Structure of the protective nematode protease complex H-gal-GP and its conservation across roundworm parasites.

Roundworm parasite infections are a major cause of human and livestock disease worldwide and a threat to global food security. Disease control currently relies on anthelmintic drugs to which roundworms are becoming increasingly resistant. An alternative approach is control by vaccination and 'hidden antigens', components of the worm gut not encountered by the infected host, have been exploited to produce Barbervax, the first commercial vaccine for a gut dwelling nematode of any host. Here we present the structure of H-gal-GP, a hidden antigen from Haemonchus contortus, the Barber's Pole worm, and a major component of Barbervax. We demonstrate its novel architecture, subunit composition and topology, flexibility and heterogeneity using cryo-electron microscopy, mass spectrometry, and modelling. Importantly, we demonstrate that complexes with the same architecture are present in other Strongylid roundworm parasites including human hookworm. This suggests a common ancestry and the potential for development of a unified hidden antigen vaccine.

Structure of isolated Z-disks from honeybee flight muscle.

The Z-disk is a complex structure comprising some 40 proteins that are involved in the transmission of force developed during muscle contraction and in important signalling pathways that govern muscle homeostasis. In the Z-disk the ends of antiparallel thin filaments from adjacent sarcomeres are crosslinked by α-actinin. The structure of the Z-disk lattice varies greatly throughout the animal kingdom. In vertebrates the thin filaments form a tetragonal lattice, whereas invertebrate flight muscle has a hexagonal lattice. The width of the Z-disk varies considerably and correlates with the number of α-actinin bridges. A detailed description at a high resolution of the Z-disk lattice is needed in order to better understand muscle function and disease. The molecular architecture of the Z-disk lattice in honeybee (Apis mellifera) is known from plastic embedded thin sections to a resolution of 7 nm, which is not sufficient to dock component protein crystal structures. It has been shown that sectioning is a damaging process that leads to the loss of finer details present in biological specimens. However, the Apis Z-disk is a thin structure (120 nm) suitable for cryo EM. We have isolated intact honeybee Z-disks from indirect flight muscle, thus obviating the need of plastic sectioning. We have employed cryo electron tomography and image processing to investigate the arrangement of proteins within the hexagonal lattice of the Apis Z-disk. The resolution obtained, ~6 nm, was probably limited by damage caused by the harshness of the conditions used to extract the myofibrils and isolate the Z-disks.

Titin and Nebulin in Thick and Thin Filament Length Regulation.

In this review we discuss the history and the current state of ideas related to the mechanism of size regulation of the thick (myosin) and thin (actin) filaments in vertebrate striated muscles. Various hypotheses have been considered during of more than half century of research, recently mostly involving titin and nebulin acting as templates or 'molecular rulers', terminating exact assembly. These two giant, single-polypeptide, filamentous proteins are bound in situ along the thick and thin filaments, respectively, with an almost perfect match in the respective lengths and structural periodicities. However, evidence still questions the possibility that the proteins function as templates, or scaffolds, on which the thin and thick filaments could be assembled. In addition, the progress in muscle research during the last decades highlighted a number of other factors that could potentially be involved in the mechanism of length regulation: molecular chaperones that may guide folding and assembly of actin and myosin; capping proteins that can influence the rates of assembly-disassembly of the myofilaments; Ca2+ transients that can activate or deactivate protein interactions, etc. The entire mechanism of sarcomere assembly appears complex and highly dynamic. This mechanism is also capable of producing filaments of about the correct size without titin and nebulin. What then is the role of these proteins? Evidence points to titin and nebulin stabilizing structures of the respective filaments. This stabilizing effect, based on linear proteins of a fixed size, implies that titin and nebulin are indeed molecular rulers of the filaments. Although the proteins may not function as templates in the assembly of the filaments, they measure and stabilize exactly the same size of the functionally important for the muscles segments in each of the respective filaments.

Structure of the vacuolar H+-ATPase rotary motor reveals new mechanistic insights.

