The “Roll and Lock” Mechanism of Force Generation in Muscle

SummaryMuscle force results from the interaction of the globular heads of myosin-II with actin filaments. We studied the structure-function relationship in the myosin motor in contracting muscle fibers by using temperature jumps or length steps combined with time-resolved, low-angle X-ray diffraction. Both perturbations induced simultaneous changes in the active muscle force and in the extent of labeling of the actin helix by stereo-specifically bound myosin heads at a constant total number of attached heads. The generally accepted hypothesis assumes that muscle force is generated solely by tilting of the lever arm, or the light chain domain of the myosin head, about its catalytic domain firmly bound to actin. Data obtained suggest an additional force-generating step: the “roll and lock” transition of catalytic domains of non-stereo-specifically attached heads to a stereo-specifically bound state. A model based on this scheme is described to quantitatively explain the data

Flexibility of KorA, a plasmid-encoded, global transcription regulator, in the presence and the absence of its operator

The IncP (Incompatibility group P) plasmids are important carriers in the spread of antibiotic resistance across Gram-negative bacteria. Gene expression in the IncP-1 plasmids is stringently controlled by a network of four global repressors, KorA, KorB, TrbA and KorC interacting cooperatively. Intriguingly, KorA and KorB can act as co-repressors at varying distances between their operators, even when they are moved to be on opposite sides of the DNA. KorA is a homodimer with the 101-amino acid subunits, folding into an N-terminal DNA-binding domain and a C-terminal dimerization domain. In this study, we have determined the structures of the free KorA repressor and two complexes each bound to a 20-bp palindromic DNA duplex containing its consensus operator sequence. Using a combination of X-ray crystallography, nuclear magnetic resonance spectroscopy, SAXS and molecular dynamics calculations, we show that the linker between the two domains is very flexible and the protein remains highly mobile in the presence of DNA. This flexibility allows the DNA-binding domains of the dimer to straddle the operator DNA on binding and is likely to be important in cooperative binding to KorB. Unexpectedly, the C-terminal domain of KorA is structurally similar to the dimerization domain of the tumour suppressor p53

A Helical Structural Nucleus Is the Primary Elongating Unit of Insulin Amyloid Fibrils

Although amyloid fibrillation is generally believed to be a nucleation-dependent process, the nuclei are largely structurally uncharacterized. This is in part due to the inherent experimental challenge associated with structural descriptions of individual components in a dynamic multi-component equilibrium. There are indications that oligomeric aggregated precursors of fibrillation, and not mature fibrils, are the main cause of cytotoxicity in amyloid disease. This further emphasizes the importance of characterizing early fibrillation events. Here we present a kinetic x-ray solution scattering study of insulin fibrillation, revealing three major components: insulin monomers, mature fibrils, and an oligomeric species. Low-resolution three-dimensional structures are determined for the fibril repeating unit and for the oligomer, the latter being a helical unit composed of five to six insulin monomers. This helical oligomer is likely to be a structural nucleus, which accumulates above the supercritical concentration used in our experiments. The growth rate of the fibrils is proportional to the amount of the helical oligomer present in solution, suggesting that these oligomers elongate the fibrils. Hence, the structural nucleus and elongating unit in insulin amyloid fibrillation may be the same structural component above supercritical concentrations. A novel elongation pathway of insulin amyloid fibrils is proposed, based on the shape and size of the fibrillation precursor. The distinct helical oligomer described in this study defines a conceptually new basis of structure-based drug design against amyloid diseases

Pulmonary surfactant protein A-induced changes in the molecular conformation of bacterial deep-rough LPS lead to reduced activity on human macrophages.

The lung is constantly exposed to immune stimulation by LPS from inhaled microorganisms. A primary mechanism to maintain immune homeostasis is based on anti-inflammatory regulation by surfactant protein A (SP-A), a secreted component of lung innate immunity. The architecture of LPS aggregates is strongly associated with biological activity. We therefore investigated whether SP-A affects the physico-chemical properties of LPS. Determination of the three-dimensional aggregate structure of LPS by small-angle X-ray scattering demonstrated that SP-A induced the formation of multi-lamellar aggregate structures. Determination of the acyl-chain-fluidity of LPS aggregates by Fourier transform infrared (FTIR) spectroscopy showed that the phase transition temperature of LPS was reduced in the presence of SP-A. The phosphate groups at the diglucosamine backbone of LPS represent important functional groups for the bioactivity of LPS. FTIR analysis revealed changes in the vibrational bands νas PO-(2), indicating an interaction of SP-A with the 1-phosphate, but not with the 4'-phosphate. The physico-chemical changes induced by SP-A were associated with up to 90% reduction in LPS-induced TNF-α-production by human macrophages. In conclusion, our data demonstrate that the SP-A/LPS interaction induces conformational changes in LPS aggregates leading to biologically less active structures, thereby providing a new molecular mechanism of immune modulation by SP-A

Fusion of Legionella pneumophila outer membrane vesicles with eukaryotic membrane systems is a mechanism to deliver pathogen factors to host cell membranes.

