RESUMEN
Serial X-ray crystallography allows macromolecular structure determination at both X-ray free electron lasers (XFELs) and, more recently, synchrotron sources. The time resolution for serial synchrotron crystallography experiments has been limited to millisecond timescales with monochromatic beams. The polychromatic, "pink", beam provides a more than two orders of magnitude increased photon flux and hence allows accessing much shorter timescales in diffraction experiments at synchrotron sources. Here we report the structure determination of two different protein samples by merging pink-beam diffraction patterns from many crystals, each collected with a single 100 ps X-ray pulse exposure per crystal using a setup optimized for very low scattering background. In contrast to experiments with monochromatic radiation, data from only 50 crystals were required to obtain complete datasets. The high quality of the diffraction data highlights the potential of this method for studying irreversible reactions at sub-microsecond timescales using high-brightness X-ray facilities.
Asunto(s)
Cristalografía por Rayos X/métodos , Cristalografía por Rayos X/instrumentación , Cristalografía por Rayos X/estadística & datos numéricos , Bases de Datos de Compuestos Químicos/estadística & datos numéricos , Endopeptidasa K/química , Diseño de Equipo , Modelos Moleculares , Ficocianina/química , Conformación Proteica , Electricidad Estática , Sincrotrones , Difracción de Rayos XRESUMEN
Sophisticated molecular genetic, biochemical and biophysical studies have been used to probe the molecular mechanism of actomyosin-based motility. Recent solution measurements, high-resolution structures of recombinant myosin motor domains, and lower resolution structures of the complex formed by filamentous actin and the myosin motor domain provide detailed insights into the mechanism of chemomechanical coupling in the actomyosin system. They show how small conformational changes are amplified by a lever-arm mechanism to a working stroke of several nanometres, explain the mechanism that governs the directionality of actin-based movement, and reveal a communication pathway between the nucleotide binding pocket and the actin-binding region that explains the reciprocal relationship between actin and nucleotide affinity. Here we focus on the interacting elements in the actomyosin system and the communication pathways in the myosin motor domain that respond to actin binding.
Asunto(s)
Actomiosina/fisiología , Movimiento/fisiología , Actinas/química , Actinas/metabolismo , Actomiosina/química , Actomiosina/metabolismo , Animales , Humanos , Proteínas Motoras Moleculares , Miosinas/química , Miosinas/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas RecombinantesRESUMEN
Dynamins form a family of multidomain GTPases involved in endocytosis, vesicle trafficking and maintenance of mitochondrial morphology. In contrast to the classical switch GTPases, a force-generating function has been suggested for dynamins. Here we report the 2.3 A crystal structure of the nucleotide-free and GDP-bound GTPase domain of Dictyostelium discoideum dynamin A. The GTPase domain is the most highly conserved region among dynamins. The globular structure contains the G-protein core fold, which is extended from a six-stranded beta-sheet to an eight-stranded one by a 55 amino acid insertion. This topologically unique insertion distinguishes dynamins from other subfamilies of GTP-binding proteins. An additional N-terminal helix interacts with the C-terminal helix of the GTPase domain, forming a hydrophobic groove, which could be occupied by C-terminal parts of dynamin not present in our construct. The lack of major conformational changes between the nucleotide-free and the GDP-bound state suggests that mechanochemical rearrangements in dynamin occur during GTP binding, GTP hydrolysis or phosphate release and are not linked to loss of GDP.