Vacuolar H(+)-ATPases are multisubunit complexes that operate with rotary mechanics and are essential for membrane proton transport throughout eukaryotes. Here we report a ∼ 1 nm resolution reconstruction of a V-ATPase in a different conformational state from that previously reported for a lower-resolution yeast model. The stator network of the V-ATPase (and by implication that of other rotary ATPases) does not change conformation in different catalytic states, and hence must be relatively rigid. We also demonstrate that a conserved bearing in the catalytic domain is electrostatic, contributing to the extraordinarily high efficiency of rotary ATPases. Analysis of the rotor axle/membrane pump interface suggests how rotary ATPases accommodate different c ring stoichiometries while maintaining high efficiency. The model provides evidence for a half channel in the proton pump, supporting theoretical models of ion translocation. Our refined model therefore provides new insights into the structure and mechanics of the V-ATPases.

PA1b inhibitor binding to subunits c and e of the vacuolar ATPase reveals its insecticidal mechanism.

The vacuolar ATPase (V-ATPase) is a 1MDa transmembrane proton pump that operates via a rotary mechanism fuelled by ATP. Essential for eukaryotic cell homeostasis, it plays central roles in bone remodeling and tumor invasiveness, making it a key therapeutic target. Its importance in arthropod physiology also makes it a promising pesticide target. The major challenge in designing lead compounds against the V-ATPase is its ubiquitous nature, such that any therapeutic must be capable of targeting particular isoforms. Here, we have characterized the binding site on the V-ATPase of pea albumin 1b (PA1b), a small cystine knot protein that shows exquisitely selective inhibition of insect V-ATPases. Electron microscopy shows that PA1b binding occurs across a range of equivalent sites on the c ring of the membrane domain. In the presence of Mg·ATP, PA1b localizes to a single site, distant from subunit a, which is predicted to be the interface for other inhibitors. Photoaffinity labeling studies show radiolabeling of subunits c and e. In addition, weevil resistance to PA1b is correlated with bafilomycin resistance, caused by mutation of subunit c. The data indicate a binding site to which both subunits c and e contribute and inhibition that involves locking the c ring rotor to a static subunit e and not subunit a. This has implications for understanding the V-ATPase mechanism and that of inhibitors with therapeutic or pesticidal potential. It also provides the first evidence for the position of subunit e within the complex.

Three-dimensional organization of troponin on cardiac muscle thin filaments in the relaxed state.

Muscle contraction is regulated by troponin-tropomyosin, which blocks and unblocks myosin binding sites on actin. To elucidate this regulatory mechanism, the three-dimensional organization of troponin and tropomyosin on the thin filament must be determined. Although tropomyosin is well defined in electron microscopy helical reconstructions of thin filaments, troponin density is mostly lost. Here, we determined troponin organization on native relaxed cardiac muscle thin filaments by applying single particle reconstruction procedures to negatively stained specimens. Multiple reference models led to the same final structure, indicating absence of model bias in the procedure. The new reconstructions clearly showed F-actin, tropomyosin, and troponin densities. At the 25 Å resolution achieved, troponin was considerably better defined than in previous reconstructions. The troponin density closely resembled the shape of troponin crystallographic structures, facilitating detailed interpretation of the electron microscopy density map. The orientation of troponin-T and the troponin core domain established troponin polarity. Density attributable to the troponin-I mobile regulatory domain was positioned where it could hold tropomyosin in its blocking position on actin, thus suggesting the underlying structural basis of thin filament regulation. Our previous understanding of thin filament regulation had been limited to known movements of tropomyosin that sterically block and unblock myosin binding sites on actin. We now show how troponin, the Ca(2+) sensor, may control these movements, ultimately determining whether muscle contracts or relaxes.

¹H, ¹5N, and ¹³C backbone chemical shift assignment of titin domains A59-A60 and A60 alone.

