ABSTRACT
Microtubule dynamics is modulated by many cellular factors including stathmin family proteins. Vertebrate stathmins sequester two αß-tubulin heterodimers into a tight complex that cannot be incorporated in microtubules. Stathmins are regulated at the expression level during development and among tissues; they are also regulated by phosphorylation. Here, we study the dissociation kinetics of tubulin:stathmin assemblies in presence of different tubulin-binding proteins and identify a critical role of the C-terminus of the stathmin partner. Destabilizing this C-terminal region may represent an additional regulatory mechanism of the interaction with tubulin of stathmin proteins.
Subject(s)
Stathmin , Tubulin , Microtubule Proteins/analysis , Microtubule Proteins/metabolism , Microtubules/metabolism , Phosphoproteins/metabolism , Protein Binding , Stathmin/metabolism , Tubulin/metabolismABSTRACT
Synthetic ÉRep repeat proteins are engineered as Brick and Staple protein pairs that together self-assemble into helical filaments. In most cases, the filaments spontaneously form supercrystals. Here, we describe an expanded series of ÉRep Bricks designed to stabilize the interaction between consecutive Bricks, to control the length of the assembled multimers, or to alter the spatial distribution of the Staple on the filaments. The effects of these Brick modifications on the assembly, on the final filament structure and on the crystal symmetry are analyzed by biochemical methods, electron microscopy and small angle X-ray scattering. We further extend the concept of Brick/Staple protein origami by designing a new type of "Janus"-like Brick protein that is equally assembled by orthogonal staples binding its inner or outer surfaces and thus ending inside or outside the filaments. The relative roles of longitudinal and lateral associations in the assembly process are discussed. This set of results demonstrates important proofs-of-principle for engineering these remarkably versatile proteins toward nanometer-to-micron scale constructions.
Subject(s)
Cytoskeleton , Proteins , Proteins/genetics , Proteins/chemistry , Microscopy, ElectronABSTRACT
Microtubule dynamics is regulated by various cellular proteins and perturbed by small-molecule compounds. To what extent the mechanism of the former resembles that of the latter is an open question. We report here structures of tubulin bound to the PN2-3 domain of CPAP, a protein controlling the length of the centrioles. We show that an α-helix of the PN2-3 N-terminal region binds and caps the longitudinal surface of the tubulin ß subunit. Moreover, a PN2-3 N-terminal stretch lies in a ß-tubulin site also targeted by fungal and bacterial peptide-like inhibitors of the vinca domain, sharing a very similar binding mode with these compounds. Therefore, our results identify several characteristic features of cellular partners that bind to this site and highlight a structural convergence of CPAP with small-molecule inhibitors of microtubule assembly.
Subject(s)
Tubulin , Vinca , Microtubules/metabolism , Protein Binding , Tubulin/metabolism , Tubulin Modulators , Vinca/metabolismABSTRACT
Colchicine, the main active alkaloid from Colchicum autumnale L., is a potent tubulin binder and represents an interesting lead structure for the development of potential anticancer chemotherapeutics. We report on the synthesis and investigation of potentially reactive colchicinoids and their surprising biological activities. In particular, the previously undescribed colchicinoid PT-100, a B-ring contracted 6-exo-methylene colchicinoid, exhibits extraordinarily high antiproliferative and apoptosis-inducing effects on various types of cancer cell lines like acute lymphoblastic leukemia (Nalm6), acute myeloid leukemia (HL-60), Burkitt-like lymphoma (BJAB), human melanoma (MelHO), and human breast adenocarcinoma (MCF7) cells at low nanomolar concentrations. Apoptosis induction proved to be especially high in multidrug-resistant Nalm6-derived cancer cell lines, while healthy human leukocytes and hepatocytes were not affected by the concentration range studied. Furthermore, caspase-independent initiation of apoptosis via an intrinsic pathway was observed. PT-100 also shows strong synergistic effects in combination with vincristine on BJAB and Nalm6 cells. Cocrystallization of PT-100 with tubulin dimers revealed its (noncovalent) binding to the colchicine-binding site of ß-tubulin at the interface to the α-subunit. A pronounced effect of PT-100 on the cytoskeleton morphology was shown by fluorescence microscopy. While the reactivity of PT-100 as a weak Michael acceptor toward thiols was chemically proven, it remains unclear whether this contributes to the remarkable biological properties of this unusual colchicinoid.