Asunto(s)
Dinaminas , GTP Fosfohidrolasas , GTP Fosfohidrolasas/química , Guanosina Difosfato/química , Modelos Moleculares , Estructura Terciaria de Proteína , Sitios de Unión/fisiología , Cristalografía por Rayos X , GTP Fosfohidrolasas/metabolismo , Guanosina Difosfato/metabolismo , Datos de Secuencia Molecular , Nucleótidos/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína/fisiología , Proteínas Protozoarias , Homología de Secuencia de AminoácidoRESUMEN
The giant muscle protein titin contains a unique sequence, the PEVK domain, the elastic properties of which contribute to the mechanical behavior of relaxed cardiomyocytes. Here, human N2-B-cardiac PEVK was expressed in Escherichia coli and tested-along with recombinant cardiac titin constructs containing immunoglobulin-like or fibronectin-like domains-for a possible interaction with actin filaments. In the actomyosin in vitro motility assay, only the PEVK construct inhibited actin filament sliding over myosin. The slowdown occurred in a concentration-dependent manner and was accompanied by an increase in the number of stationary actin filaments. High [Ca(2+)] reversed the PEVK effect. PEVK concentrations >/=10 microgram/mL caused actin bundling. Actin-PEVK association was found also in actin fluorescence binding assays without myosin at physiological ionic strength. In cosedimentation assays, PEVK-titin interacted weakly with actin at 0 degrees C, but more strongly at 30 degrees C, suggesting involvement of hydrophobic interactions. To probe the interaction in a more physiological environment, nonactivated cardiac myofibrils were stretched quickly, and force was measured during the subsequent hold period. The observed force decline could be fit with a three-order exponential-decay function, which revealed an initial rapid-decay component (time constant, 4 to 5 ms) making up 30% to 50% of the whole decay amplitude. The rapid, viscous decay component, but not the slower decay components, decreased greatly and immediately on actin extraction with Ca(2+)-independent gelsolin fragment, both at physiological sarcomere lengths and beyond actin-myosin overlap. Steady-state passive force dropped only after longer exposure to gelsolin. We conclude that interaction between PEVK-titin and actin occurs in the sarcomere and may cause viscous drag during diastolic stretch of cardiac myofibrils. The interaction could also oppose shortening during contraction.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas Musculares/metabolismo , Miocardio/metabolismo , Miofibrillas/metabolismo , Proteínas Quinasas/metabolismo , Secuencias de Aminoácidos/fisiología , Animales , Unión Competitiva/fisiología , Bioensayo , Pollos , Conectina , Humanos , Técnicas In Vitro , Sustancias Macromoleculares , Proteínas Musculares/genética , Contracción Miocárdica/fisiología , Unión Proteica/fisiología , Proteínas Quinasas/genética , Estructura Terciaria de Proteína/fisiología , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sarcómeros/fisiología , Estrés Mecánico , Temperatura , ViscosidadRESUMEN
Evanescent wave microscopy, also termed total internal reflection fluorescence microscopy (TIR-FM), has shed new light on important cellular processes taking place near the plasma membrane. For example, this technique can enable the direct observation of membrane fusion of synaptic vesicles and the movement of single molecules during signal transduction. There has been a recent surge in the popularity of this technique with the advent of green-fluorescent protein (GFP) as a fluorescent marker and new technical developments. These technical developments and some of the latest applications of TIR-FM are the subject of this review.
Asunto(s)
Membrana Celular/ultraestructura , Colorantes Fluorescentes , Microscopía Fluorescente/métodos , Microscopía Confocal/tendencias , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/tendencias , Espectrometría de Fluorescencia/tendenciasRESUMEN
Dictyostelium discoideum DdRacGap1 (DRG) contains both Rho-GEF and Rho-GAP domains, a feature it shares with mammalian Bcr and Abr. To elucidate the physiological role of this multifunctional protein, we characterized the enzymatic activity of recombinant DRG fragments in vitro, created DRG-null cells, and studied the function of the protein in vivo by analysing the phenotypic changes displayed by DRG-depleted cells and DRG-null cells complemented with DRG or DRG fragments. Our results show that DRG-GEF modulates F-actin dynamics and cAMP-induced F-actin formation via Rac1-dependent signalling pathways. DRG's RacE-GAP activity is required for proper cytokinesis to occur. Additionally, we provide evidence that the specificity of DRG is not limited to members of the Rho family of small GTPases. A recombinant DRG-GAP accelerates the GTP hydrolysis of RabD 30-fold in vitro and our complementation studies show that DRG-GAP activity is required for the RabD-dependent regulation of the contractile vacuole system in Dictyostelium.