The giant protein titin is the third most abundant protein of vertebrate striated muscle. The titin molecule is >1 µm long and spans half the sarcomere, from the Z-disk to the M-line, and has important roles in sarcomere assembly, elasticity and intracellular signaling. In the A-band of the sarcomere titin is attached to the thick filaments and mainly consists immunoglobulin-like and fibronectin type III-like domains. These are mostly arranged in long-range patterns or 'super-repeats'. The large super-repeats each contain 11 domains and are repeated 11 times, thus forming nearly half the titin molecule. Through interactions with myosin and C-protein, they are involved in thick filament assembly. The importance of titin in muscle assembly is highlighted by the effect of mutations in the A-band portion, which are the commonest cause of dilated cardiomyopathy, affecting ~1 in 250 (Herman et al. in N Engl J Med 366:619-628, 2012). Here we report backbone (15)N, (13)C and (1)H chemical shift and (13)Cß assignments for the A59-A60 domain tandem from the titin A59-A69 large super-repeat, completed using triple resonance NMR. Since, some regions of the backbone remained unassigned in A60 domain of the complete A59-A60 tandem, a construct containing a single A60 domain, A60sd, was also studied using the same methods. Considerably improved assignment coverage was achieved using A60sd due to its lower mass and improved molecular tumbling rate; these assignments also allowed the analysis of inter-domain interactions using chemical shift mapping against A59-A60.

Mutagenetic and electron microscopy analysis of actin filament severing by Cordon-Bleu, a WH2 domain protein.

Cordon-Bleu (Cobl) is a regulator of actin dynamics in neural development and ciliogenesis. Its function is associated with three adjacent actin binding WASP Homology 2 (WH2) domains. We showed that these WH2 repeats confer multifunctional regulation of actin dynamics, which makes Cobl a « dynamizer ¼ of actin assembly, inducing fast turnover of actin filaments and oscillatory polymerization regime via nucleation, severing, and rapid depolymerization activities. Cobl is the most efficient severer of actin filaments characterized so far. To understand which primary sequence elements determine the filament severing activity of the WH2 repeats, here we combine a mutagenetic/domain swapping approach of the minimal fully active Cobl-KAB construct, which comprises the lysine rich region K preceding the two first WH2 domains A and B. The mutated Cobl constructs display variable loss of the original filament nucleating activities of native Cobl-KAB, without any strict correlation with a loss in actin binding, which emphasizes the functional importance of the electrostatic environment of WH2 domains. Filament severing displayed the greatest stringency and was abolished in all mutated forms of Cobl-KAB. Filament severing and re-annealing by Cobl-KAB, which is key in its rapid remodeling of a population of actin filaments, and most likely responsible for its function in ciliogenesis, was analyzed by electron microscopy in comparison with Spire and ADF.

Subunit positioning and stator filament stiffness in regulation and power transmission in the V1 motor of the Manduca sexta V-ATPase.

The vacuolar H(+)-ATPase (V-ATPase) is an ATP-driven proton pump essential to the function of eukaryotic cells. Its cytoplasmic V1 domain is an ATPase, normally coupled to membrane-bound proton pump Vo via a rotary mechanism. How these asymmetric motors are coupled remains poorly understood. Low energy status can trigger release of V1 from the membrane and curtail ATP hydrolysis. To investigate the molecular basis for these processes, we have carried out cryo-electron microscopy three-dimensional reconstruction of deactivated V1 from Manduca sexta. In the resulting model, three peripheral stalks that are parts of the mechanical stator of the V-ATPase are clearly resolved as unsupported filaments in the same conformations as in the holoenzyme. They are likely therefore to have inherent stiffness consistent with a role as flexible rods in buffering elastic power transmission between the domains of the V-ATPase. Inactivated V1 adopted a homogeneous resting state with one open active site adjacent to the stator filament normally linked to the H subunit. Although present at 1:1 stoichiometry with V1, both recombinant subunit C reconstituted with V1 and its endogenous subunit H were poorly resolved in three-dimensional reconstructions, suggesting structural heterogeneity in the region at the base of V1 that could indicate positional variability. If the position of H can vary, existing mechanistic models of deactivation in which it binds to and locks the axle of the V-ATPase rotary motor would need to be re-evaluated.

Flexibility within the rotor and stators of the vacuolar H+-ATPase.