ABSTRACT
Nucleation of microtubules (MTs) is essential for cellular activities, but its mechanism is unknown because of the difficulty involved in capturing rare stochastic events in the early stage of polymerization. Here, combining rapid flush negative stain electron microscopy (EM) and kinetic analysis, we demonstrate that the formation of straight oligomers of critical size is essential for nucleation. Both GDP and GTP tubulin form single-stranded oligomers with a broad range of curvatures, but upon nucleation, the curvature distribution of GTP oligomers is shifted to produce a minor population of straight oligomers. With tubulin having the Y222F mutation in the ß subunit, the proportion of straight oligomers increases and nucleation accelerates. Our results support a model in which GTP binding generates a minor population of straight oligomers compatible with lateral association and further growth to MTs. This study suggests that cellular factors involved in nucleation promote it via stabilization of straight oligomers.
Subject(s)
Drosophila Proteins/chemistry , Guanosine Triphosphate/chemistry , Microtubules/chemistry , Protein Multimerization , Tubulin/chemistry , Animals , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster , Guanosine Diphosphate/chemistry , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/genetics , Guanosine Triphosphate/metabolism , Microtubules/genetics , Microtubules/metabolism , Tubulin/genetics , Tubulin/metabolismABSTRACT
Motile kinesins are motor proteins that translocate along microtubules as they hydrolyze ATP. They share a conserved motor domain which harbors both ATPase and microtubule-binding activities. An ATP hydrolysis mechanism involving two water molecules has been proposed based on the structure of the kinesin-5 Eg5 bound to an ATP analog. Whether this mechanism is general in the kinesin superfamily remains uncertain. Here, we present structural snapshots of the motor domain of OSM-3 along its nucleotide cycle. OSM-3 belongs to the homodimeric kinesin-2 subfamily and is the Caenorhabditis elegans homologue of human KIF17. OSM-3 bound to ADP or devoid of a nucleotide shows features of ADP-kinesins with a docked neck linker. When bound to an ATP analog, OSM-3 adopts a conformation similar to those of several ATP-like kinesins, either isolated or bound to tubulin. Moreover, the OSM-3 nucleotide-binding site is virtually identical to that of ATP-like Eg5, demonstrating a shared ATPase mechanism. Therefore, our data extend to kinesin-2 the two-water ATP hydrolysis mechanism and further suggest that it is universal within the kinesin superfamily. PROTEIN DATABASE ENTRIES: 7A3Z, 7A40, 7A5E.
Subject(s)
Adenosine Triphosphate/metabolism , Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Kinesins/chemistry , Kinesins/metabolism , Adenosine Triphosphate/chemistry , Animals , Binding Sites , Hydrolysis , Models, Molecular , Nucleotides/metabolism , Protein Conformation , Protein DomainsABSTRACT
Microtubules are cytoskeletal components involved in pivotal eukaryotic functions such as cell division, ciliogenesis, and intracellular trafficking. They assemble from αß-tubulin heterodimers and disassemble in a process called dynamic instability, which is driven by GTP hydrolysis. Structures of the microtubule and of soluble tubulin have been determined by cryo-EM and by X-ray crystallography, respectively. Altogether, these data define the mechanism of tubulin assembly-disassembly at atomic or near-atomic level. We review here the structural changes that occur during assembly, tubulin switching from a curved conformation in solution to a straight one in the microtubule core. We also present more subtle changes associated with GTP binding, leading to tubulin activation for assembly. Finally, we show how cryo-EM and X-ray crystallography are complementary methods to characterize the interaction of tubulin with proteins involved either in intracellular transport or in microtubule dynamics regulation.
ABSTRACT
Two series of heterocyclic colchicinoids bearing ß-methylenedihydrofuran or 2H-pyran-2-one fragments were synthesized by the intramolecular Heck reaction. Methylenedihydrofuran compounds 9a and 9h were found to be the most cytotoxic among currently known colchicinoids, exhibiting outstanding antiproliferative activity on tumor cell lines in picomolar (0.01-2.1 nM) range of concentrations. Compound 9a potently and substoichiometrically inhibits microtubule formation in vitro, being an order of magnitude more active in this assay than colchicine. Derivatives 9a and 9h revealed relatively low acute toxicity in mice (LD50 ≥ 10 mg/kg i.v.). The X-Ray structure of colchicinoid 9a bound to tubulin confirmed interaction of this compound with the colchicine binding site of tubulin.
