Project description:Myosin-motors are conserved from yeast to human and transport a great variety of cargoes. Most plus-end directed myosins, which constitute the vast majority of all myosin motors, form stable dimers and interact constitutively with their cargo complexes. To date, little is known about regulatory mechanisms for cargo-complex assembly. In this study, we show that the type V myosin Myo4p binds to its cargo via two distinct binding regions, the C-terminal tail and a coiled-coil domain-containing fragment. Furthermore, we find that Myo4p is strictly monomeric at physiologic concentrations. Because type V myosins are thought to require dimerization for processive movement, a mechanism must be in place to ensure that oligomeric Myo4p is incorporated into cargo-translocation complexes. Indeed, we find that artificial dimerization of the Myo4p C-terminal tail promotes stabilization of myosin-cargo complexes, suggesting that full-length Myo4p dimerizes in the cocomplex as well. We also combined the Myo4p C-terminal tail with the coiled-coil region, lever arm, and motor domain from a different myosin to form constitutively dimeric motor proteins. This heterologous motor successfully translocates its cargo in vivo, suggesting that wild-type Myo4p may also function as a dimer during cargo-complex transport.
Project description:The myosin superfamily is a versatile group of molecular motors involved in the transport of specific biomolecules, vesicles and organelles in eukaryotic cells. The processivity of myosins along an actin filament and transport of intracellular 'cargo' are achieved by generating physical force from chemical energy of ATP followed by appropriate conformational changes. The typical myosin has a head domain, which harbors an ATP binding site, an actin binding site, and a light-chain bound 'lever arm', followed often by a coiled coil domain and a cargo binding domain. Evolution of myosins started at the point of evolution of eukaryotes, S. cerevisiae being the simplest one known to contain these molecular motors. The coiled coil domain of the myosin classes II, V and VI in whole genomes of several model organisms display differences in the length and the strength of interactions at the coiled coil interface. Myosin II sequences have long-length coiled coil regions that are predicted to have a highly stable dimeric interface. These are interrupted, however, by regions that are predicted to be unstable, indicating possibilities of alternate conformations, associations to make thick filaments, and interactions with other molecules. Myosin V sequences retain intermittent regions of strong and weak interactions, whereas myosin VI sequences are relatively devoid of strong coiled coil motifs. Structural deviations at coiled coil regions could be important for carrying out normal biological function of these proteins.
Project description:Parasites of the phylum Apicomplexa are responsible for significant morbidity and mortality on a global scale. Central to the virulence of these pathogens are the phylum-specific, unconventional class XIV myosins that power the essential processes of parasite motility and host cell invasion. Notably, class XIV myosins differ from human myosins in key functional regions, yet they are capable of fast movement along actin filaments with kinetics rivaling previously studied myosins. Toward establishing a detailed molecular mechanism of class XIV motility, we determined the 2.6-Å resolution crystal structure of the Toxoplasma gondii MyoA (TgMyoA) motor domain. Structural analysis reveals intriguing strategies for force transduction and chemomechanical coupling that rely on a divergent SH1/SH2 region, the class-defining "HYAG"-site polymorphism, and the actin-binding surface. In vitro motility assays and hydrogen-deuterium exchange coupled with MS further reveal the mechanistic underpinnings of phosphorylation-dependent modulation of TgMyoA motility whereby localized regions of increased stability and order correlate with enhanced motility. Analysis of solvent-accessible pockets reveals striking differences between apicomplexan class XIV and human myosins. Extending these analyses to high-confidence homology models of Plasmodium and Cryptosporidium MyoA motor domains supports the intriguing potential of designing class-specific, yet broadly active, apicomplexan myosin inhibitors. The successful expression of the functional TgMyoA complex combined with our crystal structure of the motor domain provides a strong foundation in support of detailed structure-function studies and enables the development of small-molecule inhibitors targeting these devastating global pathogens.