The V-ATPase is a membrane-bound protein complex which pumps protons across the membrane to generate a large proton motive force through the coupling of an ATP-driven 3-stroke rotary motor (V1) to a multistroke proton pump (Vo). This is done with near 100% efficiency, which is achieved in part by flexibility within the central rotor axle and stator connections, allowing the system to flex to minimise the free energy loss of conformational changes during catalysis. We have used electron microscopy to reveal distinctive bending along the V-ATPase complex, leading to angular displacement of the V1 domain relative to the Vo domain to a maximum of ~30°. This has been complemented by elastic network normal mode analysis that shows both flexing and twisting with the compliance being located in the rotor axle, stator filaments, or both. This study provides direct evidence of flexibility within the V-ATPase and by implication in related rotary ATPases, a feature predicted to be important for regulation and their high energetic efficiencies.

Making muscle elastic: the structural basis of myomesin stretching.

Skeletal and cardiac muscles are remarkable biological machines that support and move our bodies and power the rhythmic work of our lungs and hearts. As well as producing active contractile force, muscles are also passively elastic, which is essential to their performance. The origins of both active contractile and passive elastic forces can be traced to the individual proteins that make up the highly ordered structure of muscle. In this Primer, we describe the organization of sarcomeres--the structural units that produce contraction--and the nature of the proteins that make muscle elastic. In particular, we focus on an elastic protein called myomesin, whose novel modular architecture helps explain elasticity.

Structure and operation of the DNA-translocating type I DNA restriction enzymes.

Type I DNA restriction/modification (RM) enzymes are molecular machines found in the majority of bacterial species. Their early discovery paved the way for the development of genetic engineering. They control (restrict) the influx of foreign DNA via horizontal gene transfer into the bacterium while maintaining sequence-specific methylation (modification) of host DNA. The endonuclease reaction of these enzymes on unmethylated DNA is preceded by bidirectional translocation of thousands of base pairs of DNA toward the enzyme. We present the structures of two type I RM enzymes, EcoKI and EcoR124I, derived using electron microscopy (EM), small-angle scattering (neutron and X-ray), and detailed molecular modeling. DNA binding triggers a large contraction of the open form of the enzyme to a compact form. The path followed by DNA through the complexes is revealed by using a DNA mimic anti-restriction protein. The structures reveal an evolutionary link between type I RM enzymes and type II RM enzymes.

¹H, ¹5N and ¹³C backbone chemical shift assignment of the titin A67-A68 domain tandem.

Single molecules of the giant protein titin extend across half of the muscle sarcomere, from the Z-line to the M-line, and have roles in muscle assembly and elasticity. In the A-band titin is attached to thick filaments and here the domain arrangement occurs in regular patterns of eleven called the large super-repeat. The large super-repeat itself occurs eleven times and forms nearly half the titin molecule. Interactions of the large super-repeats with myosin are consistent with a role in thick filament assembly. Here we report backbone assignments of the titin A67-A68 domain tandem (Fn-Ig) from the third super-repeat (A65-A75) completed using triple resonance NMR experiments.

Structural divergence of the rotary ATPases.

The rotary ATPase family of membrane protein complexes may have only three members, but each one plays a fundamental role in biological energy conversion. The F1F(o)-ATPase (F-ATPase) couples ATP synthesis to the electrochemical membrane potential in bacteria, mitochondria and chloroplasts, while the vacuolar H⁺-ATPase (V-ATPase) operates as an ATP-driven proton pump in eukaryotic membranes. In different species of archaea and bacteria, the A1A(o)-ATPase (A-ATPase) can function as either an ATP synthase or an ion pump. All three of these multi-subunit complexes are rotary molecular motors, sharing a fundamentally similar mechanism in which rotational movement drives the energy conversion process. By analogy to macroscopic systems, individual subunits can be assigned to rotor, axle or stator functions. Recently, three-dimensional reconstructions from electron microscopy and single particle image processing have led to a significant step forward in understanding of the overall architecture of all three forms of these complexes and have allowed the organisation of subunits within the rotor and stator parts of the motors to be more clearly mapped out. This review describes the emerging consensus regarding the organisation of the rotor and stator components of V-, A- and F-ATPases, examining core similarities that point to a common evolutionary origin, and highlighting key differences. In particular, it discusses how newly revealed variation in the complexity of the inter-domain connections may impact on the mechanics and regulation of these molecular machines.