Subject(s)
Antimitotic Agents/chemistry , Antimitotic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Colchicine/analogs & derivatives , Colchicine/pharmacology , Animals , Antimitotic Agents/toxicity , Antineoplastic Agents/toxicity , Cell Line, Tumor , Cell Proliferation/drug effects , Colchicine/toxicity , Crystallography, X-Ray , Drug Screening Assays, Antitumor , Furans/chemistry , Furans/pharmacology , Furans/toxicity , Humans , Mice, Inbred BALB C , Mice, Inbred C57BL , Molecular Docking Simulation , Neoplasms/drug therapy , Neoplasms/metabolism , Tubulin/metabolism , Tubulin Modulators/chemistry , Tubulin Modulators/pharmacology , Tubulin Modulators/toxicityABSTRACT
Nucleation is one of the least understood steps of microtubule dynamics. It is a kinetically unfavorable process that is templated in the cell by the γ-tubulin ring complex or by preexisting microtubules; it also occurs in vitro from pure tubulin. Here we study the nucleation inhibition potency of natural or artificial proteins in connection with their binding mode to the longitudinal surface of α- or ß-tubulin. The structure of tubulin-bound CopN, a Chlamydia protein that delays nucleation, suggests that this protein may interfere with two protofilaments at the (+) end of a nucleus. Designed ankyrin repeat proteins that share a binding mode similar to that of CopN also impede nucleation, whereas those that target only one protofilament do not. In addition, an αRep protein predicted to target two protofilaments at the (-) end does not delay nucleation, pointing to different behaviors at both ends of the nucleus. Our results link the interference with protofilaments at the (+) end and the inhibition of nucleation.
Subject(s)
Bacterial Proteins/metabolism , Microtubules/metabolism , Tubulin/metabolism , Chlamydophila pneumoniaeABSTRACT
Tau is a microtubule-associated protein involved in the regulation of axonal microtubules in neurons. In pathological conditions, it forms fibrils that are molecular hallmarks of neurological disorders known as tauopathies. In the last 2 years, cryo-EM has given unprecedented high-resolution views of Tau in both physiological and pathological conditions. We review here these new findings and put them into the context of the knowledge about Tau before this structural breakthrough. The first structures of Tau fibrils, a molecular hallmark of Alzheimer's disease (AD), were based on fibrils from the brain of an individual with AD and, along with similar patient-derived structures, have set the gold standard for the field. Cryo-EM structures of Tau fibers in three distinct diseases, AD, Pick's disease, and chronic traumatic encephalopathy, represent the end points of Tau's molecular trajectory. We propose that the recent Tau structures may call for a re-examination of databases that link different Tau variants to various forms of dementia. We also address the question of how this structural information may link Tau's functional and pathological aspects. Because this structural information on Tau was obtained in a very short period, the new structures should be viewed in light of earlier structural observations and past and present functional data to shed additional light on Tau function and dysfunction.
Subject(s)
Alzheimer Disease/pathology , Cryoelectron Microscopy/methods , Tauopathies/pathology , tau Proteins/metabolism , tau Proteins/ultrastructure , Alzheimer Disease/metabolism , Animals , Humans , Tauopathies/metabolismABSTRACT
Microtubules are cytoskeletal filaments of eukaryotic cells made of αß-tubulin heterodimers. Structural studies of non-microtubular tubulin rely mainly on molecules that prevent its self-assembly and are used as crystallization chaperones. Here we identified artificial proteins from an αRep library that are specific to α-tubulin. Turbidity experiments indicate that these αReps impede microtubule assembly in a dose-dependent manner and total internal reflection fluorescence microscopy further shows that they specifically block growth at the microtubule (-) end. Structural data indicate that they do so by targeting the α-tubulin longitudinal surface. Interestingly, in one of the complexes studied, the α subunit is in a conformation that is intermediate between the ones most commonly observed in X-ray structures of tubulin and those seen in the microtubule, emphasizing the plasticity of tubulin. These α-tubulin-specific αReps broaden the range of tools available for the mechanistic study of microtubule dynamics and its regulation.
