RESUMEN
Recent breakthroughs in the structural biology of cytoskeletal motor proteins show that two distinct families of motors--kinesins and myosins - use a similar mechanism of conformational switching for converting small structural changes in their nucleotide-binding sites into larger movements to provide force generation and motion. This mechanism is found to be similar to that employed by G proteins, the well-known molecular switches that regulate protein-protein interactions in many biological systems.
Asunto(s)
Cinesinas/fisiología , Proteínas Motoras Moleculares/fisiología , Miosinas/fisiología , Dominio Catalítico , Predicción , Nucleótidos , Conformación ProteicaRESUMEN
Kinesin motors are specialized enzymes that use hydrolysis of ATP to generate force and movement along their cellular tracks, the microtubules. Although numerous biochemical and biophysical studies have accumulated much data that link microtubule-assisted ATP hydrolysis to kinesin motion, the structural view of kinesin movement remains unclear. This study of the monomeric kinesin motor KIF1A combines X-ray crystallography and cryo-electron microscopy, and allows analysis of force-generating conformational changes at atomic resolution. The motor is revealed in its two functionally critical states-complexed with ADP and with a non-hydrolysable analogue of ATP. The conformational change observed between the ADP-bound and the ATP-like structures of the KIF1A catalytic core is modular, extends to all kinesins and is similar to the conformational change used by myosin motors and G proteins. Docking of the ADP-bound and ATP-like crystallographic models of KIF1A into the corresponding cryo-electron microscopy maps suggests a rationale for the plus-end directional bias associated with the kinesin catalytic core.
Asunto(s)
Cinesinas/fisiología , Proteínas Motoras Moleculares , Proteínas del Tejido Nervioso/fisiología , Adenosina Difosfato/fisiología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/química , Adenosina Trifosfato/fisiología , Dominio Catalítico , Microscopía por Crioelectrón , Cristalografía por Rayos X , Cinesinas/química , Microtúbulos/fisiología , Modelos Biológicos , Modelos Moleculares , Proteínas del Tejido Nervioso/química , Conformación Proteica , Relación Estructura-ActividadRESUMEN
Atomic resolution three-dimensional structures of two oppositely directed kinesin motors - conventional kinesin and non-claret disjunctional (ncd) protein - are now available in their functional dimeric form. A detailed model of the microtubule has also been recently obtained by docking the 3.7 A structure of tubulin into a 20 A map of the microtubule. Recent structural studies of kinesin motors and their microtubule tracks are contributing to our current understanding of kinesin motor mechanisms.
Asunto(s)
Proteínas de Drosophila , Cinesinas/química , Cinesinas/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/metabolismo , Secuencia de Aminoácidos , Animales , Dimerización , Datos de Secuencia Molecular , Conformación ProteicaRESUMEN
Ncd is a microtubule minus-end directed motor of the kinesin superfamily. Previously it has been shown that ncd and kinesin motor domains share the same major binding site on microtubules. Here we report a three-dimensional EM reconstruction of negatively stained two-dimensional Zn-induced tubulin crystal sheets (Zn-sheets) decorated with the ncd motor domain at a resolution of 16 A. This work has revealed a second specific binding site for the ncd motor domain. The motor binding site on the tubulin Zn-sheets spans both alpha and beta tubulin subunits. This binding site is located at a position different from the previously identified ncd binding site on microtubules and may play a role in motor function.
Asunto(s)
Proteínas de Drosophila , Cinesinas/química , Tubulina (Proteína)/química , Animales , Sitios de Unión , Cristalografía/métodos , Drosophila , Electrones , Microscopía Electrónica , Microtúbulos/química , Modelos Moleculares , Conformación Proteica , Zinc/químicaRESUMEN
Motor proteins of the kinesin superfamily transport intracellular cargo along microtubules. Although different kinesin proteins share 30-50% amino-acid identity in their motor catalytic cores, some move to the plus end of microtubules whereas others travel in the opposite direction. Crystal structures of the catalytic cores of conventional kinesin (a plus-end-directed motor involved in organelle transport) and ncd (a minus-end-directed motor involved in chromosome segregation) are nearly identical; therefore, the structural basis for their opposite directions of movement is unknown. Here we show that the ncd 'neck' made up of 13 class-specific residues next to the superfamily-conserved catalytic core, is essential for minus-end-directed motility, as mutagenesis of these neck residues reverses the direction of ncd motion. By solving the 2.5 A structure of a functional ncd dimer, we show that the ncd neck (a coiled-coil) differs from the corresponding region in the kinesin neck (an interrupted beta-strand), although both necks interact with similar elements in the catalytic cores. The distinct neck architectures also confer different symmetries to the ncd and kinesin dimers and position these motors with appropriate directional bias on the microtubule.
