translocated preferentially to axons and five translocated into both axons and dendrites. None translocated selectively to dendrites. Highly homologous members of the same subfamily displayed distinctly different translocation preferences and were differentially regulated during development. By expressing chimeric kinesins, we identified two microtubule‐binding elements within the motor domain that are important for selective translocation. We also discovered elements in the dimerization domain of kinesin‐2 motors that contribute to their selective translocation. These observations indicate that selective interactions between kinesin motor domains and microtubules can account for polarized transport to the axon, but not for selective dendritic transport.