The Mechanism of Tubulin Assembly into Microtubules: Insights from Structural Studies.

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.

Destabilizing an interacting motif strengthens the association of a designed ankyrin repeat protein with tubulin.

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.

New Insights into the Coupling between Microtubule Depolymerization and ATP Hydrolysis by Kinesin-13 Protein Kif2C.

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.

Kinesin, 30 years later: Recent insights from structural studies.

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.

The structure of apo-kinesin bound to tubulin links the nucleotide cycle to movement.

Kinesin-1 is a dimeric ATP-dependent motor protein that moves towards microtubules (+) ends. This movement is driven by two conformations (docked and undocked) of the two motor domains carboxy-terminal peptides (named neck linkers), in correlation with the nucleotide bound to each motor domain. Despite extensive data on kinesin-1, the structural connection between its nucleotide cycle and movement has remained elusive, mostly because the structure of the critical tubulin-bound apo-kinesin state was unknown. Here we report the 2.2 Å structure of this complex. From its comparison with detached kinesin-ADP and tubulin-bound kinesin-ATP, we identify three kinesin motor subdomains that move rigidly along the nucleotide cycle. Our data reveal how these subdomains reorient on binding to tubulin and when ATP binds, leading respectively to ADP release and to neck linker docking. These results establish a framework for understanding the transformation of chemical energy into mechanical work by (+) end-directed kinesins.

Mechanism of Tau-promoted microtubule assembly as probed by NMR spectroscopy.

Determining the molecular mechanism of the neuronal Tau protein in the tubulin heterodimer assembly has been a challenge owing to the dynamic character of the complex and the large size of microtubules. We use here defined constructs comprising one or two tubulin heterodimers to characterize their association with a functional fragment of Tau, named TauF4. TauF4 binds with high affinities to the tubulin heterodimer complexes, but NMR spectroscopy shows that it remains highly dynamic, partly because of the interaction with the acidic C-terminal tails of the tubulin monomers. When bound to a single tubulin heterodimer, TauF4 is characterized by an overhanging peptide corresponding to the first of the four microtubule binding repeats of Tau. This peptide becomes immobilized in the complex with two longitudinally associated tubulin heterodimers. The longitudinal associations are favored by the fragment and contribute to Tau's functional role in microtubule assembly.

Biochemical and structural insights into microtubule perturbation by CopN from Chlamydia pneumoniae.

Although the actin network is commonly hijacked by pathogens, there are few reports of parasites targeting microtubules. The proposed member of the LcrE protein family from some Chlamydia species (e.g. pCopN from C. pneumoniae) binds tubulin and inhibits microtubule assembly in vitro. From the pCopN structure and its similarity with that of MxiC from Shigella, we definitively confirm CopN as the Chlamydia homolog of the LcrE family of bacterial proteins involved in the regulation of type III secretion. We have also investigated the molecular basis for the pCopN effect on microtubules. We show that pCopN delays microtubule nucleation and acts as a pure tubulin-sequestering protein at steady state. It targets the ß subunit interface involved in the tubulin longitudinal self-association in a way that inhibits nucleotide exchange. pCopN contains three repetitions of a helical motif flanked by disordered N- and C-terminal extensions. We have identified the pCopN minimal tubulin-binding region within the second and third repeats. Together with the intriguing observation that C. trachomatis CopN does not bind tubulin, our data support the notion that, in addition to the shared function of type III secretion regulation, these proteins have evolved different functions in the host cytosol. Our results provide a mechanistic framework for understanding the C. pneumoniae CopN-specific inhibition of microtubule assembly.

Structure of a kinesin-tubulin complex and implications for kinesin motility.

The typical function of kinesins is to transport cargo along microtubules. Binding of ATP to microtubule-attached motile kinesins leads to cargo displacement. To better understand the nature of the conformational changes that lead to the power stroke that moves a kinesin's load along a microtubule, we determined the X-ray structure of human kinesin-1 bound to αß-tubulin. The structure defines the mechanism of microtubule-stimulated ATP hydrolysis, which releases the kinesin motor domain from microtubules. It also reveals the structural linkages that connect the ATP nucleotide to the kinesin neck linker, a 15-amino acid segment C terminal to the catalytic core of the motor domain, to result in the power stroke. ATP binding to the microtubule-bound kinesin favors neck-linker docking. This biases the attachment of kinesin's second head in the direction of the movement, thus initiating each of the steps taken.

Structural plasticity of tubulin assembly probed by vinca-domain ligands.