Asunto(s)
Proteínas de Drosophila , Cinesinas/química , Clonación Molecular , Cristalografía por Rayos X , Dimerización , Cinesinas/genética , Cinesinas/fisiología , Modelos Moleculares , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/genética , Proteínas Motoras Moleculares/fisiología , Mutagénesis Sitio-Dirigida , Conformación ProteicaRESUMEN
Kinesin is the founding member of a superfamily of microtubule based motor proteins that perform force-generating tasks such as organelle transport and chromosome segregation. It has two identical approximately 960-amino-acid chains containing an amino-terminal globular motor domain, a central alpha-helical region that enables dimer formation through a coiled-coil, and a carboxy-terminal tail domain that binds light chains and possibly an organelle receptor. The kinesin motor domain of approximately 340 amino acids, which can produce movement in vitro, is much smaller than that of myosin (approximately 850 amino acids) and dynein (1,000 amino acids), and is the smallest known molecular motor. Here, we report the crystal structure of the human kinesin motor domain with bound ADP determined to 1.8-A resolution by X-ray crystallography. The motor consists primarily of a single alpha/beta arrowhead-shaped domain with dimensions of 70 x 45 x 45 A. Unexpectedly, it has a striking structural similarity to the core of the catalytic domain of the actin-based motor myosin. Although kinesin and myosin have virtually no amino-acid sequence++ identity, and exhibit distinct enzymatic and motile properties, our results suggest that these two classes of mechanochemical enzymes evolved from a common ancestor and share a similar force-generating strategy.
Asunto(s)
Cinesinas/química , Miosinas/química , Adenosina Difosfato/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Alineación de SecuenciaRESUMEN
Microtubule-based ATPases of the kinesin superfamily provide the motile force for many animated features of living cells. Kinesin motors differ in their direction of movement along microtubules. Kinesin and ncd, a kinesin-related motor involved in formation and maintenance of mitotic and meiotic spindles, move in opposite directions along microtubules, even though their motor domains are 40% identical in amino-acid sequence. Here we report the crystal structure of the MgADP complex of the Drosophila ncd motor domain determined to 2.5A by X-ray crystallography, and compare it to the kinesin structure. The ncd and kinesin motor domains are remarkably similar in structure, and the locations of conserved surface amino acids suggest these motors share a common microtubule-binding site. Moreover, structural and functional comparisons of ncd, kinesin, myosin and G proteins indicate that these NTPases may have a similar strategy of changing conformation between NTP and NDP states. We propose a general model for converting a common gamma-phosphate-sensing mechanism into opposite polarities of movement for kinesin and ncd.
Asunto(s)
Proteínas de Drosophila , Cinesinas/química , Proteínas de Microtúbulos/química , Adenosina Difosfato/química , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Cristalografía por Rayos X , Drosophila , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
The shapes of the motor domains of kinesin and ncd, which move in opposite directions along microtubules, have been investigated. Using proteins expressed in Escherichia coli, it was found that at high salt (> 200 mM) Drosophila ncd motor domain (R335-K700) and human kinesin motor domain (M1-E349) were both sufficiently monomeric to allow an accurate determination of their radii of gyration (Rg) and their molecular weights. The measured Rg values of the ncd and kinesin motor domains in D2O were 2.06 +/- 0.06 and 2.05 +/- 0.04 nm, respectively, and the molecular weights were consistent with those computed from the amino acid compositions. Fitting of the scattering curves to approximately 3.5 nm resolution showed that the ncd and kinesin motor domains can be described adequately by triaxial ellipsoids having half-axes of 1.42 +/- 0.38, 2.24 +/- 0.44, and 3.65 +/- 0.22 nm, and half-axes of 1.52 +/- 0.23, 2.00 +/- 0.25, and 3.73 +/- 0.10 nm, respectively. Both motor domains are described adequately as somewhat flattened prolate ellipsoids with a maximum dimension of approximately 7.5 nm. Thus, it appears that the overall shapes of these motor domains are not the major determinants of the directionality of their movement along microtubules.
Asunto(s)
Proteínas de Drosophila , Cinesinas/química , Proteínas de Microtúbulos/química , Conformación Proteica , Animales , Clonación Molecular , Drosophila/enzimología , Escherichia coli , Humanos , Cinesinas/biosíntesis , Cinesinas/aislamiento & purificación , Proteínas de Microtúbulos/biosíntesis , Proteínas de Microtúbulos/aislamiento & purificación , Peso Molecular , Neutrones , Fragmentos de Péptidos/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Dispersión de RadiaciónRESUMEN
We have synthesized 2'-deoxy-2'-iodoadenosine-5'-triphosphate (2'-IATP), a heavy-atom analog of adenosine-5'-triphosphate. This compound was made for X-ray structural studies to target the nucleotide site of ATP binding proteins. It was diffused successfully into crystals of the microtubule-based motor proteins ncd (non-claret disjunctional protein from Drosophila melanogaster) and kinesin. With ncd, the nucleotide binding site was 70% occupied and the crystals were able to diffract X-rays to 2.5 A. The iodo-analog provided a useful isomorphous derivative with overall phasing power 1.89 in the range of 25.0-2.5 A. With kinesin, 2'-IATP co-crystallized with the protein. The crystals diffracted to at least 2.8 A with a phasing power of 1.73 in the range of 20.0-5.0 A. The analog was also found to be a substrate for all of the enzymes tested, including creatine kinase, pyruvate kinase, hexokinase, and myosin, with values of Km and Vmax that were within a factor of 10 of those for ATP. The analog supported muscle contraction, relaxing fibers, and producing active tension with values not statistically different from those obtained with ATP. These results all suggest that this analog should be useful for providing a heavy-atom derivative for crystals of enzymes that bind ATP.
Asunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfato/análogos & derivados , Proteínas de Drosophila , Cinesinas/química , Proteínas de Microtúbulos/química , Nucleótidos/metabolismo , Proteínas/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/síntesis química , Adenosina Trifosfato/metabolismo , Sitios de Unión , Creatina Quinasa/metabolismo , Cristalización , Cristalografía por Rayos X , Hexoquinasa/metabolismo , Cinesinas/metabolismo , Proteínas de Microtúbulos/metabolismo , Estructura Molecular , Miosinas/metabolismo , Proteínas/metabolismo , Piruvato Quinasa/metabolismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The kinesin superfamily is a class of microtubule-based mechano-enzymes involved in intracellular transport and chromosome movements. Molecules that move towards either the plus end or the minus end of microtubules are represented within the family. The motor domains of these molecules exhibit considerable sequence homology and contain both the ATP- and microtubule-binding sites (reviewed in refs 1, 2). Here we focus on non-claret disjunctional (ncd), a minus-end-directed motor involved in chromosome segregation in meiosis and early mitosis in Drosophila. We have calculated a three-dimensional map of tubulin sheets decorated with monomeric recombinant ncd motor domains by negative-stain electron microscopy and image analysis. Comparisons with a control structure of tubulin alone reveal that each motor domain binds to the crest of a single protofilament, making extensive contacts with both the alpha and beta tubulin monomers. Binding of the motor domain results in significant conformational changes in both of the tubulin monomers.
Asunto(s)
Tubulina (Proteína)/ultraestructura , Animales , Drosophila , Procesamiento de Imagen Asistido por Computador , Conformación Proteica , Proteínas Recombinantes/ultraestructuraRESUMEN
The effects of selected ligands on the structure of the truncated heavy-chain chemomechanical motor domains of Drosophila ncd and human kinesin were compared using the technique of transient electric birefringence. The 366-amino acid C-terminal motor domain of Drosophila nonclaret disjunctional, ncd(335-700), and the 349-amino acid N-terminal motor domain of human kinesin, kinesin(349), were studied at 4 degrees C in neutral buffers with ionic strength of 100 mM to form complexes with either MgADP or MgADP.Vi. The rotational diffusion time adjusted to 20 degrees C and water, tau 20,W, for ncd(335-700).MgADP is 32.8 ns, and for ncd(335-700).MgADP.Vi is 34.8 ns, suggesting prolate ellipsoids with dimensions 9.40 x 3.77 nm and 9.73 x 3.70 nm, respectively. The specific Kerr constant, Ksp, of ncd is -1.65 x 10(-12) cm2V-2 for the MgADP complex and -1.15 x 10(-12) cm2V-2 for the MgADP.Vi complex. The large negative Ksp for a prolate protein suggests an unusual charge distribution with two long surfaces with opposite charge. The tau 20,W for kinesin(349).MgADP is longer than the corresponding ncd motor and shows a decrease with increased electric field. The kinesin(349).MgADP.Vi complex has a longer tau 20,W. The Ksp for kinesin(349) is 0.36 x 10(-12) cm2V-2 for each complex.
Asunto(s)
Proteínas de Drosophila , Cinesinas/química , Proteínas de Microtúbulos/química , Animales , Fenómenos Biofísicos , Biofisica , Birrefringencia , Difusión , Drosophila , Humanos , Técnicas In Vitro , Cinesinas/genética , Cinesinas/fisiología , Proteínas de Microtúbulos/genética , Proteínas de Microtúbulos/fisiología , Modelos Químicos , Estructura Molecular , Rotación , SolucionesRESUMEN
The motor domain of the kinesin homolog ncd has been crystallized in the presence of MgATP by the vapor diffusion method using polyethylene glycol as the precipitant. The crystals belong to the orthorhombic space group I222 with unit cell dimensions a = 127.1 A, b = 122.3 A, c = 68.0 A, and there is one ncd molecule per asymmetric unit. The crystals diffract X-ray to at least 2.3 A and are appropriate for high-resolution structure determination.