Project description:The stressosome transduces environmental stress signals to SigB to upregulate SigB-dependent transcription, which is required for bacterial viability. The stressosome core is composed of RsbS and at least one of the RsbR paralogs. A previous cryo-electron microscopy (cryo-EM) structure of the RsbRA-RsbS complex determined under a D2 symmetry restraint showed that the stressosome core forms a pseudo-icosahedron consisting of 60 STAS domains of RsbRA and RsbS. However, it is still unclear how RsbS and one of the RsbR paralogs assemble into the stressosome. Here, an assembly model of the stressosome is presented based on the crystal structure of the RsbS icosahedron and cryo-EM structures of the RsbRA-RsbS complex determined under diverse symmetry restraints (nonsymmetric C1, dihedral D2 and icosahedral I envelopes). 60 monomers of the crystal structure of RsbS fitted well into the I-restrained cryo-EM structure determined at 4.1 Å resolution, even though the STAS domains in the I envelope were averaged. This indicates that RsbS and RsbRA share a highly conserved STAS fold. 22 protrusions observed in the C1 envelope, corresponding to dimers of the RsbRA N-domain, allowed the STAS domains of RsbRA and RsbS to be distinguished in the stressosome core. Based on these, the model of the stressosome core was reconstructed. The mutation of RsbRA residues at the binding interface in the model (R189A/Q191A) significantly reduced the interaction between RsbRA and RsbS. These results suggest that nonconserved residues in the conserved STAS folds between RsbS and RsbR paralogs determine stressosome assembly.
Project description:The apelin receptor (APJR) emerges as a promising drug target for cardiovascular health and muscle regeneration. While prior research unveiled the structural versatility of APJR in coupling to Gi proteins as a monomer or dimer, the dynamic regulation within the APJR dimer during activation remains poorly understood. In this study, we present the structures of the APJR dimer and monomer complexed with its endogenous ligand apelin-13. In the dimeric structure, apelin-13 binds exclusively to one protomer that is coupled with Gi proteins, revealing a distinct ligand-binding behavior within APJR homodimers. Furthermore, binding of an antagonistic antibody induces a more compact dimerization by engaging both protomers. Notably, structural analyses of the APJR dimer complexed with an agonistic antibody, with or without Gi proteins, suggest that G protein coupling may promote the dissociation of the APJR dimer during activation. These findings underscore the intricate interplay between ligands, dimerization, and G protein coupling in regulating APJR signaling pathways.
Project description:Biogenesis of photosystem II (PSII), nature's water-splitting catalyst, is assisted by auxiliary proteins that form transient complexes with PSII components to facilitate stepwise assembly events. Using cryo-electron microscopy, we solved the structure of such a PSII assembly intermediate from Thermosynechococcus elongatus at 2.94 Å resolution. It contains three assembly factors (Psb27, Psb28 and Psb34) and provides detailed insights into their molecular function. Binding of Psb28 induces large conformational changes at the PSII acceptor side, which distort the binding pocket of the mobile quinone (QB) and replace the bicarbonate ligand of non-haem iron with glutamate, a structural motif found in reaction centres of non-oxygenic photosynthetic bacteria. These results reveal mechanisms that protect PSII from damage during biogenesis until water splitting is activated. Our structure further demonstrates how the PSII active site is prepared for the incorporation of the Mn4CaO5 cluster, which performs the unique water-splitting reaction.