Vinca-domain ligands are compounds that bind to tubulin at its inter-heterodimeric interface and favour heterogeneous protofilament-like assemblies, giving rise to helices and rings. This is the basis for their inhibition of microtubule assembly, for their antimitotic activities and for their use in anticancer chemotherapy. Ustiloxins are vinca-domain ligands with a well established total synthesis. A 2.7 Å resolution structure of ustiloxin D bound to the vinca domain embedded in the complex of two tubulins with the stathmin-like domain of RB3 (T(2)R) has been determined. This finding precisely defines the interactions of ustiloxins with tubulin and, taken together with structures of other vinca-ligand complexes, allows structure-based suggestions to be made for improved activity. These comparisons also provide a rationale for the large-scale polymorphism of the protofilament-like assemblies mediated by vinca-domain ligands based on local differences in their interactions with the two tubulin heterodimers constituting their binding site.

A designed ankyrin repeat protein selected to bind to tubulin caps the microtubule plus end.

Microtubules are cytoskeleton filaments consisting of αß-tubulin heterodimers. They switch between phases of growth and shrinkage. The underlying mechanism of this property, called dynamic instability, is not fully understood. Here, we identified a designed ankyrin repeat protein (DARPin) that interferes with microtubule assembly in a unique manner. The X-ray structure of its complex with GTP-tubulin shows that it binds to the ß-tubulin surface exposed at microtubule (+) ends. The details of the structure provide insight into the role of GTP in microtubule polymerization and the conformational state of tubulin at the very microtubule end. They show in particular that GTP facilitates the tubulin structural switch that accompanies microtubule assembly but does not trigger it in unpolymerized tubulin. Total internal reflection fluorescence microscopy revealed that the DARPin specifically blocks growth at the microtubule (+) end by a selective end-capping mechanism, ultimately favoring microtubule disassembly from that end. DARPins promise to become designable tools for the dissection of microtubule dynamic properties selective for either of their two different ends.

Design and characterization of modular scaffolds for tubulin assembly.

In cells, microtubule dynamics is regulated by stabilizing and destabilizing factors. Whereas proteins in both categories have been identified, their mechanism of action is rarely understood at the molecular level. This is due in part to the difficulties faced in structural approaches to obtain atomic models when tubulin is involved. Here, we design and characterize new stathmin-like domain (SLD) proteins that sequester tubulins in numbers different from two, the number of tubulins bound by stathmin or by the SLD of RB3, two stathmin family members that have been extensively studied. We established rules for the design of tight tubulin-SLD assemblies and applied them to complexes containing one to four tubulin heterodimers. Biochemical and structural experiments showed that the engineered SLDs behaved as expected. The new SLDs will be tools for structural studies of microtubule regulation. The larger complexes will be useful for cryo-electron microscopy, whereas crystallography or nuclear magnetic resonance will benefit from the 1:1 tubulin-SLD assembly. Finally, our results provide new insight into SLD function, suggesting that a major effect of these phosphorylatable proteins is the programmed release of sequestered tubulin for microtubule assembly at the specific cellular locations of members of the stathmin family.

Kif2C minimal functional domain has unusual nucleotide binding properties that are adapted to microtubule depolymerization.

The kinesin-13 Kif2C hydrolyzes ATP and uses the energy released to disassemble microtubules. The mechanism by which this is achieved remains elusive. Here we show that Kif2C-(sN+M), a monomeric construct consisting of the motor domain with the proximal part of the N-terminal Neck extension but devoid of its more distal, unstructured, and highly basic part, has a robust depolymerase activity. When detached from microtubules, the Kif2C-(sN+M) nucleotide-binding site is occupied by ATP at physiological concentrations of adenine nucleotides. As a consequence, Kif2C-(sN+M) starts its interaction with microtubules in that state, which differentiates kinesin-13s from motile kinesins. Moreover, in this ATP-bound conformational state, Kif2C-(sN+M) has a higher affinity for soluble tubulin compared with microtubules. We propose a mechanism in which, in the first step, the specificity of ATP-bound Kif2C for soluble tubulin causes it to stabilize a curved conformation of tubulin heterodimers at the ends of microtubules. Data from an ATPase-deficient Kif2C mutant suggest that, then, ATP hydrolysis precedes and is required for tubulin release to take place. Finally, comparison with Kif2C-Motor indicates that the binding specificity for curved tubulin and, accordingly, the microtubule depolymerase activity are conferred to the motor domain by its N-terminal Neck extension.

The determinants that govern microtubule assembly from the atomic structure of GTP-tubulin.

