Inhibition of Arp2/3 Complex after ADP-Ribosylation of Arp2 by Binary Toxins.

Clostridioides bacteria are responsible for life threatening infections. Here, we show that in addition to actin, the binary toxins CDT, C2I, and Iota from , , and , respectively, ADP-ribosylate the actin-related protein Arp2 of Arp2/3 complex and its additional components ArpC1, ArpC2, and ArpC4/5. The Arp2/3 complex is composed of seven subunits and stimulates the formation of branched actin filament networks.

Functional Characterization of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilated Cardiomyopathy (p.R312H and p.E361G).

Human wild type (wt) cardiac α-actin and its mutants p.A295S or p.R312H and p.

Interaction of isolated cross-linked short actin oligomers with the skeletal muscle myosin motor domain.

The cyclical interaction between F-actin and myosin in muscle cells generates contractile force. The myosin motor domain hydrolyses ATP, resulting in conformational changes that are amplified by the myosin lever arm that links the motor domain to the rod domain. Recent cryo-electron microscopic data have provided a clear picture of the myosin-ATP-F-actin complex, but structural insights into other stages of the myosin-actin interaction have been less forthcoming.

Force-producing ADP state of myosin bound to actin.

Molecular motors produce force when they interact with their cellular tracks. For myosin motors, the primary force-generating state has MgADP tightly bound, whereas myosin is strongly bound to actin. We have generated an 8-Å cryoEM reconstruction of this state for myosin V and used molecular dynamics flexed fitting for model building.

Structural basis for the allosteric interference of myosin function by reactive thiol region mutations G680A and G680V.

The cold-sensitive single-residue mutation of glycine 680 in the reactive thiol region of Dictyostelium discoideum myosin-2 or the corresponding conserved glycine in other myosin isoforms has been reported to interfere with motor function. Here we present the x-ray structures of myosin motor domain mutants G680A in the absence and presence of nucleotide as well as the apo structure of mutant G680V. Our results show that the Gly-680 mutations lead to uncoupling of the reactive thiol region from the surrounding structural elements.

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.

Functional characterization of the N-terminal region of myosin-2.

All class 2 myosins contain an N-terminal extension of approximately 80 residues that includes an Src homology 3 (SH3)-like subdomain. To explore the functional importance of this region, which is also present in most other myosin classes, we generated truncated constructs of Dictyostelium discoideum myosin-2. Truncation at position 80 resulted in the complete loss of myosin-2 function in vivo.

The actin-binding cleft: functional characterisation of myosin II with a strut mutation.

The myosin cross-bridge has two essential properties: to undergo the "power stroke" and to bind and release from actin - both under control of ATP binding and hydrolysis. In the absence of ATP the cross-bridge binds to actin with high affinity: the binding of ATP causes rapid release of the cross-bridge from actin. The actin binding-site is split by a deep cleft that closes on strong binding to actin.

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.

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.

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 movement. 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 arm. Myosins move on polarized actin filaments either forwards to the barbed (+) or backwards to the pointed (-) end.

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.

PEVK domain of titin: an entropic spring with actin-binding properties.

The PEVK domain of the giant muscle protein titin is a proline-rich sequence with unknown secondary/tertiary structure. Here we compared the force-extension behavior of cloned cardiac PEVK titin measured by single-molecule atomic force spectroscopy with the extensibility of the PEVK domain measured in intact cardiac muscle sarcomeres. The analysis revealed that cardiac PEVK titin acts as an entropic spring with the properties of a random coil exhibiting mechanical conformations of different flexibility.

Toxoplasma gondii myosin A and its light chain: a fast, single-headed, plus-end-directed motor.

Successful host cell invasion is a prerequisite for survival of the obligate intracellular apicomplexan parasites and establishment of infection. Toxoplasma gondii penetrates host cells by an active process involving its own actomyosin system and which is distinct from induced phagocytosis. Toxoplasma gondii myosin A (TgMyoA) is presumed to achieve power gliding motion and host cell penetration by the capping of apically released adhesins towards the rear of the parasite.