Asunto(s)
Proteínas Activadoras de GTPasa/fisiología , Factores de Intercambio de Guanina Nucleótido/fisiología , Proteínas Tirosina Quinasas , Proteínas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Transducción de Señal , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Animales , Dictyostelium , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcr , Factores de Intercambio de Guanina Nucleótido RhoRESUMEN
Molecular motors move unidirectionally along polymer tracks, producing movement and force in an ATP-dependent fashion. They achieve this by amplifying small conformational changes in the nucleotide-binding region into force-generating movements of larger protein domains. We present the 2.8 A resolution crystal structure of an artificial actin-based motor. By combining the catalytic domain of myosin II with a 130 A conformational amplifier consisting of repeats 1 and 2 of alpha-actinin, we demonstrate that it is possible to genetically engineer single-polypeptide molecular motors with precisely defined lever arm lengths and specific motile properties. Furthermore, our structure shows the consequences of mutating a conserved salt bridge in the nucleotide-binding region. Disruption of this salt bridge, which is known to severely inhibit ATP hydrolysis activity, appears to interfere with formation of myosin's catalytically active 'closed' conformation. Finally, we describe the structure of alpha-actinin repeats 1 and 2 as being composed of two rigid, triple-helical bundles linked by an uninterrupted alpha-helix. This fold is very similar to the previously described structures of alpha-actinin repeats 2 and 3, and alpha-spectrin repeats 16 and 17.
Asunto(s)
Actinina/química , Proteínas Motoras Moleculares/química , Actinina/genética , Cristalografía por Rayos X , Modelos Moleculares , Conformación Proteica , Ingeniería de Proteínas/métodos , Estructura Secundaria de Proteína , Proteínas Recombinantes/químicaRESUMEN
We combined protein engineering and single molecule measurements to directly record the step size of a series of myosin constructs with shortened and elongated artificial neck domains. Our results show that the step size has a clear linear dependence on the length of the neck domain and we also established that mechanical amplification in the myosin motor is based on a rotation of the neck domain relative to the actin-bound head. For all our constructs, including those with artificial necks, the magnitude of the neck rotation concurrent with the displacement step was approximately 30 degrees. The engineered change in the step size of myosin marks a significant advance in our ability to selectively modify the functional properties of molecular motors.
Asunto(s)
Dictyostelium/química , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/metabolismo , Miosinas/química , Miosinas/metabolismo , Ingeniería de Proteínas , Animales , Dictyostelium/genética , Glicina/metabolismo , Rayos Láser , Proteínas Motoras Moleculares/genética , Miosinas/genética , Estructura Terciaria de Proteína , Conejos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , RotaciónRESUMEN
Sequence comparisons of members of the myosin superfamily show a high degree of charge conservation in a surface exposed helix (Dictyostelium discoideum myosin II heavy chain residues S510 to K546). Most myosins display a triplet of acidic residues at the equivalent positions to D. discoideummyosin II residues D530, E531, and Q532. The high degree of charge conservation suggests strong evolutionary constrain and that this region is important for myosin function. Mutations at position E531 were shown to strongly affect actin binding [Giese, K. C., and Spudich, J. A. (1997) Biochemistry 36, 8465-8473]. Here, we used steady-state and transient kinetics to characterize the enzymatic competence of mutant constructs E531Q and Q532E, and their properties were compared with those of a loop 2 mutant with a 20 amino acid insertion containing 12 positive charges (20/+12) [Furch et al. (1998) Biochemistry 37, 6317-6326], double mutant Q532E(20/+12), and the native motor domain constructs. Our results confirm that charge changes at residues 531 and 532 primarily affect actin binding with little change being communicated to the nucleotide pocket. Mutation D531Q reduces actin affinity (K(A)) 10-fold, while Q532E leads to a 5-fold increase. The observed changes in K(A)() stem almost exclusively from variations in the dissociation rate constant (k(-A)), with the introduction of a single negative charge at position 532 having the same effect on k(-A) as the introduction of 12 positive charges in the loop 2 region.
Asunto(s)
Actomiosina/química , Miosinas/química , Actinas/metabolismo , Actomiosina/metabolismo , Adenosina Trifosfato/química , Animales , Activación Enzimática/genética , Cinética , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/genética , Proteínas Motoras Moleculares/metabolismo , Subfragmentos de Miosina/genética , Subfragmentos de Miosina/metabolismo , Miosinas/genética , Miosinas/aislamiento & purificación , Miosinas/metabolismo , Mutación Puntual , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Conejos , Electricidad EstáticaRESUMEN
The thermal unfolding of Dictyostelium discoideum myosin head fragments with alterations in the actin-binding surface loop 2 was studied by differential scanning calorimetry. Lengthening of loop 2 without concomitant charge changes led to decreases in the transition temperature of not more than 1.8 degrees C. Insertions with multiple positive or negative charges had a stronger destabilizing effect and led to reductions in the thermal transition temperature of up to 3.7 degrees C. In the presence of nucleotide, most mutants displayed similar or higher transition temperatures than M765. Only constructs M765(11/+6) and M765(20/+12) with long positively charged inserts showed transition temperatures that were more than 2 degrees C below the values measured for M765 in the presence of ADP, ADP-V(i), and ADP-BeF(3). Interaction with F-actin in the presence of ADP shifted the thermal transition of M765 by 6 degrees C, from 49.1 to 55.1 degrees C. The actin-induced increase in thermal stability varied between 1.2 and 9.1 degrees C and showed a strong correlation with the mutant constructs' affinity for actin. Our results show that length and charge changes in loop 2 do not significantly affect nucleotide-induced structural changes in the myosin motor domain, but they affect structural changes that occur when the motor domain is strongly bound to actin and affect the coupling between the actin- and nucleotide-binding sites.
