Dictyostelium Myosin-IE Is a Fast Molecular Motor Involved in Phagocytosis

Class I myosins are single-headed motor proteins, implicated in various motile processes including organelle translocation, ion-channel gating, and cytoskeleton reorganization. Here we describe the cellular localization of myosin-IE and its role in the phagocytic uptake of solid particles and cells. A complete analysis of the kinetic and motor properties of Dictyostelium discoideum myosin-IE was achieved by the use of motor domain constructs with artificial lever arms. Class I myosins belonging to subclass IC like myosin-IE are thought to be tuned for tension maintenance or stress sensing. In contrast to this prediction, our results show myosin-IE to be a fast motor. Myosin-IE motor activity is regulated by myosin heavy chain phosphorylation, which increases the coupling efficiency between the actin and nucleotide binding sites tenfold and the motile activity more than fivefold. Changes in the level of free Mg(2+) ions, which are within the physiological range, are shown to modulate the motor activity of myosin-IE by inhibiting the release of adenosine diphosphate

Molecular engineering of a backwards−moving myosin motor

All members of the diverse myosin superfamily have a highly conserved globular motor domain that contains the actin− and nucleotide−binding sites and produces force and movement1, 2. The light−chain−binding domain connects the motor domain to a variety of functionally specialized tail domains and amplifies small structural changes in the motor domain through rotation of a lever arm3, 4. Myosins move on polarized actin filaments either forwards to the barbed (+ ) or backwards to the pointed (− ) end5, 6. Here, we describe the engineering of an artificial backwards−moving myosin from three pre−existing molecular building blocks. These blocks are: a forward−moving class I myosin motor domain, a directional inverter formed by a four−helix bundle segment of human guanylate−binding protein−1 and an artificial lever arm formed by two −actinin repeats. Our results prove that reverse−direction movement of myosins can be achieved simply by rotating the direction of the lever arm 180°. Most myosins move towards the barbed (+ ) end of actin filaments, but recent studies have established that at least one member of the family, myosin VI, moves towards the pointed (− ) end5. The structural basis for reverse−direction movement has not been established. Two mechanisms for achieving reversal of myosin motility on the inherently polar actin filament have been suggested. On the basis of direct functional assays, electron microscopy and sequence analysis, Sweeney and co−workers proposed a model whereby reversal is achieved by rotating the lever arm in the opposite direction to conventional myosin lever arm movement5 (Fig. 1). Ikebe and co−workers, however, measured the motile properties of chimaeric constructs and proposed that the core of the motor domain is the sole determinant of directionality6

Reconstitution of Rabbit Skeletal Muscle Troponin from the Recombinant Subunits All Expressed in and Purified from E. coli

Three subunits of rabbit skeletal muscle troponin were expressed in and purified from Escherichia coli. The procedures were optimized, and the reconstituted troponin complex is highly homogeneous, stable, and obtainable in large quantities, allowing us to conduct crystallization studies of the troponin complex. The three subunits expressed and purified are β-TnT(N'–208), TnI(C64A, C133S), and the wild type TnC. β–TnT(N'–208) is a 25 kDa fragment of y9-troponin T, which consists of 208 amino acids and lacks 58 residues in the N–terminal variable region. TnI(C64A, C133S) is a mutant troponin I, in which Cys–64 and Cys–133 are replaced by Ala and Ser, respectively. Each subunit was separately expressed in E. coli, purified by column chromatography including HPLC, and reassembled to form troponin complex. The reconstituted troponin complex was not distinguishable from authentic troponin prepared from rabbit skeletal muscle; the acto-Sl ATPase rate, as well as the superprecipitation, was calcium-sensitive. Small flat crystals up to 0.2 mm long have been reproducibly obtained in preliminary crystallization trials

Structure of a genetically engineered molecular motor

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 Å resolution crystal structure of an artificial actin-based motor. By combining the catalytic domain of myosin II with a 130 Å conformational amplifier consisting of repeats 1 and 2 of α-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 myosins catalytically active closed conformation. Finally, we describe the structure of -actinin repeats 1 and 2 as being composed of two rigid, triple-helical bundles linked by an uninterrupted -helix. This fold is very similar to the previously described structures of -actinin repeats 2 and 3, and -spectrin repeats 16 and 17