Tubulin alternates between a soluble curved structure and a microtubule straight conformation. GTP binding to αß-tubulin is required for microtubule assembly, but whether this triggers conversion into a straighter structure is still debated. This is due, at least in part, to the lack of structural data for GTP-tubulin before assembly. Here, we report atomic-resolution crystal structures of soluble tubulin in the GDP and GTP nucleotide states in a complex with a stathmin-like domain. The structures differ locally in the neighborhood of the nucleotide. A loop movement in GTP-bound tubulin favors its recruitment to the ends of growing microtubules and facilitates its curved-to-straight transition, but this conversion has not proceeded yet. The data therefore argue for the conformational change toward the straight structure occurring as microtubule-specific contacts are established. They also suggest a model for the way the tubulin structure is modified in relation to microtubule assembly.

Systematic identification of tubulin-interacting fragments of the microtubule-associated protein Tau leads to a highly efficient promoter of microtubule assembly.

Tau is a microtubule-associated protein that stabilizes microtubules and stimulates their assembly. Current descriptions of the tubulin-interacting regions of Tau involve microtubules as the target and result mainly from deletions of Tau domains based on sequence analysis and from NMR spectroscopy experiments. Here, instead of microtubules, we use the complex of two tubulin heterodimers with the stathmin-like domain of the RB3 protein (T(2)R) to identify interacting Tau fragments generated by limited proteolysis. We show that fragments in the proline-rich region and in the microtubule-binding repeats domain each interact on their own not only with T(2)R but also with microtubules, albeit with moderate affinity. NMR analysis of the interaction with T(2)R of constructs in these two regions leads to a fragment, composed of adjacent parts of the microtubule-binding repeat domain and of the proline-rich region, that binds tightly to stabilized microtubules. This demonstrates the synergy of the two Tau regions we identified in the Tau-microtubule interaction. Moreover, we show that this fragment, which binds to two tubulin heterodimers, stimulates efficiently microtubule assembly.

Stathmin and interfacial microtubule inhibitors recognize a naturally curved conformation of tubulin dimers.

Tubulin is able to switch between a straight microtubule-like structure and a curved structure in complex with the stathmin-like domain of the RB3 protein (T(2)RB3). GTP hydrolysis following microtubule assembly induces protofilament curvature and disassembly. The conformation of the labile tubulin heterodimers is unknown. One important question is whether free GDP-tubulin dimers are straightened by GTP binding or if GTP-tubulin is also curved and switches into a straight conformation upon assembly. We have obtained insight into the bending flexibility of tubulin by analyzing the interplay of tubulin-stathmin association with the binding of several small molecule inhibitors to the colchicine domain at the tubulin intradimer interface, combining structural and biochemical approaches. The crystal structures of T(2)RB3 complexes with the chiral R and S isomers of ethyl-5-amino-2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-yl-carbamate, show that their binding site overlaps with colchicine ring A and that both complexes have the same curvature as unliganded T(2)RB3. The binding of these ligands is incompatible with a straight tubulin structure in microtubules. Analytical ultracentrifugation and binding measurements show that tubulin-stathmin associations (T(2)RB3, T(2)Stath) and binding of ligands (R, S, TN-16, or the colchicine analogue MTC) are thermodynamically independent from one another, irrespective of tubulin being bound to GTP or GDP. The fact that the interfacial ligands bind equally well to tubulin dimers or stathmin complexes supports a bent conformation of the free tubulin dimers. It is tempting to speculate that stathmin evolved to recognize curved structures in unassembled and disassembling tubulin, thus regulating microtubule assembly.

The binding of vinca domain agents to tubulin: structural and biochemical studies.

Vinca domain ligands are small molecules that interfere with the binding of vinblastine to tubulin and inhibit microtubule assembly. Many such compounds cause isodesmic association which results in difficulties in biochemical or structural studies of their interaction with tubulin. The complex of two tubulins with the stathmin-like domain of the RB3 protein (T(2)R) is a protofilament-like short assembly that does not assemble further. This has allowed structural studies of the binding of several vinca domain ligands by X-ray crystallography as crystals of the corresponding complexes diffract to near atomic resolution. This proved that their sites are located at the interface of two tubulin molecules arranged as in a curved protofilament. These sites overlap with that of vinblastine. Structural data are generally consistent with the results of available structure-function studies, though subtle differences exist. Binding in solution to the vinca domain displayed in T(2)R is conveniently studied by fluorescence spectroscopy or by monitoring inhibition of the T(2)R GTPase activity. In addition, inhibition of nucleotide exchange allows characterization of the binding to the vinca domain moiety displayed by the beta-subunit of an isolated tubulin molecule. T(2)R is therefore a useful tool to characterize and dissect the binding of vinca domain ligands to tubulin. In addition, these studies have provided new information on the interaction of tubulin with guanine nucleotides, namely on the mechanisms of nucleotide exchange and hydrolysis.