Asunto(s)
Actinas/metabolismo , Dictyostelium , Proteínas Motoras Moleculares/metabolismo , Miosinas/química , Miosinas/metabolismo , Pliegue de Proteína , Adenosina Difosfato/metabolismo , Adenosina Difosfato/farmacología , Animales , Sitios de Unión/efectos de los fármacos , Rastreo Diferencial de Calorimetría , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/genética , Peso Molecular , Mutagénesis Insercional/genética , Miosinas/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosfatos/metabolismo , Unión Proteica/efectos de los fármacos , Desnaturalización Proteica/efectos de los fármacos , Estructura Terciaria de Proteína/efectos de los fármacos , Electricidad Estática , Temperatura , TermodinámicaRESUMEN
The role of the interaction between actin and the secondary actin binding site of myosin (segment 565-579 of rabbit skeletal muscle myosin, referred to as loop 3 in this work) has been studied with proteolytically generated smooth and skeletal muscle myosin subfragment 1 and recombinant Dictyostelium discoideum myosin II motor domain constructs. Carbodiimide-induced cross-linking between filamentous actin and myosin loop 3 took place only with the motor domain of skeletal muscle myosin and not with those of smooth muscle or D. discoideum myosin II. Chimeric constructs of the D. discoideum myosin motor domain containing loop 3 of either human skeletal muscle or nonmuscle myosin were generated. Significant actin cross-linking to the loop 3 region was obtained only with the skeletal muscle chimera both in the rigor and in the weak binding states, i.e., in the absence and in the presence of ATP analogues. Thrombin degradation of the cross-linked products was used to confirm the cross-linking site of myosin loop 3 within the actin segment 1-28. The skeletal muscle and nonmuscle myosin chimera showed a 4-6-fold increase in their actin dissociation constant, due to a significant increase in the rate for actin dissociation (k(-)(A)) with no significant change in the rate for actin binding (k(+A)). The actin-activated ATPase activity was not affected by the substitutions in the chimeric constructs. These results suggest that actin interaction with the secondary actin binding site of myosin is specific for the loop 3 sequence of striated muscle myosin isoforms but is apparently not essential either for the formation of a high affinity actin-myosin interface or for the modulation of actomyosin ATPase activity.
Asunto(s)
Actinas/química , Actinas/metabolismo , Miosinas/química , Miosinas/metabolismo , Actomiosina/química , Actomiosina/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Catálisis , Pollos , Humanos , Hidrólisis , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/metabolismo , Músculo Esquelético/química , Músculo Liso/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Isoformas de Proteínas/metabolismo , ConejosRESUMEN
We created a Dictyostelium discoideum myosin II mutant in which the highly conserved residue Trp-501 was replaced by a tyrosine residue. The mutant myosin alone, when expressed in a Dictyostelium strain lacking the functional myosin II heavy chain gene, supported cytokinesis and multicellular development, processes which require a functional myosin in Dictyostelium. Additionally, we expressed the W501 Y mutant in the soluble myosin head fragment M761-2R (W501Y-2R) to characterise the kinetic properties of the mutant myosin motor domain. The affinity of the mutant myosin for actin was approximately 6-fold decreased, but other kinetic properties of the protein were changed less than 2-fold by the W501Y mutation. Based on spectroscopic studies and structural considerations, Trp-501, corresponding to Trp-510 in chicken fast skeletal muscle myosin, has been proposed to be the primary ATP-sensitive tryptophanyl residue. Our results confirm these conclusions. While the wild-type construct displayed a 10% fluorescence increase, addition of ATP to W501Y-2R was not followed by an increase in tryptophan fluorescence emission.