The formation and release of outer membrane vesicles (OMVs) is a phenomenon observed in many bacteria, including Legionella pneumophila. During infection this human pathogen primarily invades alveolar macrophages and replicates within a unique membrane-bound compartment termed Legionella-containing vacuole. In the current study we analyzed the membrane architecture of L.âpneumophila OMVs by small-angle X-ray scattering and biophysically characterized OMV membranes. We investigated the interaction of L. pneumophila OMVs with model membranes by Förster resonance energy transfer and Fourier-transform infrared spectroscopy. These experiments demonstrated the incorporation of OMV membrane material into liposomes composed of different eukaryotic phospholipids, revealing an endogenous property of OMVs to fuse with eukaryotic membranes. Cellular co-incubation experiments showed a dose- and time-dependent binding of fluorophore-labeled OMVs to macrophages. Trypan blue quenching experiments disclosed a rapid internalization of OMVs into macrophages at 37°C and 4°C. Purified OMVs induced TNFα production in human macrophages at concentrations starting at 300âng/ml. Experiments on HEK293-TLR2 and TLR4/MD-2 cell lines demonstrated a dominance of TLR2-dependent signaling pathways. In summary, we demonstrate binding, internalization and biological activity of L. pneumophila OMVs on human macrophages. Our data support OMV membrane fusion as a mechanism for the remote delivery of virulence factors to host cells

Comparative Characterization of Apo-, Reconstituted- and In Vivo-Folded forms of a Durum Wheat Metallothionein

Durum wheat metallothionein (dMT), a plant type 1 metallothionein, with a long “hinge” region between metal coordinating cysteine clusters, is efficient cadmium (Cd) chelator. In this work, biophysical features of purified recombinant holo-dMT, its demetallated form (apo-dMT) and the reconstituted Cd5-dMT are compared to obtain insight into the structure and metal binding features of this protein. Results show that the purified holo-dMT is polydisperse and has 5.3±0.5 Cd2+ ions per molecule. Demetallation followed by size exclusion chromatography yields homogeneous apo-dMT which can be reconstituted with Cd2+. Synchrotron small angle X-ray scattering (SAXS) demonstrates that apo-dMT, at pH 2.0, is flexible and extended in solution. According to UV-vis, CD and native-PAGE data conformation of apo-dMT is sensitive to pH changes in the range 2.0 to 8.0. Reconstitution of the apo-protein at pH 8.0, with Cd2+ appears to take place in two phases during which first the monomer is folded to accommodate 5 Cd2+ ions and then reorganization into oligomeric forms allows incorporation of further metal ions. SAXS data indicate that holo-dMT has limited flexibility in structure, but its conformation is significantly more compact than that of apo-dMT. Results of UV-vis and circular dichroism spectroscopy show that the in vitro folded protein is structurally different from the purified holo-dMT with the same number of Cd2+ ions.This work was supported by Sabanci University Internal Research Fund Project No. IACF08-00514 and by the bilateral program TUBITAK-Julich Research Center projects TBAG-U-155 and TBAG-U-157

Variations of subunit Varepsilon of the Mycobacterium tuberculosis F-1Fo ATP Synthase and Novel Model for Mechanism of Action of the Tuberculosis Drug TMC207.

Subunit ε of bacterial F-ATP synthases plays an important role in regulation by affecting the coupling, catalytic pathway, and inhibition of ATP hydrolysis, achieved through conformational transitions of its C-terminal domain. Here we present the low resolution solution structure of ε of Mycobacterium tuberculosis (Mtε) F-ATP synthase and the NMR structure of its C-terminal segment (Mt103-120), displaying an α-helical region between residues 104-119. The overall length of Mtε (61.6 Å) is significantly shorter compared to other bacterial forms, reflected by the missing C-terminal segment, described to be important in coupling processes via the catalytic β subunit. The surface potential of Mt103-120 reveals highly positive surface charge due to the presence of arginine residues. The unique structural traits of the C-terminus of Mtε make this segment a promising epitope for compounds to bind. Using NMR- and fluorescence spectroscopy, we demonstrate that the TB drug TMC207, proposed to bind to the proton translocating c-ring, does also bind to Mtε. W15 has been identified as one of the interacting residues. A model for the interaction of TMC207 with both the ε and the c-ring is presented, showing that TMC207 forms a wedge between the two rotating subunits by interacting with the residues W15 and F50 of ε and the c-ring, respectively. The other two residues, T19 and R37 of ε give the necessary polar interaction to the drug molecule. The new mechanism of TMC207 binding opens a promising platform for the design of antimycobacterials that effictively inhibit the ATP synthase in M. tuberculosis