Advances in Structural Biology and the Application to Biological Filament Systems.

Structural biology has experienced several transformative technological advances in recent years. These include: development of extremely bright X-ray sources (microfocus synchrotron beamlines and free electron lasers) and the use of electrons to extend protein crystallography to ever decreasing crystal sizes; and an increase in the resolution attainable by cryo-electron microscopy. Here we discuss the use of these techniques in general terms and highlight their application for biological filament systems, an area that is severely underrepresented in atomic resolution structures. We assemble a model of a capped tropomyosin-actin minifilament to demonstrate the utility of combining structures determined by different techniques. Finally, we survey the methods that attempt to transform high resolution structural biology into more physiological environments, such as the cell. Together these techniques promise a compelling decade for structural biology and, more importantly, they will provide exciting discoveries in understanding the designs and purposes of biological machines.

The myosin start-of-power stroke state and how actin binding drives the power stroke.

We propose that on binding to actin at the start of the power stroke the myosin cross-bridge takes on the rigor configuration at the actin interface. Starting from the prepower stroke state, this can be achieved by a small movement (16° rotation) of the lower 50K domain without twisting the central ß-sheet or opening switch-1 or switch-2. The movement of the lower 50K domain puts a strain on the W-helix. This strain tries to twist the ß-sheet, which could drive the power stroke. This would provide a coupling between actin binding and the execution of the power stroke. During the power stroke the ß-sheet twists, moving the P-loop away from switch-2, which opens the nucleotide binding pocket and separates ADP from Pi . The power stroke is different from the recovery stroke because the upper and lower 50K domains are tethered in the rigor configuration.

Structural modeling and molecular dynamics simulation of the actin filament.

Actin is a major structural protein of the eukaryotic cytoskeleton and enables cell motility. Here, we present a model of the actin filament (F-actin) that not only incorporates the global structure of the recently published model by Oda et al. but also conserves internal stereochemistry. A comparison is made using molecular dynamics simulation of the model with other recent F-actin models. A number of structural determents such as the protomer propeller angle, the number of hydrogen bonds, and the structural variation among the protomers are analyzed. The MD comparison is found to reflect the evolution in quality of actin models over the last 6 years. In addition, simulations of the model are carried out in states with both ADP or ATP bound and local hydrogen-bonding differences characterized.

Actin structure and function.

Actin is the most abundant protein in most eukaryotic cells. It is highly conserved and participates in more protein-protein interactions than any known protein. These properties, along with its ability to transition between monomeric (G-actin) and filamentous (F-actin) states under the control of nucleotide hydrolysis, ions, and a large number of actin-binding proteins, make actin a critical player in many cellular functions, ranging from cell motility and the maintenance of cell shape and polarity to the regulation of transcription. Moreover, the interaction of filamentous actin with myosin forms the basis of muscle contraction. Owing to its central role in the cell, the actin cytoskeleton is also disrupted or taken over by numerous pathogens. Here we review structures of G-actin and F-actin and discuss some of the interactions that control the polymerization and disassembly of actin.

The actin-myosin interface.

In order to understand the mechanism of muscle contraction at the atomic level, it is necessary to understand how myosin binds to actin in a reversible way. We have used a novel molecular dynamics technique constrained by an EM map of the actin-myosin complex at 13-A resolution to obtain an atomic model of the strong-binding (rigor) actin-myosin interface. The constraining force resulting from the EM map during the molecular dynamics simulation was sufficient to convert the myosin head from the initial weak-binding state to the strong-binding (rigor) state. Our actin-myosin model suggests extensive contacts between actin and the myosin head (S1). S1 binds to two actin monomers. The contact surface between actin and S1 has increased dramatically compared with previous models. A number of loops in S1 and actin are involved in establishing the interface. Our model also suggests how the loop carrying the critical Arg 405 Glu mutation in S1 found in a familial cardiomyopathy might be functionally involved.

50 years of fiber diffraction.

In 1955 Ken Holmes started working on tobacco mosaic virus (TMV) as a research student with Rosalind Franklin at Birkbeck College, London. Afterward he spent 18months as a post doc with Don Caspar and Carolyn Cohen at the Children's Hospital, Boston where he continued the work on TMV and also showed that the core of the thick filament of byssus retractor muscle from mussels is made of two-stranded alpha-helical coiled-coils. Returning to England he joined Aaron Klug's group at the newly founded Laboratory of Molecular Biology in Cambridge. Besides continuing the TMV studies, which were aimed at calculating the three-dimensional density map of the virus, he collaborated with Pringle's group in Oxford to show that two conformation of the myosin cross-bridge could be identified in insect flight muscle. In 1968 he opened the biophysics department at the Max Planck Institute for Medical Research in Heidelberg, Germany. With Gerd Rosenbaum he initiated the use of synchrotron radiation as a source for X-ray diffraction. In his lab the TMV structure was pushed to 4A resolution and showed how the RNA binds to the protein. With his co-workers he solved the structure of g-actin as a crystalline complex and then solved the structure of the f-actin filament by orientating the g-actin structure so as to give the f-actin fiber diffraction pattern. He was also able to solve the structure of the complex of actin with tropomyosin from fiber diffraction.

