The myosin interacting-heads motif present in live tarantula muscle explains tetanic and post-tetanic phosphorylation mechanisms

Striated muscle contraction involves sliding of actin thin filaments along myosin thick filaments, controlled by calcium through thin filament activation. In relaxed muscle, the two heads of myosin interact with each other on the filament surface to form the interacting-heads motif (IHM). A key question is how both heads are released from the surface to approach actin and produce force. We used time-resolved synchrotron X-ray diffraction to study tarantula muscle before and after tetani. The patterns showed that the IHM is present in live relaxed muscle. Tetanic contraction produced only a very small backbone elongation, implying that mechanosensing - proposed in vertebrate muscle - is not of primary importance in tarantula. Rather, thick filament activation results from increases in myosin phosphorylation that release a fraction of heads to produce force, with the remainder staying in the ordered IHM configuration. After the tetanus the released heads slowly recover towards the resting, helical ordered state. During this time the released heads remain close to actin and can quickly rebind, enhancing the force produced by post-tetanic twitches, structurally explaining post-tetanic potentiation. Taken together, these results suggest that, in addition to stretch activation in insects, two other mechanisms for thick filament activation have evolved to disrupt …

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Nebulin mutations causing nemaline myopathy in humans change the structure of thin filaments in muscle

Compound nebulin mutations cause changes in thin filament structure.

Nebulin is a giant protein that winds around the actin filaments in the sarcomeres of skeletal muscle. Mutations in the nebulin gene (NEB) cause typical nemaline myopathy (NM), a muscle disorder characterized by muscle weakness that are difficult to treat. Here, the authors of this study created a mouse model that mimics the typical nebulin-based NM patient with compound-heterozygous mutations.

Functional, structural, and biochemical studies revealed altered thin filament structure, increased myofilament lattice spacing, a reduced myofibrillar fractional area, and reduced force production. In particular, X-ray diffraction studies on the BioCAT beamline 18ID revealed that the actin filament is twisted with a larger radius, that tropomyosin and troponin behavior is altered, and that the myofilament spacing is increased providing a structural explanation for muscle weakness. This new Compound-Het mouse model will be useful for testing experimental therapies for typical NM.

See: Johan Lindquist, Weikang Ma, Frank Li, Yaeren Hernandez, Justin Kolb, Balazs Kiss, Poala Tonino, Robbert van der Piil, Esmat Karini, Henry Gong, Josh Strom, Zaynab Hourani, John E Smith III, Coen Ottenheijm, Thomas Irving and Henk Granzier. Triggering typical nemaline myopathy with compound heterozygous nebulin mutations reveals myofilament structural changes …

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Structure-Function Studies Elucidate GPCR-Independent Regulation of G-proteins

Guanine nucleotide binding proteins popularly known as G-proteins, involved in a variety of cellular signal transduction pathways are heterotrimeric proteins consisting of α, β, and γ subunits. Ric8A is known to be both a chaperone for the assembly of the α-subunit of G-proteins, and a Guanine nucleotide Exchange Factor (GEF). McClelland et al., have conducted a detailed structural analysis on the complex between Ric8A and Gαi1 using cryoEM, X-ray crystallography, and SAXS. Constructs of Ric8A and Gαi1 optimized for structure determination and to reduce conformational heterogeneity were used to assemble the Ric8A-Gαi1 complex. They were able to determine the interface between the two proteins which consists of three separate surface contacts which essentially stabilize Gα in its nucleotide-free state. Furthermore, it was found that a specific Casein Kinase mediated phosphorylation of Ric8A stimulated the GEF activity by structurally stabilizing the Ric8A-Gα interface. Ric8A binding to the disrupted Guanine nucleotide binding site on Gα was determined to be critical for the GEF function as point mutants at residues of Ric8A involved in this interaction compromised the GEF activity. A significant amount of re-organization of a particular helical domain in Gα relative to the GTPase domain is involved in providing a means …

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How Prion-like domains Drive Liquid-Liquid Phase Transitions in Cells

Single chain of the model (top), microscopy of liquid-liquid pase separation (middle), and images from phase separating simulations (bottom).