Subject(s)
Recombinant Fusion Proteins/pharmacology , Tubulin/chemistry , Tubulin/metabolism , Animals , Crystallography, X-Ray , Dose-Response Relationship, Drug , Humans , Microtubules/drug effects , Microtubules/metabolism , Models, Molecular , Peptide Library , Protein Conformation , Recombinant Fusion Proteins/chemistry , Repetitive Sequences, Amino AcidABSTRACT
We report the synthesis and metabolic and biological evaluation of a series of 17 novel heterocyclic derivatives of isocombretastatin-A4 (iso-CA-4) and their structure-activity relationships. Among these derivatives, the most active compound, 4f, inhibited the growth of a panel of seven cancer cell lines with an IC50 in the low nanomolar range. In addition, 4f showed interesting activity against CA-4-resistant colon-carcinoma cells and multidrug-resistant leukemia cells. It also induced G2/M cell-cycle arrest. Structural data indicated binding of 4f to the colchicine site of tubulin, likely preventing the curved-to-straight tubulin structural changes that occur during microtubule assembly. Also, 4f disrupted the blood-vessel-like assembly formed by human umbilical-vein endothelial cells in vitro, suggesting its function as a vascular-disrupting agent. An in vitro metabolism study of 4f showed its high human-microsomal stability in comparison with that of iso-CA-4. The physicochemical properties of 4f may be conducive to CNS permeability, suggesting that this compound may be a possible candidate for the treatment of glioblastoma.
Subject(s)
Carbazoles/pharmacology , Quinaldines/pharmacology , Tubulin Modulators/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Carbazoles/chemical synthesis , Carbazoles/metabolism , Cell Line, Tumor , Drug Screening Assays, Antitumor , G2 Phase Cell Cycle Checkpoints/drug effects , Human Umbilical Vein Endothelial Cells , Humans , Microsomes, Liver/metabolism , Molecular Structure , Polymerization/drug effects , Protein Binding , Quinaldines/chemical synthesis , Quinaldines/metabolism , Rats , Structure-Activity Relationship , Tubulin/metabolism , Tubulin Modulators/chemical synthesis , Tubulin Modulators/metabolismABSTRACT
Kinesin-13s are critical microtubule regulators which induce microtubule disassembly in an ATP dependent manner. To clarify their mechanism, we report here the crystal structure of a functional construct of the kinesin-13 Kif2C/MCAK in an ATP-like state and bound to the αß-tubulin heterodimer, a complex mimicking the species that dissociates from microtubule ends during catalytic disassembly. Our results picture how Kif2C stabilizes a curved tubulin conformation. The Kif2C α4-L12-α5 region undergoes a remarkable 25° rotation upon tubulin binding to target the αß-tubulin hinge. This movement leads the ß5a-ß5b motif to interact with the distal end of ß-tubulin, whereas the neck and the KVD motif, two specific elements of kinesin-13s, target the α-tubulin distal end. Taken together with the study of Kif2C mutants, our data suggest that stabilization of a curved tubulin is an important contribution to the Kif2C mechanism.Kinesin-13s are microtubule depolymerizing enzymes. Here the authors present the crystal structure of a DARPin fused construct comprising the short neck region and motor domain of kinesin-13 in complex with an αß-tubulin heterodimer, which shows that kinesin-13 functions by stabilizing a curved tubulin conformation.
Subject(s)
Kinesins/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Crystallization , Escherichia coli , Gene Expression Regulation, Enzymologic , Kinesins/chemistry , Microtubules , Mutation , Protein ConformationABSTRACT
As a major component of the cytoskeleton, microtubules consist of αß-tubulin heterodimers and have been recognized as attractive targets for cancer chemotherapy. Microtubule-stabilizing agents (MSAs) promote polymerization of tubulin and stabilize the polymer, preventing depolymerization. The molecular mechanisms by which MSAs stabilize microtubules remain elusive. Here we report a 2.05 Å crystal structure of tubulin complexed with taccalonolide AJ, a newly identified taxane-site MSA. Taccalonolide AJ covalently binds to ß-tubulin D226. On AJ binding, the M-loop undergoes a conformational shift to facilitate tubulin polymerization. In this tubulin-AJ complex, the E-site of tubulin is occupied by GTP rather than GDP. Biochemical analyses confirm that AJ inhibits the hydrolysis of the E-site GTP. Thus, we propose that the ß-tubulin E-site is locked into a GTP-preferred status by AJ binding. Our results provide experimental evidence for the connection between MSA binding and tubulin nucleotide state, and will help design new MSAs to overcome taxane resistance.