Project description:The structural dynamics of actin, including the tilting motion between the small and large domains, are essential for proper interactions with actin-binding proteins. Gly146 is situated at the hinge between the two domains, and we previously showed that a G146V mutation leads to severe motility defects in skeletal myosin but has no effect on motility of myosin V. The present study tested the hypothesis that G146V mutation impaired rotation between the two domains, leading to such functional defects. First, our study showed that depolymerization of G146V filaments was slower than that of wild-type filaments. This result is consistent with the distinction of structural states of G146V filaments from those of the wild type, considering the recent report that stabilization of actin filaments involves rotation of the two domains. Next, we measured intramolecular FRET efficiencies between two fluorophores in the two domains with or without skeletal muscle heavy meromyosin or the heavy meromyosin equivalent of myosin V in the presence of ATP. Single-molecule FRET measurements showed that the conformations of actin subunits of control and G146V actin filaments were different in the presence of skeletal muscle heavy meromyosin. This altered conformation of G146V subunits may lead to motility defects in myosin II. In contrast, distributions of FRET efficiencies of control and G146V subunits were similar in the presence of myosin V, consistent with the lack of motility defects in G146V actin with myosin V. The distribution of FRET efficiencies in the presence of myosin V was different from that in the presence of skeletal muscle heavy meromyosin, implying that the roles of actin conformation in myosin motility depend on the type of myosin.
Project description:In all herpesviruses, the capsid is icosahedral in shape, composed of 162 capsomers, and assembled in the infected cell nucleus. Once a closed capsid has formed, it is packaged with the virus DNA and transported to the cytoplasm where further morphogenetic events take place. Herpesvirus capsid populations are highly uniform in shape, and this property has made them attractive for structural analysis particularly by cryo electron microscopy followed by three-dimensional image reconstruction. Here we describe what is known about herpesvirus capsid structure and assembly with emphasis on herpes simplex virus and on the contribution of structural studies. The overall analysis has demonstrated that herpesvirus capsids are formed by a pathway resembling that established for dsDNA bacteriophage such as P22 and HK97. For example herpes capsid assembly is found to: (1) involve a scaffolding protein not present in the mature virus; (2) proceed through a fragile, spherical procapsid intermediate; and (3) result in incorporation of a portal complex at a unique capsid vertex.
Project description:Class V myosins are actin-dependent motors, which recognize numerous cellular cargos mainly via the C-terminal globular tail domain (GTD). Myo2, a yeast class V myosin, can transport a broad range of organelles. However, little is known about the capacity of Myo2-GTD to recognize such a diverse array of cargos specifically at the molecular level. Here, we solved crystal structures of Myo2-GTD (at 1.9-3.1 Å resolutions) in complex with three cargo adaptor proteins: Smy1 (for polarization of secretory vesicles), Inp2 (for peroxisome transport), and Mmr1 (for mitochondria transport). The structures of Smy1- and Inp2-bound Myo2-GTD, along with site-directed mutagenesis experiments, revealed a binding site in subdomain-I having a hydrophobic groove with high flexibility enabling Myo2-GTD to accommodate different protein sequences. The Myo2-GTD-Mmr1 complex structure confirmed and complemented a previously identified mitochondrion/vacuole-specific binding region. Moreover, differences between the conformations and locations of cargo-binding sites identified here for Myo2 and those reported for mammalian MyoVA (MyoVA) suggest that class V myosins potentially have co-evolved with their specific cargos. Our structural and biochemical analysis not only uncovers a molecular mechanism that explains the diverse cargo recognition by Myo2-GTD, but also provides structural information useful for future functional studies of class V myosins in cargo transport.
Project description:Myosin V (MyoV) motors have been implicated in the intracellular transport of diverse cargoes including vesicles, organelles, RNA-protein complexes, and regulatory proteins. Here, we have solved the cargo-binding domain (CBD) structures of the three human MyoV paralogs (Va, Vb, and Vc), revealing subtle structural changes that drive functional differentiation and a novel redox mechanism controlling the CBD dimerization process, which is unique for the MyoVc subclass. Moreover, the cargo- and motor-binding sites were structurally assigned, indicating the conservation of residues involved in the recognition of adaptors for peroxisome transport and providing high resolution insights into motor domain inhibition by CBD. These results contribute to understanding the structural requirements for cargo transport, autoinhibition, and regulatory mechanisms in myosin V motors.