Asunto(s)
Dictyostelium/metabolismo , Miosinas/metabolismo , Triptófano/química , Tirosina/química , Sustitución de Aminoácidos , Animales , Modelos Moleculares , Miosinas/química , Espectrometría de FluorescenciaRESUMEN
The kinetic and functional consequences of deleting nine residues from an actin-binding surface loop (loop 2) were examined to investigate the role of this region in myosin function. The nucleotide binding properties of myosin were not altered by the deletion. However, the deletion affected actin binding and the communication between the actin- and nucleotide-binding sites. The affinity of M765NL for actin (644 nM) was approximately 100-fold lower than that of wild-type construct M765 (5.8 nM). Despite this reduction in affinity, actin binding weakened the affinity of ADP for the motor to a similar extent for both mutant and wild-type constructs. The addition of 0.5 microM actin decreased ADP affinity from 0.6 to 34 microM for M765NL and from 1.6 to 39 microM for M765. In contrast, communication between the actin- and nucleotide-binding sites appears disturbed in regard to phosphate release: thus, basal ATPase activity for M765NL (0.19 s-1) was 3-fold larger than for M765 (0.06 s-1), and the stimulation of ATPase activity by actin was 5-fold lower for M765NL. These results indicate different paths of communication between the actin- and nucleotide-binding sites, in regard to ADP and Pi release, and they confirm that loop 2 is involved in high affinity actin binding.
Asunto(s)
Actinas/metabolismo , Adenosina Difosfato/metabolismo , Miosinas/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Cartilla de ADN , Dictyostelium/metabolismo , Activación Enzimática , Cinética , Mutagénesis Sitio-Dirigida , Miosinas/química , Miosinas/genética , ConejosRESUMEN
Motifs N2 and N3, also referred to as switch-1 and switch-2, form part of the active site of molecular motors such as myosins and kinesins. In the case of myosin, N3 is thought to act as a gamma-phosphate sensor and moves almost 6 A relative to N2 during the catalysed turnover of ATP, opening and closing the active site surrounding the gamma-phosphate. The closed form seems to be necessary for hydrolysis and is stabilised by the formation of a salt-bridge between an arginine residue in N2 and a glutamate residue in N3. We examined the role of this salt-bridge in Dictyostelium discoideum myosin. Myosin motor domains with mutations E459R or R238E, that block salt-bridge formation, show defects in nucleotide-binding, reduced rates of ATP hydrolysis and a tenfold reduction in actin affinity. Inversion of the salt-bridge in double-mutant M765-IS eliminates most of the defects observed for the single mutants. With the exception of a 2,500-fold higher KMvalue for ATP, the double-mutant displayed enzymatic and functional properties very similar to those of the wild-type protein. Our results reveal that, independent of its orientation, the salt-bridge is required to support efficient ATP hydrolysis, normal communication between different functional regions of the myosin head, and motor function.
Asunto(s)
Dictyostelium/química , Miosinas/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Colorantes Fluorescentes , Hidrólisis , Cinética , Modelos Moleculares , Miosinas/química , Miosinas/genética , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Three conserved glycine residues in the reactive thiol region of Dictyostelium discoideummyosin II were replaced by alanine residues. The resulting mutants G680A, G684A, and G691A were expressed in the soluble myosin head fragment M761-2R [Anson, M., Geeves, M. A., Kurzawa, S. E., and Manstein, D. J. (1996) EMBO J. 15, 6069-6074] and characterized using transient kinetic methods. Mutant G691A showed no major alterations except for a marked increase in basal Mg2+-ATPase activity. Phosphate release seemed to be facilitated by this mutation, and the addition of actin to G691A stimulated ATP turnover not more than 3-fold. In comparison to M761-2R, mutant constructs G691A and G684A showed a 4-fold reduction in the rate of the ATP cleavage step. Most other changes in the kinetic properties of G684A were small ( approximately 2-fold). In contrast, substitution of G680 by an alanine residue led to large changes in nucleotide binding. Compared to M761-2R, rates of nucleotide binding were 20-30-fold slower and the affinity for mantADP was approximately 10-fold increased due to a 200-fold reduction in the dissociation rate constant of mantADP. The ATP-induced dissociation of actin from the acto.680A complex was normal, but the communication between ADP and actin binding was altered such that the two sites are thermodynamically uncoupled but kinetically actin still accelerates ADP release.
