Different Myosin Head Conformations in Bony Fish Muscles Put into Rigor at Different Sarcomere Lengths

At a resting sarcomere length of approximately 2.2 µm bony fish muscles put into rigor in the presence of BDM (2,3-butanedione monoxime) to reduce rigor tension generation show the normal arrangement of myosin head interactions with actin filaments as monitored by low-angle X-ray diffraction. However, if the muscles are put into rigor using the same protocol but stretched to 2.5 µm sarcomere length, a markedly different structure is observed. The X-ray diffraction pattern is not just a weaker version of the pattern at full overlap, as might be expected, but it is quite different. It is compatible with the actin-attached myosin heads being in a different conformation on actin, with the average centre of cross-bridge mass at a higher radius than in normal rigor and the myosin lever arms conforming less to the actin filament geometry, probably pointing back to their origins on their parent myosin filaments. The possible nature of this new rigor cross-bridge conformation is discussed in terms of other well-known states such as the weak binding state and the ‘roll and lock’ mechanism; we speculate that we may have trapped most myosin heads in an early attached strong actin-binding state in the cross-bridge cycle on actin

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

Recombinant production of Yersinia enterocolitica pyruvate kinase isoenzymes PykA and PykF

The glycolytic enzyme pyruvate kinase (PK) generates ATP from ADP through substrate-level phosphorylation powered by the conversion of phosphoenolpyruvate to pyruvate. In contrast to other bacteria, Enterobacteriaceae, such as pathogenic yersiniae, harbour two pyruvate kinases encoded by pykA and pykF. The individual roles of these isoenzymes are poorly understood. In an attempt to make the Yersinia enterocolitica pyruvate kinases PykA and PykF amenable to structural and functional characterisation, we produced them untagged in Escherichia coli and purified them to near homogeneity through a combination of ion exchange and size exclusion chromatography, yielding more than 180 mg per litre of batch culture. The solution structure of PykA and PykF was analysed through small angle X-ray scattering which revealed the formation of PykA and PykF tetramers and confirmed the binding of the allosteric effector fructose-1,6-bisphosphate (FBP) to PykF but not to Pyk