Liquid-liquid phase separation (LLPS) provides a way for cells to create membraneless micro-environments (“condensates”) that have been proposed to be involved in diverse cellular processes including stress responses, RNA splicing, mitosis, chromatin organization, and the clustering of receptors at membranes. Proteins driving LLPS often contain intrinsically disordered prion like domains (PLD’s) that appear to be necessary and sufficient to produce LLPS. PLDS’s have a high proportion of polar amino acids mixed in with aromatic residues. In a recent paper in the journal Science, researchers from St. Jude’s Children’s Research Hospital and Washington University at St. Louis used a combination of NMR, multiscale simulations and Size Exclusion Chromatography SAXS experiments at the BioCAT Beamline 18ID to discover sequence features that determine the phase behavior of PLD’s. They used their experimental findings to develop a “sticker-and-spacers” model that can predict the phase behavior of PLDs on the basis of their sequence. Phase condensation is driven by noncovalent, intra- and intermolecular cross-links between stickers (largely aromatic residues and other hydrophobic motifs), whereas spacers either facilitate or inhibit the formation …

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Sarcomere Structure and Nemaline Myopathy

NEM6 mutations cause changes in thin filament structure.

Nemaline myopathy (NM) is one of the most common congenital non-dystrophic myopathies and is characterized by severe hypotonia, muscle weakness, feeding difficulties, respiratory failure, and the presence of nemaline bodies (rods) in skeletal muscle biopsies. One form of nemaline myopathy is caused by mutations in the KBTBD13 (NEM6) gene. In addition to weakness, NEM6 patients have slow muscle relaxation, compromising contractility and daily-life activities. The role of KBTBD13 in muscle is unknown, and the p athomechanism underlying NEM6 is undetermined. A combination of transcranial magnetic stimulation-induced muscle relaxation, muscle fiber- and sarcomere-contractility assays, super-resolution microscopy, and low angle X-ray diffraction on the BioCAT Beamline 18ID revealed that the impaired muscle relaxation kinetics in NEM6 patients are caused by structural changes in the thin filament, a sarcomeric microstructure. Using homology modeling, binding- and contractility assays with recombinant KBTBD13, novel Kbtbd13-knockout and Kbtbd13R408C-knockin mouse models and a transgenic zebrafish model the authors discovered that KBTBD13 binds to actin – a major constituent of the thin filament - and that mutations in KBTBD13 cause structural changes impairing muscle relaxation kinetics. The authors propose that this actin-based impaired relaxation is central to NEM6 pathology.

See: Josine M. de …

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Structure-Function Understanding of aGPCR ECRs Critical for Drug-Design

Cellular communication mediated by a variety of cell-surface receptors involves ligand induced conformational changes in the extracellular region (ECR). A variety of drugs such as cetuximab (Epidermal Growth Factor Receptor), etrolizumab (Integrins), and erenumab (calcitonin receptor-like receptor) function by trapping ECRs in specific conformations and have proved to be effective therapeutic agents in several cancers, bowel diseases, and migraine. Leon et al., have addressed a class of relatively understudied G-protein couple receptors (GPCRs) called adhesion-GPCRs (aGPCRs) which have a structurally unique ECR with a diverse set of mechanistic possibilities. Specifically, they study the Gpr126, which is known to be essential for Schwann cell myelination, involved in heart development in the mouse model, and inner ear development in Zebra fish. Determination of the high-resolution structure of the Zebra fish Gpr126 ECR revealed the involvement of a heretofore undefined domain that has splice variants with or without a 23aa stretch in different isotypes (-ss or +ss) and is the primary determinant of whether the ECR is in the open active state or the closed inactive state. Also remarkable was the calcium dependent site at the tip of the ECR which promoted the closed inactive state of Gpr126. The closed conformation observed in …

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Cold temperatures put myosin in a refractory state unable to bind to actin

Low temperatures shift myosin heads from an ordered relaxed state to a disordered state that cannot bind actin in response to stimulation.

