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1.
J Biol Chem ; 290(30): 18721-31, 2015 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-26055718

RESUMO

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.


Assuntos
Trifosfato de Adenosina/metabolismo , Cinesinas/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Substituição de Aminoácidos/genética , Sítios de Ligação , Cristalografia por Raios X , Humanos , Hidrólise , Cinesinas/química , Cinesinas/genética , Microtúbulos/química , Microtúbulos/genética , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Serina/genética , Relação Estrutura-Atividade , Tubulina (Proteína)/química , Tubulina (Proteína)/genética , Valina/genética
2.
J Biol Chem ; 289(36): 25199-210, 2014 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-25056950

RESUMO

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.


Assuntos
Proteínas de Bactérias/metabolismo , Chlamydophila pneumoniae/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Chlamydophila pneumoniae/genética , Cristalografia por Raios X , Microtúbulos/química , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Ovinos , Espectrometria de Fluorescência , Tubulina (Proteína)/química
3.
Proc Natl Acad Sci U S A ; 109(30): 12011-6, 2012 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-22778434

RESUMO

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.


Assuntos
Repetição de Anquirina/fisiologia , Microtúbulos/metabolismo , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Repetição de Anquirina/genética , Cristalografia por Raios X , Primers do DNA/genética , Polarização de Fluorescência , Guanosina Trifosfato/metabolismo , Microscopia de Fluorescência , Engenharia de Proteínas/métodos , Xenopus
4.
J Am Chem Soc ; 136(36): 12615-23, 2014 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-25162583

RESUMO

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.


Assuntos
Microtúbulos/metabolismo , Ressonância Magnética Nuclear Biomolecular , Proteínas tau/metabolismo , Microtúbulos/química , Modelos Moleculares , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Proteínas tau/química
5.
J Biol Chem ; 287(18): 15143-53, 2012 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-22403406

RESUMO

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.


Assuntos
Trifosfato de Adenosina/metabolismo , Cinesinas/metabolismo , Microtúbulos/metabolismo , Modelos Biológicos , Multimerização Proteica , Tubulina (Proteína)/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Animais , Sítios de Ligação , Humanos , Hidrólise , Cinesinas/química , Cinesinas/genética , Microtúbulos/química , Microtúbulos/genética , Ligação Proteica , Suínos , Tubulina (Proteína)/química , Tubulina (Proteína)/genética
6.
J Biol Chem ; 287(37): 31085-94, 2012 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-22791712

RESUMO

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.


Assuntos
Microtúbulos/química , Modelos Químicos , Multimerização Proteica/fisiologia , Tubulina (Proteína)/química , Animais , Microtúbulos/genética , Microtúbulos/metabolismo , Engenharia de Proteínas , Estrutura Terciária de Proteína , Estatmina/química , Estatmina/genética , Estatmina/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
7.
J Biol Chem ; 286(38): 33358-68, 2011 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-21757739

RESUMO

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.


Assuntos
Microtúbulos/metabolismo , Fragmentos de Peptídeos/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas tau/metabolismo , Sequência de Aminoácidos , Animais , Ligação Competitiva , Cinética , Espectroscopia de Ressonância Magnética , Microtúbulos/ultraestrutura , Dados de Sequência Molecular , Prolina/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Ovinos , Proteínas tau/química
8.
Acta Crystallogr D Biol Crystallogr ; 68(Pt 8): 927-34, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22868758

RESUMO

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.


Assuntos
Tubulina (Proteína)/química , Animais , Antineoplásicos/farmacologia , Sítios de Ligação , Encéfalo/metabolismo , Dimerização , Ligantes , Microtúbulos/química , Mitose , Micotoxinas/química , Peptídeos Cíclicos/química , Ligação Proteica , Estrutura Terciária de Proteína , Ovinos , Vimblastina/química
9.
Proc Natl Acad Sci U S A ; 106(33): 13775-9, 2009 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-19666559

RESUMO

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.


Assuntos
Colchicina/química , Tubulina (Proteína)/química , Animais , Antineoplásicos/farmacologia , Encéfalo/metabolismo , Dimerização , Ligantes , Microtúbulos/metabolismo , Modelos Químicos , Conformação Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ovinos
10.
J Biol Chem ; 285(41): 31672-81, 2010 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-20675373

RESUMO

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.