Variations in the colchicine-binding domain provide insight into the structural switch of tubulin.

Structural changes occur in the alphabeta-tubulin heterodimer during the microtubule assembly/disassembly cycle. Their most prominent feature is a transition from a straight, microtubular structure to a curved structure. There is a broad range of small molecule compounds that disturbs the microtubule cycle, a class of which targets the colchicine-binding site and prevents microtubule assembly. This class includes compounds with very different chemical structures, and it is presently unknown whether they prevent tubulin polymerization by the same mechanism. To address this issue, we have determined the structures of tubulin complexed with a set of such ligands and show that they interfere with several of the movements of tubulin subunits structural elements upon its transition from curved to straight. We also determined the structure of tubulin unliganded at the colchicine site; this reveals that a beta-tubulin loop (termed T7) flips into this site. As with colchicine site ligands, this prevents a helix which is at the interface with alpha-tubulin from stacking onto a beta-tubulin beta sheet as in straight protofilaments. Whereas in the presence of these ligands the interference with microtubule assembly gets frozen, by flipping in and out the beta-subunit T7 loop participates in a reversible way in the resistance to straightening that opposes microtubule assembly. Our results suggest that it thereby contributes to microtubule dynamic instability.

The PN2-3 domain of centrosomal P4.1-associated protein implements a novel mechanism for tubulin sequestration.

Microtubules are cytoskeletal components involved in multiple cell functions such as mitosis, motility, or intracellular traffic. In vivo, these polymers made of alphabeta-tubulin nucleate mostly from the centrosome to establish the interphasic microtubule network or, during mitosis, the mitotic spindle. Centrosomal P4.1-associated protein (CPAP; also named CENPJ) is a centrosomal protein involved in the assembly of centrioles and important for the centrosome function. This protein contains a microtubule-destabilizing region referred to as PN2-3. Here we decrypt the microtubule destabilization activity of PN2-3 at the molecular level and show that it results from the sequestration of tubulin by PN2-3 in a non-polymerizable 1:1 complex. We also map the tubulin/PN2-3 interaction both on the PN2-3 sequence and on the tubulin surface. NMR and CD data on free PN2-3 in solution show that this is an intrinsically unstructured protein that comprises a 23-amino acid residue alpha-helix. This helix is embedded in a 76-residue region that interacts strongly with tubulin. The interference of PN2-3 with well characterized tubulin properties, namely GTPase activity, nucleotide exchange, vinblastine-induced self-assembly, and stathmin family protein binding, highlights the beta subunit surface located at the intermolecular longitudinal interface when tubulin is embedded in a microtubule as a tubulin/PN2-3 interaction area. These findings characterize the PN2-3 fragment of CPAP as a protein with an unprecedented tubulin sequestering mechanism distinct from that of stathmin family proteins.

Synthesis and biological evaluation of vinca alkaloids and phomopsin hybrids.

Ten hybrids of vinca alkaloids and phomopsin A have been synthesized by linking the octahydrophomopsin lateral chain to the tertiary amine of the cleavamine moiety of anhydrovinblastine (AVLB) and vinorelbine. These compounds have been elaborated in order to obtain original products that may interfere with both binding sites of vinblastine (VLB) and phomopsin in tubulin. Although NMR and molecular modeling studies have shown that the orientation of the added peptide chains of these hybrids is not the same as those of phomopsin A, most of them are very potent inhibitors of microtubules assembly and they present good cytotoxicity against KB cell line. These interesting biological activities may eventually be explained by the fact that their lateral chain resides in a pocket distinct from that of the phomopsin A peptide, at the interface of tubulins beta and alpha.

Structural insight into the inhibition of tubulin by vinca domain peptide ligands.

The tubulin vinca domain is the target of widely different microtubule inhibitors that interfere with the binding of vinblastine. Although all these ligands inhibit the hydrolysis of GTP, they affect nucleotide exchange to variable extents. The structures of two vinca domain antimitotic peptides--phomopsin A and soblidotin (a dolastatin 10 analogue)--bound to tubulin in a complex with a stathmin-like domain show that their sites partly overlap with that of vinblastine and extend the definition of the vinca domain. The structural data, together with the biochemical results from the ligands we studied, highlight two main contributors in nucleotide exchange: the flexibility of the tubulin subunits' arrangement at their interfaces and the residues in the carboxy-terminal part of the beta-tubulin H6-H7 loop. The structures also highlight common features of the mechanisms by which vinca domain ligands favour curved tubulin assemblies and destabilize microtubules.