Asunto(s)
Dictyostelium/química , Miosinas/química , Compuestos de Sulfhidrilo/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Alanina/química , Animales , Dictyostelium/metabolismo , Glicina/química , Cinética , Mutación , Miosinas/genética , Miosinas/metabolismoRESUMEN
Changes in the actin-myosin interface are thought to play an important role in microfilament-linked cellular movements. In this study, we compared the actin binding properties of the motor domain of Dictyostelium discoideum (M765) and rabbit skeletal muscle myosin subfragment-1 (S1). The Dictyostelium motor domain resembles S1(A2) (S1 carrying the A2 light chain) in its interaction with G-actin. Similar to S1(A2), none of the Dictyostelium motor domain constructs induced G-actin polymerization. The affinity of monomeric actin (G-actin) was 20-fold lower for M765 than for S1(A2) but increasing the number of positive charges in the loop 2 region of the D. discoideum motor domain (residues 613-623) resulted in equivalent affinities of G-actin for M765 and for S1. Proteolytic cleavage and cross-linking approaches were used to show that M765, like S1, interacts via the loop 2 region with filamentous actin (F-actin). For both types of myosin, F-actin prevents trypsin cleavage in the loop 2 region and F-actin segment 1-28 can be cross-linked to loop 2 residues by a carbodiimide-induced reaction. In contrast with the S1, loop residues 559-565 of D. discoideum myosin was not cross-linked to F-actin, probably due to the lower number of positive charges. These results confirm the importance of the loop 2 region of myosin for the interaction with both G-actin and F-actin, regardless of the source of myosin. The differences observed in the way in which M765 and S1 interact with actin may be linked to more general differences in the structure of the actomyosin interface of muscle and nonmuscle myosins.
Asunto(s)
Actinas/metabolismo , Músculo Esquelético/metabolismo , Miosinas/metabolismo , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Dictyostelium , Activación Enzimática , Proteínas Motoras Moleculares/metabolismo , Datos de Secuencia Molecular , Conformación Proteica , Conejos , Electricidad EstáticaRESUMEN
The identification and functional characterization of Dictyostelium discoideum dynamin A, a protein composed of 853 amino acids that shares up to 44% sequence identity with other dynamin-related proteins, is described. Dynamin A is present during all stages of D. discoideum development and is found predominantly in the cytosolic fraction and in association with endosomal and postlysosomal vacuoles. Overexpression of the protein has no adverse effect on the cells, whereas depletion of dynamin A by gene-targeting techniques leads to multiple and complex phenotypic changes. Cells lacking a functional copy of dymA show alterations of mitochondrial, nuclear, and endosomal morphology and a defect in fluid-phase uptake. They also become multinucleated due to a failure to complete normal cytokinesis. These pleiotropic effects of dynamin A depletion can be rescued by complementation with the cloned gene. Morphological studies using cells producing green fluorescent protein-dynamin A revealed that dynamin A associates with punctate cytoplasmic vesicles. Double labeling with vacuolin, a marker of a postlysosomal compartment in D. discoideum, showed an almost complete colocalization of vacuolin and dynamin A. Our results suggest that that dynamin A is likely to function in membrane trafficking processes along the endo-lysosomal pathway of D. discoideum but not at the plasma membrane.