The shape and flexibility of tropomyosin coiled coils: implications for actin filament assembly and regulation.

Wrapped superhelically around actin filaments, the coiled-coil alpha-helices of tropomyosin regulate muscle contraction by cooperatively blocking or exposing myosin-binding sites on actin. In non-muscle cells, tropomyosin additionally controls access of actin-binding proteins involved in cytoskeletal actin filament maintenance and remodeling. Tropomyosin's global shape and flexibility play a key role in the assembly, maintenance, and regulatory switching of thin filaments yet remain insufficiently characterized. Here, electron microscopy and molecular dynamics simulations yielded conformations of tropomyosin closely resembling each other. The electron microscopy and simulations show that isolated tropomyosin has an average curved conformation with a design well matched to its superhelical shape on F-actin. In addition, they show that tropomyosin bends smoothly yet anisotropically about its distinctive helically curved conformation, without any signs of unfolding, chain separation, localized kinks, or joints. Previous measurements, assuming tropomyosin to be straight on average, mistakenly suggested considerable flexibility (with persistence lengths only approximately 3 times the protein's length). However, taking the curved average structure determined here as reference for the flexibility measurements yields a persistence length of approximately 12 lengths, revealing that tropomyosin actually is semirigid. Corresponding simulation of a triple mutant (A74L-A78V-A81L) with weak actin affinity shows that it lacks shape complementarity to F-actin. Thus, tropomyosin's pre-shaped semirigid architecture is essential for the assembly of actin filaments. Further, we propose that once bound to thin filaments, tropomyosin will be stiff enough to act as a cooperative unit and move on actin in a concerted way between known regulatory states.

Curvature variation along the tropomyosin molecule.

Complementarity between the tropomyosin supercoil and the helical contour of actin-filaments is required for the binding interaction of actin and tropomyosin (Li et al., 2010). Clusters of small alanine residues in place of canonical leucines along coiled-coil tropomyosin may be responsible for pre-shaping tropomyosin and promoting conformational complementarity to F-actin. A longitudinal displacement between the two chains of the tropomyosin coiled-coil induced by the alanine clusters could produce localized bending or limited flexibility along tropomyosin needed to shape tropomyosin (Brown and Cohen, 2005). To evaluate the influence of alanine clusters on tropomyosin curvature, we calculated the longitudinal displacement between amino acid residues on adjacent chains of the tropomyosin coiled-coil and related this "Z-displacement" to the position of the alanine clusters. Measurements were made on high-resolution crystal structures of tropomyosin fragments and on trajectories from molecular dynamics simulations of full-length alphaalpha-tropomyosin. We found no strict one-for-one spatial correlation between alanine cluster position and the Z-displacement. Neither did we find any direct correspondence between the clusters and the local curvature of tropomyosin. Rather than just causing specific local structural effects, the overall influence of alanine clusters is complex and delocalized, leading to a gradually changing bending pattern along the length of tropomyosin.

Gestalt-binding of tropomyosin to actin filaments.

We argue that the overall behavior of tropomyosin on F-actin cannot be easily discerned by examining thin filaments reduced to their smallest interacting units. In isolation, the individual interactions of actin and tropomyosin, by themselves, are too weak to account for the specificity of the system. Instead the association of tropomyosin on actin can only be fully explained after considering the concerted action of the entire acto-tropomyosin system. We propose that the low K ( a ) describing tropomyosin:actin interaction, when taken together with the form-fitting complementarity of tropomyosin strands on F-actin and the tendency for tropomyosin to polymerize end-to-end, make possible unique thin filament functions both locally and at higher levels of filament organization.

A comparison of muscle thin filament models obtained from electron microscopy reconstructions and low-angle X-ray fibre diagrams from non-overlap muscle.