Changes in Mg2+ ion concentration and heavy chain phosphorylation regulate the motor activity of a class I myosin

Class I myosins are single-headed motor proteins implicated in various motile processes including organelle translocation, ion channel gating, and cytoskeleton reorganization. Dictyostelium discoideum myosin-ID belongs to subclass 1alpha, whose members are thought to be tuned for rapid sliding. The direct analysis of myosin-ID motor activity is made possible by the production of single polypeptide constructs carrying an artificial lever arm. Using these constructs, we show that the motor activity of myosin-ID is activated 80-fold by phosphorylation at the TEDS site. TEDS site phosphorylation acts by stabilizing the actomyosin complex and increasing the coupling between actin binding and the release of hydrolysis products. A surprising effect of Mg(2+) ions on in vitro motility was discovered. Changes in the level of free Mg(2+) ions within the physiological range are shown to modulate motor activity by inhibiting ADP release. Our results indicate that higher concentrations of free Mg(2+) ions stabilize the tension-bearing actin myosin ADP state and shift the system from the production of rapid movement toward the generation of tension

Mechanism, regulation, and functional properties of Dictyostelium myosin-1B

Myosin-1B is one of three long tailed class-1 myosins containing an ATP-insensitive actin-binding site in the tail region that are produced in Dictyostelium discoideum. Myosin-1B localizes to actin-rich structures at the leading edge of migrating cells where it has been implicated in the formation and retraction of membrane projections, the recycling of plasma membrane components, and intracellular particle transport. Here, we have used a combination of molecular engineering approaches to describe the kinetic and motile properties of the myosin-1B motor and its regulation by TEDS site phosphorylation. Our results show that myosin-1B is a low duty ratio motor and displays the fastest nucleotide binding kinetics of any of the Dictyostelium class-1 myosins studied so far. Different from Dictyostelium myosin-1D and myosin-1E, dephosphorylated myosin-1B is not inactivated but moves actin filaments efficiently, albeit at an up to 8-fold slower velocity in the in vitro motility assay. A further difference is that myosin-1B lacks the ability to switch between rapid movement and bearing tension upon physiological changes of free Mg2+ ions. In this respect, its motor properties appear to be more closely related to Dictyostelium myosin-2 and rabbit skeletal muscle myosin

Structural Basis for the Allosteric Interference of Myosin Function by Reactive Thiol Region Mutations G680A and G680V*

Background: Cold-sensitive mutations in the reactive thiol region of myosin interfere with motor function

Dictyostelium myosin-IE is a fast molecular motor involved in phagocytosis

Class I myosins are single-headed motor proteins, implicated in various motile processes including organelle translocation, ion-channel gating, and cytoskeleton reorganization. Here we describe the cellular localization of myosin-IE and its role in the phagocytic uptake of solid particles and cells. A complete analysis of the kinetic and motor properties of Dictyostelium discoideum myosin-IE was achieved by the use of motor domain constructs with artificial lever arms. Class I myosins belonging to subclass IC like myosin-IE are thought to be tuned for tension maintenance or stress sensing. In contrast to this prediction, our results show myosin-IE to be a fast motor. Myosin-IE motor activity is regulated by myosin heavy chain phosphorylation, which increases the coupling efficiency between the actin and nucleotide binding sites tenfold and the motile activity more than fivefold. Changes in the level of free Mg(2+) ions, which are within the physiological range, are shown to modulate the motor activity of myosin-IE by inhibiting the release of adenosine diphosphate

Mutations in the relay loop region result in dominant-negative inhibition of myosin II function in Dictyostelium

Dominant-negative inhibition is a powerful genetic tool for the characterization of gene function in vivo, based on the specific impairment of a gene product by the coexpression of a mutant version of the same gene product. We describe the detailed characterization of two myosin constructs containing either point mutations F487A or F506G in the relay region. Dictyostelium cells transformed with F487A or F506G myosin are unable to undergo processes that require myosin II function, including fruiting-body formation, normal cytokinesis and growth in suspension. Our results show that the dominant-negative inhibition of myosin function is caused by disruption of the communication between active site and lever arm, which blocks motor activity completely, and perturbation of the communication between active site and actin-binding site, leading to an approximately 100-fold increase in the mutants' affinity for actin in the presence of ATP