Assuntos
Microtúbulos , Multimerização Proteica , Estatmina/química , Tubulina (Proteína)/química , Animais , Cristalografia por Raios X , Humanos , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Ovinos , Estatmina/agonistas , Estatmina/metabolismo , Tubulina (Proteína)/agonistas , Tubulina (Proteína)/metabolismo
11.
Nature ; 435(7041): 519-22, 2005 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-15917812

RESUMO

Vinblastine is one of several tubulin-targeting Vinca alkaloids that have been responsible for many chemotherapeutic successes since their introduction in the clinic as antitumour drugs. In contrast with the two other classes of small tubulin-binding molecules (Taxol and colchicine), the binding site of vinblastine is largely unknown and the molecular mechanism of this drug has remained elusive. Here we report the X-ray structure of vinblastine bound to tubulin in a complex with the RB3 protein stathmin-like domain (RB3-SLD). Vinblastine introduces a wedge at the interface of two tubulin molecules and thus interferes with tubulin assembly. Together with electron microscopical and biochemical data, the structure explains vinblastine-induced tubulin self-association into spiral aggregates at the expense of microtubule growth. It also shows that vinblastine and the amino-terminal part of RB3-SLD binding sites share a hydrophobic groove on the alpha-tubulin surface that is located at an intermolecular contact in microtubules. This is an attractive target for drugs designed to perturb microtubule dynamics by interfacial interference, for which tubulin seems ideally suited because of its propensity to self-associate.


Assuntos
Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Vimblastina/química , Vimblastina/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Interações Hidrofóbicas e Hidrofílicas , Cinética , Proteínas dos Microtúbulos/química , Modelos Moleculares , Fosfoproteínas/química , Estrutura Terciária de Proteína , Estatmina , Relação Estrutura-Atividade , Vimblastina/metabolismo
12.
EMBO Rep ; 9(11): 1101-6, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18787557

RESUMO

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.


Assuntos
Tubulina (Proteína)/metabolismo , Ligantes , Microtúbulos/metabolismo , Modelos Moleculares , Micotoxinas/metabolismo , Oligopeptídeos/metabolismo , Estrutura Terciária de Proteína , Tubulina (Proteína)/química , Tubulina (Proteína)/farmacologia , Vimblastina/metabolismo
13.
Nature ; 428(6979): 198-202, 2004 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-15014504

RESUMO

Microtubules are cytoskeletal polymers of tubulin involved in many cellular functions. Their dynamic instability is controlled by numerous compounds and proteins, including colchicine and stathmin family proteins. The way in which microtubule instability is regulated at the molecular level has remained elusive, mainly because of the lack of appropriate structural data. Here, we present the structure, at 3.5 A resolution, of tubulin in complex with colchicine and with the stathmin-like domain (SLD) of RB3. It shows the interaction of RB3-SLD with two tubulin heterodimers in a curved complex capped by the SLD amino-terminal domain, which prevents the incorporation of the complexed tubulin into microtubules. A comparison with the structure of tubulin in protofilaments shows changes in the subunits of tubulin as it switches from its straight conformation to a curved one. These changes correlate with the loss of lateral contacts and provide a rationale for the rapid microtubule depolymerization characteristic of dynamic instability. Moreover, the tubulin-colchicine complex sheds light on the mechanism of colchicine's activity: we show that colchicine binds at a location where it prevents curved tubulin from adopting a straight structure, which inhibits assembly.


Assuntos
Colchicina/química , Colchicina/metabolismo , Proteínas dos Microtúbulos , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/metabolismo , Fosfoproteínas/química , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Colchicina/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas , Estatmina , Moduladores de Tubulina
14.
iScience ; 23(9): 101511, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32920486

RESUMO

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.

15.
Ann N Y Acad Sci ; 1112: 67-75, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17947587

RESUMO

beta-thymosins are acknowledged G-actin sequesterers. However, in the recent years, the conserved beta-thymosins/WH2 actin-binding module, has been identified in a large number of proteins that all interact with actin and play diverse functions in cell motility. The functional evolution of the WH2 domain has been approached by a combination of structural and biochemical methods, using thymosin beta4 (Tbeta4) and Ciboulot, a 3 beta-thymosin repeat protein from Drosophila as models. Ciboulot binds actin like Tbeta4 but promotes actin assembly like profilin. The first repeat of Ciboulot (D1) has the profilin function of the whole protein. The crystal structure of Ciboulot-actin shows that the major interaction with G-actin lies in the N-terminal amphipathic helix of D1. By point mutagenesis the sequestering activity of Tbeta4 can be changed into a profilin activity. ((1)H, (15)N)-NMR studies show that the functional switch from inhibition to promotion of actin assembly is linked to a change in the dynamics of interaction of the central and C-terminal regions of the WH2 domain with subdomains 1 and 2 of G-actin. Further systematic mutagenesis studies have been performed by engineering a series of chimeras of Ciboulot and Tbeta4. Proteins displaying either profilin function or enhanced sequestering activity compared to Tbeta4 have been characterized. The results provide insight into the structural basis for the regulation of the multiple functions of the WH2 domain.