The regulation of striated muscle contraction involves changes in the interactions of troponin and tropomyosin with actin thin filaments. In resting muscle, myosin-binding sites on actin are thought to be blocked by the coiled-coil protein tropomyosin. During muscle activation, Ca2+ binding to troponin alters the tropomyosin position on actin, resulting in cyclic actin-myosin interactions that accompany muscle contraction. Evidence for this steric regulation by troponin-tropomyosin comes from X-ray data [Haselgrove, J.C., 1972. X-ray evidence for a conformational change in the actin-containing filaments of verterbrate striated muscle. Cold Spring Habor Symp. Quant. Biol. 37, 341-352; Huxley, H.E., 1972. Structural changes in actin and myosin-containing filaments during contraction. Cold Spring Habor Symp. Quant. Biol. 37, 361-376; Parry, D.A., Squire, J.M., 1973. Structural role of tropomyosin in muscle regulation: analysis of the X-ray diffraction patterns from relaxed and contracting muscles. J. Mol. Biol. 75, 33-55] and electron microscope (EM) data [Spudich, J.A., Huxley, H.E., Finch, J., 1972. Regulation of skeletal muscle contraction. II. Structural studies of the interaction of the tropomyosin-troponin complex with actin. J. Mol. Biol. 72, 619-632; O'Brien, E.J., Gillis, J.M., Couch, J., 1975. Symmetry and molecular arrangement in paracrystals of reconstituted muscle thin filaments. J. Mol. Biol. 99, 461-475; Lehman, W., Craig, R., Vibert, P., 1994. Ca2+-induced tropomyosin movement in Limulus thin filaments revealed by three-dimensional reconstruction. Nature 368, 65-67] each with its own particular strengths and limitations. Here we bring together some of the latest information from EM analysis of single thin filaments from Pirani et al. [Pirani, A., Xu, C., Hatch, V., Craig, R., Tobacman, L.S., Lehman, W. (2005). Single particle analysis of relaxed and activated muscle thin filaments. J. Mol. Biol. 346, 761-772], with synchrotron X-ray data from non-overlapped muscle fibres to refine the models of the striated muscle thin filament. This was done by incorporating current atomic-resolution structures of actin, tropomyosin, troponin and myosin subfragment-1. Fitting these atomic coordinates to EM reconstructions, we present atomic models of the thin filament that are entirely consistent with a steric regulatory mechanism. Furthermore, fitting the atomic models against diffraction data from skinned muscle fibres, stretched to non-overlap to preclude crossbridge binding, produced very similar results, including a large Ca2+-induced shift in tropomyosin azimuthal location but little change in the actin structure or apparent alteration in troponin position.

The actin-binding cleft: functional characterisation of myosin II with a strut mutation.

The myosin cross-bridge has two essential properties: to undergo the "power stroke" and to bind and release from actin - both under control of ATP binding and hydrolysis. In the absence of ATP the cross-bridge binds to actin with high affinity: the binding of ATP causes rapid release of the cross-bridge from actin. The actin binding-site is split by a deep cleft that closes on strong binding to actin. The cleft is straddled by a short polypeptide known as the "strut". In the following we summarise the structural basis of the power stroke and the control of actin affinity and then present data on the effects on actin affinity of replacing the strut by a flexible linker.

Structural mechanism of the recovery stroke in the myosin molecular motor.

The power stroke pulling myosin along actin filaments during muscle contraction is achieved by a large rotation ( approximately 60 degrees ) of the myosin lever arm after ATP hydrolysis. Upon binding the next ATP, myosin dissociates from actin, but its ATPase site is still partially open and catalytically off. Myosin must then close and activate its ATPase site while returning the lever arm for the next power stroke. A mechanism for this coupling between the ATPase site and the distant lever arm is determined here by generating a continuous series of optimized intermediates between the crystallographic end-states of the recovery stroke. This yields a detailed structural model for communication between the catalytic and the force-generating regions that is consistent with experimental observations. The coupling is achieved by an amplifying cascade of conformational changes along the relay helix lying between the ATPase and the domain carrying the lever arm.

Electron cryo-microscopy shows how strong binding of myosin to actin releases nucleotide.

Muscle contraction involves the cyclic interaction of the myosin cross-bridges with the actin filament, which is coupled to steps in the hydrolysis of ATP. While bound to actin each cross-bridge undergoes a conformational change, often referred to as the "power stroke", which moves the actin filament past the myosin filaments; this is associated with the release of the products of ATP hydrolysis and a stronger binding of myosin to actin. The association of a new ATP molecule weakens the binding again, and the attached cross-bridge rapidly dissociates from actin. The nucleotide is then hydrolysed, the conformational change reverses, and the myosin cross-bridge reattaches to actin. X-ray crystallography has determined the structural basis of the power stroke, but it is still not clear why the binding of actin weakens that of the nucleotide and vice versa. Here we describe, by fitting atomic models of actin and the myosin cross-bridge into high-resolution electron cryo-microscopy three-dimensional reconstructions, the molecular basis of this linkage. The closing of the actin-binding cleft when actin binds is structurally coupled to the opening of the nucleotide-binding pocket.