Subject(s)
Microtubules/drug effects , Steroids/chemistry , Steroids/pharmacology , Tubulin/chemistry , Crystallography, X-Ray , Guanosine Triphosphate/metabolism , Hep G2 Cells , Humans , Hydrogen Bonding , Magnetic Resonance Spectroscopy , Microtubules/metabolism , Molecular Docking Simulation , Nerve Growth Factors/metabolism , Stathmin/metabolism , Tubulin/metabolismABSTRACT
Kinesin-1 is an ATP-dependent motor protein that moves towards microtubules (+)-ends. Whereas structures of isolated ADP-kinesin and of complexes with tubulin of apo-kinesin and of ATP-like-kinesin are available, structural data on apo-kinesin-1 in the absence of tubulin are still missing, leaving the role of nucleotide release in the structural cycle unsettled. Here, we identified mutations in the kinesin nucleotide-binding P-loop motif that interfere with ADP binding. These mutations destabilize the P-loop (T87A mutant) or magnesium binding (T92V), highlighting a dual mechanism for nucleotide release. The structures of these mutants in their apo form are either isomorphous to ADP-kinesin-1 or to tubulin-bound apo-kinesin-1. Remarkably, both structures are also obtained from the nucleotide-depleted wild-type protein. Our results lead to a model in which, when detached from microtubules, apo-kinesin possibly occupies the two conformations we characterized, whereas, upon microtubule binding, ADP-kinesin converts to the tubulin-bound apo-kinesin conformation and releases ADP. This conformation is primed to bind ATP and, therefore, to run through the natural nucleotide cycle of kinesin-1.
Subject(s)
Kinesins/metabolism , Adenosine Diphosphate/chemistry , Adenosine Diphosphate/metabolism , Humans , Kinesins/chemistry , Kinesins/genetics , Microtubules/chemistry , Microtubules/metabolism , Models, Biological , Models, Molecular , Molecular Conformation , Mutation , Nucleotides/chemistry , Nucleotides/metabolism , Protein Binding , Tubulin/chemistry , Tubulin/metabolismABSTRACT
Affinity maturation by random mutagenesis and selection is an established technique to make binding molecules more suitable for applications in biomedical research, diagnostics and therapy. Here we identified an unexpected novel mechanism of affinity increase upon in vitro evolution of a tubulin-specific designed ankyrin repeat protein (DARPin). Structural analysis indicated that in the progenitor DARPin the C-terminal capping repeat (C-cap) undergoes a 25° rotation to avoid a clash with tubulin upon binding. Additionally, the C-cap appears to be involved in electrostatic repulsion with tubulin. Biochemical and structural characterizations demonstrated that the evolved mutants achieved a gain in affinity through destabilization of the C-cap, which relieves the need of a DARPin conformational change upon tubulin binding and removes unfavorable interactions in the complex. Therefore, this specific case of an order-to-disorder transition led to a 100-fold tighter complex with a subnanomolar equilibrium dissociation constant, remarkably associated with a 30% decrease of the binding surface.
Subject(s)
Ankyrin Repeat , Escherichia coli Proteins/chemistry , Escherichia coli/metabolism , Tubulin/chemistry , Amino Acid Motifs , Ankyrins/chemistry , Circular Dichroism , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Kinetics , Models, Molecular , Mutagenesis , Mutation , Protein Binding , Protein Engineering , Ribosomes/chemistry , Spectrometry, Fluorescence , Surface Plasmon ResonanceABSTRACT
In this review, we focus on what we have learned from Nuclear Magnetic Resonance (NMR) studies on the neuronal microtubule-associated protein Tau. We consider both the mechanistic details of Tau: the tubulin relationship and its aggregation process. Phosphorylation of Tau is intimately linked to both aspects. NMR spectroscopy has depicted accurate phosphorylation patterns by different kinases, and its non-destructive character has allowed functional assays with the same samples. Finally, we will discuss other post-translational modifications of Tau and its interaction with other cellular factors in relationship to its (dys)function.
Subject(s)
Nuclear Magnetic Resonance, Biomolecular , tau Proteins/chemistry , Intrinsically Disordered Proteins/chemistry , Intrinsically Disordered Proteins/metabolism , Protein Processing, Post-Translational , Tubulin/chemistry , Tubulin/metabolism , tau Proteins/metabolismABSTRACT
UNLABELLED: Microtubules are dynamic assemblies of αß-tubulin heterodimers and have been recognized as highly attractive targets for cancer chemotherapy. A broad range of agents bind to tubulin and interfere with microtubule assembly. Despite having a long history of characterization, colchicine binding site inhibitors (CBSIs) have not yet reached the commercial phase as anti-cancer drugs to date. We determined the structures of tubulin complexed with a set of structurally diverse CBSIs (lexibulin, nocodazole, plinabulin and tivantinib), among which nocodazole and tivantinib are both binary-function inhibitors targeting cancer-related kinases and microtubules simultaneously. High resolution structures revealed the detailed interactions between these ligands and tubulin. Our results showed that the binding modes of the CBSIs were different from previous docking models, highlighting the importance of crystal structure information in structure-based drug design. A real structure-based pharmacophore was proposed to rationalize key common interactions of the CBSIs at the colchicine domain. Our studies provide a solid structural basis for developing new anti-cancer agents for the colchicine binding site. DATABASE: The atomic coordinates and structure factors for tubulin complexed with lexibulin, nocodazole, plinabulin and tivantinib have been deposited in the Protein Data Bank under accession codes 5CA0, 5CA1, 5C8Y and 5CB4, respectively.