Asunto(s)
Dictyostelium/fisiología , Dictyostelium/ultraestructura , Dinaminas , Proteínas de Unión al GTP/fisiología , Proteínas Protozoarias/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , División Celular/fisiología , Clonación Molecular , Cartilla de ADN/genética , Dictyostelium/genética , Endocitosis/fisiología , Proteínas de Unión al GTP/genética , Marcación de Gen , Genes Protozoarios , Prueba de Complementación Genética , Microscopía Confocal , Microscopía Electrónica , Datos de Secuencia Molecular , Orgánulos/ultraestructura , Proteínas Protozoarias/genética , Equilibrio HidroelectrolíticoRESUMEN
We have separately expressed the Dictyosteliumdiscoideum myosin II nonhydrolyzer point mutations E459V and E476K [Ruppel, K. M., and Spudich, J. A. (1996) Mol. Biol. Cell 7, 1123-1136] in the soluble myosin head fragment M761-1R [Anson et al. (1996) EMBO J. 15, 6069-6074] and performed transient kinetic analyses to characterize the ATPase cycles of the mutant proteins. While the mutations cause some changes in mantATP [2'(3')-O-(N-methylanthraniloyl)-ATP] and mantADP binding, the most dramatic effect is on the hydrolysis step of the ATPase cycle, which is reduced by 4 (E476K) and 6 (E459V) orders of magnitude. Thus, both mutant myosin constructs do in fact catalyze ATP hydrolysis but have very long-lived myosin.ATP states. The E459V mutation allowed for a direct measurement of the ATP off rate constant from myosin, which was found to be 2 x 10(-)5 s-1. Actin accelerated ATP release from this E459V construct by at least 100-fold. Additionally, we found that the affinity of the E476K construct for actin is significantly weaker than for the wild-type construct, while the E459V mutant interacts with actin normally. Their functional properties and the fact that they can be produced and purified in large amounts make the E476K and E459V constructs ideal tools to elucidate key structural features of the myosin ATPase cycle. These constructs should allow us to address important questions, including how binding of ATP to myosin heads results in a >3 order of magnitude reduction in actin affinity.
Asunto(s)
Adenosina Trifosfato/metabolismo , Miosinas/metabolismo , Actinas/metabolismo , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/metabolismo , Animales , Dictyostelium , Ácido Glutámico/genética , Hidrólisis , Cinética , Mutagénesis Sitio-Dirigida , Miosinas/genética , Unión Proteica , Espectrometría de Fluorescencia , Valina/genética , ortoaminobenzoatos/metabolismoRESUMEN
The effects of mutations in an actin-binding surface loop of myosin (loop 2) are described. Part of loop 2, the segment between myosin residues 618 and 622, was replaced with sequences enlarged by the introduction of positively charged GKK or neutral GNN motifs. Constructs with loops carrying up to 20 additional amino acids and charge variations from -1 to +12 were produced. Steady-state and transient kinetics were used to characterize the enzymatic behavior of the mutant motor domains. Binding of nucleotide was not affected by any of the alterations in loop 2. In regard to their interaction with actin, constructs with moderate charge changes (-1 to +2) displayed wild-type-like behavior. Introduction of more than one GKK motif led to stronger coupling between the actin- and nucleotide-binding sites of myosin and an up to 1000-fold increased affinity for actin in the absence of ATP and at zero ionic strength. In comparison to the wild-type construct M765, constructs with 4-12 extra charges displayed an increased dependence on ionic strength in their interaction with actin, a 2-3-fold increase in kcat, a more than 10-fold reduction in Kapp for actin, and a 34-70-fold increase in catalytic efficiency.
Asunto(s)
Actinas/metabolismo , Actomiosina/metabolismo , Adenosina Trifosfatasas/metabolismo , Miosinas/metabolismo , Secuencia de Aminoácidos , Animales , Dictyostelium , Cinética , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Concentración OsmolarRESUMEN
The thermal unfolding of two recombinant fragments of the head of Dictyostelium discoideum myosin II was studied by differential scanning calorimetry. These fragments M754 and M761 correspond to the globular motor portion of the myosin head that contains ATP- and actin-binding sites but lacks the light chain binding domain. Our results show that M754 is less thermostable than M761: the maximum of the thermal transition occurred at 41.7 degrees C for M754 and at 45.6 degrees C for M761, and the calorimetric enthalpy value determined for M754 (677 kJ/mol) was about half of that for M761 (1417 kJ/mol). This indicates that the region containing residues 755-761 plays a very important role in the structural stabilization of the entire globular motor part of the myosin head. ADP binding induces structural changes in both myosin fragments which are reflected in a 2-3.5 degrees C shift of the thermal transitions to higher temperature. The formation of stable ternary complexes of these myosin fragments with ADP and phosphate analogues such as orthovanadate, beryllium fluoride or aluminium fluoride causes additional structural changes which are reflected in a pronounced increase of thermal stability. The effect of beryllium fluoride was less distinct than that of aluminium fluoride or orthovanadate. In general, the changes caused by various phosphate analogues were similar to those observed with skeletal myosin subfragment 1. Thus, structural changes revealed by differential scanning calorimetry in the myosin head, that are due to the formation of stable ternary complexes with ADP and Pi analogues, occur mainly in the globular motor portion of the head.