Nucleotide-dependent shape changes in the reverse direction motor, myosin VI.

We have studied the shape of myosin VI, the actin minus-end directed motor, by negative stain and metal shadow electron microscopy. Single particle processing was used to make two-dimensional averages of the stain images, which greatly increases the clarity and allows detailed comparisons with crystal structures. A total of 169,964 particle images were obtained from two different constructs in six different states (four nucleotide states and with and without Ca(2+)). The shape of truncated apo myosin VI was very similar to the apo crystal structure, with the lever arm bent strongly backward and around the motor domain. In the full-length molecule, the C-terminal part of the tail has an additional bend taking it back across the motor domain, which may reflect a regulated state. Addition of ATP, ADP, or ATP-γS resulted in a large change, straightening the molecule from the bent shape and swinging the lever by ∼140°. Although these nucleotides would not be expected to produce the pre-powerstroke state, myosin VI in their presence was most similar to the truncated crystal structure with bound ADP-VO(4), which is thought to show the pre-powerstroke shape. The nucleotide data were therefore substantially different from expectation based on crystal structures. The full-length molecule was almost completely monomeric; only ∼1% were dimers, joined through the ends of the tail. Addition of calcium ions appeared to result in release of the second calmodulin light chain. In negatively stained molecules there was little indication of extended α-helical structure in the tail, but molecules viewed by metal shadowing had a tail ∼3× longer, 29 vs. 9 nm, part of which is likely to be a single α-helix.

An approach to automated acquisition of cryoEM images from lacey carbon grids.

An approach to automated acquisition of cryoEM image data from lacey carbon grids using the Leginon program is described. Automated liquid nitrogen top up of the specimen holder dewar was used as a step towards full automation, without operator intervention during the course of data collection. During cryoEM studies of actin labelled with myosin V, we have found it necessary to work with lacey grids rather than Quantifoil or C-flat grids due to interaction of myosin V with the support film. Lacey grids have irregular holes of variable shape and size, in contrast to Quantifoil or C-flat grids which have a regular array of similar circular holes on each grid square. Other laboratories also prefer to work with grids with irregular holes for a variety of reasons. Therefore, it was necessary to develop a different strategy from normal Leginon usage for working with lacey grids for targeting holes for image acquisition and suitable areas for focussing prior to image acquisition. This approach was implemented by using the extensible framework provided by Leginon and by developing a new MSI application within that framework which includes a new Leginon node (for a novel method for finding focus targets).

Roles of titin in the structure and elasticity of the sarcomere.

The giant protein titin is thought to play major roles in the assembly and function of muscle sarcomeres. Structural details, such as widths of Z- and M-lines and periodicities in the thick filaments, correlate with the substructure in the respective regions of the titin molecule. Sarcomere rest length, its operating range of lengths, and passive elastic properties are also directly controlled by the properties of titin. Here we review some recent titin data and discuss its implications for sarcomere architecture and elasticity.

Shape and flexibility in the titin 11-domain super-repeat.

Titin is a giant protein of striated muscle with important roles in the assembly, intracellular signalling and passive mechanical properties of sarcomeres. The molecule consists principally of approximately 300 immunoglobulin and fibronectin domains arranged in a chain more than 1 mum long. The isoform-dependent N-terminal part of the molecule forms an elastic connection between the end of the thick filament and the Z-line. The larger, constitutively expressed C-terminal part is bound to the thick filament. Through most of the thick filament part, the immunoglobulin and fibronectin domains are arranged in a repeating pattern of 11 domains termed the 'large super-repeat'. There are 11 contiguous copies of the large super-repeat making up a segment of the molecule nearly 0.5 mum long. We have studied a set of two-domain and three-domain recombinant fragments from the large super-repeat region by electron microscopy, synchrotron X-ray solution scattering and analytical ultracentrifugation, with the goal of reconstructing the overall structure of this part of titin. The data illustrate different average conformations in different domain pairs, which correlate with differences in interdomain linker lengths. They also illustrate interdomain bending and flexibility around average conformations. Overall, the data favour a helical conformation in the super-repeat. They also suggest that this region of titin is dimerized when bound to the thick filament.