Assuntos
Actinas/metabolismo , Timosina/fisiologia , Actinas/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Endotélio Vascular/fisiologia , Epiderme/fisiologia , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Timosina/química
16.
Methods Mol Med ; 137: 235-43, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-18085233

RESUMO

Tubulin, the microtubule building-block, is the target of numerous small molecule compounds that interfere with microtubule dynamics. Several of these ligands are in clinical use as antitumor drugs. There have been numerous studies on these molecules, with two main objectives: to determine their mechanism of action and to find new compounds that would expand the arsenal available for cancer chemotherapy. Although these studies would undoubtedly benefit from structural data on tubulin, this protein has long resisted crystallization attempts. We have used stathmin-like domains (SLDs) of stathmin family proteins as a tool to crystallize tubulin and have obtained three-dimensional crystals of the tubulin:SLD complexes. As many tubulin ligands bind to these complexes, the crystals are valuable tools to study tubulin-drug interactions by X-ray crystallography. They open the way to a structure-based drug design approach.


Assuntos
Colchicina/química , Tubulina (Proteína)/química , Alcaloides de Vinca/química , Colchicina/farmacologia , Cristalização , Cristalografia por Raios X , Humanos , Ligantes , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/efeitos dos fármacos , Estrutura Terciária de Proteína , Estatmina/química , Estatmina/efeitos dos fármacos , Relação Estrutura-Atividade , Tubulina (Proteína)/efeitos dos fármacos , Alcaloides de Vinca/farmacologia
17.
Sci Rep ; 6: 28922, 2016 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-27380724

RESUMO

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.


Assuntos
Repetição de Anquirina , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Tubulina (Proteína)/química , Motivos de Aminoácidos , Anquirinas/química , Dicroísmo Circular , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Cinética , Modelos Moleculares , Mutagênese , Mutação , Ligação Proteica , Engenharia de Proteínas , Ribossomos/química , Espectrometria de Fluorescência , Ressonância de Plasmônio de Superfície
18.
Biochem J ; 378(Pt 3): 877-88, 2004 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-14670078

RESUMO

Stathmin is a ubiquitous 17 kDa cytosolic phosphoprotein proposed to play a general role in the integration and relay of intracellular signalling pathways. It is believed to regulate microtubule dynamics by sequestering tubulin in a complex made of two tubulin heterodimers per stathmin molecule (T2S complex). The other proteins of the stathmin family can also bind two tubulin heterodimers through their SLD (stathmin-like domain), but the different tubulin:SLD complexes display varying stabilities. In this study, we analysed the relative influence of three regions of SLDs on the interaction with tubulin and the mechanistic processes that lead to its sequestration. Tubulin-binding properties of fragments and chimaeras of stathmin and RB3(SLD) were studied in vitro by tubulin polymerization, size-exclusion chromatography and surface plasmon resonance assays. Our results show that the N-terminal region of SLDs favours the binding of the first tubulin heterodimer and that the second C-terminal tubulinbinding site confers the specific stability of a given tubulin:SLD complex. Our results highlight the molecular processes by which tubulin co-operatively interacts with the SLDs. This knowledge may contribute to drug development aimed at disturbing microtubules that could be used for the treatment of cancer.


Assuntos
Proteínas dos Microtúbulos , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Tubulina (Proteína)/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Substâncias Macromoleculares , Metilaminas/farmacologia , Dados de Sequência Molecular , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Fosfoproteínas/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Estatmina
19.
Protein Sci ; 24(7): 1047-56, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25975756

RESUMO

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.


Assuntos
Cinesinas/química , Cinesinas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Cinesinas/antagonistas & inibidores , Modelos Moleculares , Miosinas/metabolismo , Conformação Proteica , Tubulina (Proteína)/metabolismo
20.
Nat Commun ; 5: 5364, 2014 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-25395082

RESUMO

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.


Assuntos
Cinesinas/metabolismo , Nucleotídeos/metabolismo , Tubulina (Proteína)/metabolismo , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/fisiologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/fisiologia , Humanos , Cinesinas/fisiologia , Microtúbulos/metabolismo , Microtúbulos/fisiologia , Simulação de Acoplamento Molecular , Movimento/fisiologia , Nucleotídeos/fisiologia , Estrutura Terciária de Proteína/fisiologia , Tubulina (Proteína)/fisiologia
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