Subject(s)
Antineoplastic Agents/pharmacology , Colchicine/pharmacology , Drug Discovery , Tubulin Modulators/pharmacology , Tubulin/metabolism , Animals , Antineoplastic Agents/chemistry , Binding Sites/drug effects , Colchicine/chemistry , Diketopiperazines/chemistry , Diketopiperazines/pharmacology , Ligands , Microtubules/drug effects , Models, Molecular , Molecular Structure , Nocodazole/chemistry , Nocodazole/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Kinases/metabolism , Pyrrolidinones/chemistry , Pyrrolidinones/pharmacology , Quinolines/chemistry , Quinolines/pharmacology , Structure-Activity Relationship , Swine , Tubulin/chemistry , Tubulin Modulators/chemistryABSTRACT
Kinesin-13 proteins depolymerize microtubules in an ATP hydrolysis-dependent manner. The coupling between these two activities remains unclear. Here, we first studied the role of the kinesin-13 subfamily-specific loop 2 and of the KVD motif at the tip of this loop. Shortening the loop, the lysine/glutamate interchange and the additional Val to Ser substitution all led to Kif2C mutants with decreased microtubule-stimulated ATPase and impaired depolymerization capability. We rationalized these results based on a structural model of the Kif2C-ATP-tubulin complex derived from the recently determined structures of kinesin-1 bound to tubulin. In this model, upon microtubule binding Kif2C undergoes a conformational change governed in part by the interaction of the KVD motif with the tubulin interdimer interface. Second, we mutated to an alanine the conserved glutamate residue of the switch 2 nucleotide binding motif. This mutation blocks motile kinesins in a post-conformational change state and inhibits ATP hydrolysis. This Kif2C mutant still depolymerized microtubules and yielded complexes of one Kif2C with two tubulin heterodimers. These results demonstrate that the structural change of Kif2C-ATP upon binding to microtubule ends is sufficient for tubulin release, whereas ATP hydrolysis is not required. Overall, our data suggest that the conformation reached by kinesin-13s upon tubulin binding is similar to that of tubulin-bound, ATP-bound, motile kinesins but that this conformation is adapted to microtubule depolymerization.
Subject(s)
Adenosine Triphosphate/metabolism , Kinesins/metabolism , Microtubules/metabolism , Tubulin/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/genetics , Amino Acid Substitution/genetics , Binding Sites , Crystallography, X-Ray , Humans , Hydrolysis , Kinesins/chemistry , Kinesins/genetics , Microtubules/chemistry , Microtubules/genetics , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Serine/genetics , Structure-Activity Relationship , Tubulin/chemistry , Tubulin/genetics , Valine/geneticsABSTRACT
Motile kinesins are motor proteins that move unidirectionally along microtubules as they hydrolyze ATP. They share a conserved motor domain (head) which harbors both the ATP- and microtubule-binding activities. The kinesin that has been studied most moves toward the microtubule (+)-end by alternately advancing its two heads along a single protofilament. This kinesin is the subject of this review. Its movement is associated to alternate conformations of a peptide, the neck linker, at the C-terminal end of the motor domain. Recent progress in the understanding of its structural mechanism has been made possible by high-resolution studies, by cryo electron microscopy and X-ray crystallography, of complexes of the motor domain with its track protein, tubulin. These studies clarified the structural changes that occur as ATP binds to a nucleotide-free microtubule-bound kinesin, initiating each mechanical step. As ATP binds to a head, it triggers orientation changes in three rigid motor subdomains, leading the neck linker to dock onto the motor core, which directs the other head toward the microtubule (+)-end. The relationship between neck linker docking and the orientations of the motor subdomains also accounts for kinesin's processivity, which is remarkable as this motor protein only falls off from a microtubule after taking about a hundred steps. As tools are now available to determine high-resolution structures of motor domains complexed to their track protein, it should become possible to extend these studies to other kinesins and relate their sequence variations to their diverse properties.