For many years, contraction in skeletal muscle was assumed to be primarily regulated by the binding of calcium to troponin on the thin filaments allowing tropomyosin to move unblocking the binding sites for myosin heads on the thin filament. More recently, it has been recognized that an additional thick filament base regulatory system coexists with thin filament regulation. The myosin heads in relaxed muscle are in an ordered quasi-helical arrangement around the thick filament backbone where they are unable to bind to actin. Current models propose that strain developed in the thick filament backbone generated by a small number of disordered, constitutively active myosin heads, once the strain surpasses some threshold, releases myosin heads form the ordered inactive heads to become disordered active heads. At this point, it is commonly assumed that ordered heads are in the OFF state, unable to bind to actin, while disordered heads are in the ON state, able to bind to actin and generate force. In a recent paper in J. General Physiology, researchers from the University Florence used the BioCAT Beamline 18ID …

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Frustration and Folding of a TIM Barrel Protein

Multiple folding pathways discovered by simulations, validated against the time resolved SAXS and FRET data.

In their continuing endeavor to understand misfolding proteins as part of the etiology of a variety of diseases, the Matthews lab particularly focuses on the different factors that impede a protein’s path from the unfolded state to the global free energy minimum. The complexity of the folding trajectory understandably depends on the size of the protein mostly because of the formation of intermediates many of which often stall the formation of an optimal native conformation.

The triosphosphate isomerase (TIM) barrel family of proteins have very well characterized and conserved folding path despite large variations in sequence which make it an ideal group of proteins to obtain widely applicable insights into the folding process. Like other proteins studied before, S.solfataricus indole-3-glycerol phosphate synthase (SsIGPS) a TIM barrel protein goes through a burst phase followed by a relaxation phase and then eventually folds into the native conformation. Mutational and hydrogen exchange experiments have helped characterize the species found in the few millisecond time range of the folding process. In this study, Halloran et al., have used a combination of continuous flow FRET, SAXS and simulations …

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Structure of BS Ric8A, a regulator of G-protein Biology

A model of the Ric8a protein structure showing the best scoring flexible tail (right), and the validation of the model against the SAXS data (left).

G-protein signaling has been the dominant theme in the Artemyev lab and their recent work specifically addresses Ric8A (Resistance to inhibitors of cholinesterase 8A) structure and function. Ric8A is a well-known regulator of G-protein biology and belongs to a class of proteins different from the G protein-coupled receptors (GPCRs), which act via interactions with monomeric Gα subunits as opposed to heterotrimeric Gαβγ proteins. SAXS was used in combination with crystallography and biochemical studies to show that the flexible C-terminal tail is important for the overall stability of Ric8A and the function as a guanine nucleotide exchange factor (GEF). The crystal structure revealed that Ric8A belongs to a functionally diverse class of proteins with what is known as an armadillo-fold (ARM) characterized by two layers of alpha helices arranged in a right handed superhelix. Ric8A diverged from the class in terms of the number of ARM repeats (8 as opposed to 10) and is further followed by a flexible region spanning ~ 70 residues.

Differential scanning fluorimetry (DSF) was used to determine that a construct without the …

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Protein-Folding Mechanisms Elucidated Using Chaotic-Flow SAXS

The unfolded-state ensemble (USE) undergoes a continuous reduction in chain dimensions as a function of decreased denaturant concentration.

While there is emerging consensus in the protein folding community concerning the behavior of proteins under unfolding conditions, the occurrence of unfolded states under physiological (native) conditions and their propensity to aggregate are the basis of several human pathologies. Valuable insights into these transient species were obtained by taking advantage of the temporal resolution afforded by combining time-resolved fluorescence and continuous (in this case chaotic) flow SAXS (CF-SAXS) with all atom simulations and polymer theory. A group of researchers led by the Raleigh lab (Stony Brook University) used the 59 amino acid N-terminal domain of the ribosomal protein L9 (NTL9), which has a well-studied two state folding mechanism. By introducing FRET pairs several pairwise distance distributions were measured in the unfolded and native conditions in equilibrium and also the unfolded states in native conditions using a continuous flow mixer Interestingly chain contraction as indicated by fluorescence decay was observed well within the dead time of the mixer (~40 µs) showing that chain collapse happens considerably faster than the time-scale required for completion of the folding process (2.5 ms for NTL9 …

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