Crystal structure of the dynamin tetramer

The mechanochemical protein dynamin is the prototype of the dynamin superfamily of large GTPases, which shape and remodel membranes in diverse cellular processes. Dynamin forms predominantly tetramers in the cytosol, which oligomerize at the neck of clathrin-coated vesicles to mediate constriction and subsequent scission of the membrane. Previous studies have described the architecture of dynamin dimers, but the molecular determinants for dynamin assembly and its regulation have remained unclear. Here we present the crystal structure of the human dynamin tetramer in the nucleotide-free state. Combining structural data with mutational studies, oligomerization measurements and Markov state models of molecular dynamics simulations, we suggest a mechanism by which oligomerization of dynamin is linked to the release of intramolecular autoinhibitory interactions. We elucidate how mutations that interfere with tetramer formation and autoinhibition can lead to the congenital muscle disorders Charcot-Marie-Tooth neuropathy and centronuclear myopathy, respectively. Notably, the bent shape of the tetramer explains how dynamin assembles into a right-handed helical oligomer of defined diameter, which has direct implications for its function in membrane constriction

Interaction between PEVK-titin and actin filaments: origin of a viscous force component in cardiac myofibrils.

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 [Ca2+] reversed the PEVK effect. PEVK concentrations 10 µg/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°C, but more strongly at 30°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 Ca2+-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

The Activation Mechanism of 2 5 Oligoadenylate Synthetase Gives New Insights Into OAS cGAS Triggers of Innate Immunity

Summary2′-5′-Oligoadenylate synthetases (OASs) produce the second messenger 2′-5′-oligoadenylate, which activates RNase L to induce an intrinsic antiviral state. We report on the crystal structures of catalytic intermediates of OAS1 including the OAS1·dsRNA complex without substrates, with a donor substrate, and with both donor and acceptor substrates. Combined with kinetic studies of point mutants and the previously published structure of the apo form of OAS1, the new data suggest a sequential mechanism of OAS activation and show the individual roles of each component. They reveal a dsRNA-mediated push-pull effect responsible for large conformational changes in OAS1, the catalytic role of the active site Mg2+, and the structural basis for the 2′-specificity of product formation. Our data reveal similarities and differences in the activation mechanisms of members of the OAS/cyclic GMP-AMP synthase family of innate immune sensors. In particular, they show how helix 3103-α5 blocks the synthesis of cyclic dinucleotides by OAS1

Structural and biochemical characterization of a dye-decolorizing peroxidase from <em>Dictyostelium discoideum</em>.

A novel cytoplasmic dye-decolorizing peroxidase from Dictyostelium discoideum was investi-gated that oxidizes anthraquinone dyes, lignin model compounds, and general peroxidase substrates such as ABTS efficiently. Unlike related enzymes, an aspartate residue replaces the first glycine of the conserved GXXDG motif in Dictyostelium DyPA. In solution, Dictyostelium DyPA exists as a stable dimer with the side chain of Asp146 contributing to the stabilization of the dimer interface by extending the hydrogen bond network connecting two monomers. To gain mechanistic insights, we solved the Dicty-ostelium DyPA structures in the absence of substrate as well as in the presence of potassium cyanide and veratryl alcohol to 1.7, 1.85, and 1.6 Å resolution, respectively. The active site of Dictyostelium DyPA has a hexa-coordinated heme iron with a histidine residue at the proximal axial position and either an acti-vated oxygen or CN− molecule at the distal axial position. Asp149 is in an optimal conformation to accept a proton from H2O2 during the formation of compound I. Two potential distal solvent channels and a conserved shallow pocket leading to the heme molecule were found in Dictyostelium DyPA. Further, we identified two substrate-binding pockets per monomer in Dictyostelium DyPA at the dimer interface. Long-range electron transfer pathways associated with a hydrogen-bonding network that connects the substrate-binding sites with the heme moiety are described

Identification of a Novel Type of cGMP Phosphodiesterase That Is Defective in the Chemotactic stmF Mutants

StmF mutants are chemotactic mutants that are defective in a cGMP phosphodiesterase (PDE) activity. We identified a novel gene, PdeD, that harbors two cyclic nucleotide–binding domains and a metallo-β-lactamase homology domain. Similar to stmF mutants, pdeD-null mutants displayed extensively streaming aggregates, prolonged elevation of cGMP levels after chemotactic stimulation, and reduced cGMP-PDE activity. PdeD transcripts were lacking in stmF mutant NP377, indicating that this mutant carries a PdeD lesion. Expression of a PdeD-YFP fusion protein in pdeD-null cells restored the normal cGMP response and showed that PdeD resides in the cytosol. When purified by immunoprecipitation, the PdeD-YFP fusion protein displayed cGMP-PDE activity, which was retained in a truncated construct that contained only the metallo-β-lactamase domain