Experimental Investigation of the Seesaw Mechanism of the Relay Region That Moves the Myosin Lever Arm

Kintses, Bálint; Yang, Zhenhui; Málnási‐Csizmadia, András

doi:10.1074/jbc.m805848200

Cited by 20 publications

(18 citation statements)

References 27 publications

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“…The lever swing in ATP (recovery step) and ADP–P i states (futile lever swing), associated with switch 2 conformational changes, has been proposed to involve a seesaw-like movement of the long relay helix within the motor domain [12]. In the middle of the relay helix, a phenylalanine cluster forms a fulcrum around which the tilt might occur [12,13]. This cluster structurally connects the relay/converter and transducer regions.…”

Section: Futile Lever Swing Is Kinetically Blockedmentioning

confidence: 99%

Emerging complex pathways of the actomyosin powerstroke

Málnási‐Csizmadia

Kovács

2010

Trends in Biochemical Sciences

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Actomyosin powers muscle contraction and various cellular activities including cell division, differentiation, intracellular transport and sensory functions. Despite their crucial roles, key aspects of force generation have remained elusive. To perform efficient force generation, the powerstroke must occur while myosin is bound to actin. Paradoxically, this process must be initiated when myosin is in a very low actin-affinity state. Recent results shed light on a kinetic pathway selection mechanism whereby the actin-induced activation of the swing of myosin's lever enables efficient mechanical functioning. Structural elements and biochemical principles involved in this mechanism are conserved among various NTPase-effector (e.g. kinesin-microtubule, G protein-exchange factor and kinase-scaffold protein) systems that perform chemomechanical or signal transduction. Allosteric activation in motor and signaling systemsActomyosin is the best-understood of the ubiquitous mechanochemical systems of eukaryotic organisms [1,2]. The key action of the actomyosin mechanochemical cycle is the powerstroke, a rowing action of the myosin head which results from the swing of its lever (Box 1). To produce effective translocation along actin, the lever swing must occur with the myosin head in an actin-bound state (Fig. 1); the occurrence of a futile lever swing in an actin-detached state leads to an ATP-wasting cycle (Fig. 1). The powerstroke can be viewed as the result of three linked events: binding of the myosin head to actin, structural changes in the head leading to a strong actomyosin interaction, and the lever swing. These events lead to the release of ATP hydrolysis products, P i (inorganic phosphate) and ADP. Box 1 Myosin structural elementsMyosin is a P-loop NTPase, and diverse isoforms of myosin are divided into at least 35 classes [5]. Myosin has three functional parts: the motor domain, the lever and the tail region (Fig. IA). The heavy chain subunit forms the motor domain, the axis of the lever, and the tail, whereas the light chains stabilize the structure of the lever. In some myosin classes, further light chains bind to the tail region. The tail generally confers effector functions and, in some classes, is responsible for heavy chain dimerization. In the myosin II class, which includes the muscle myosins, the tail is a long coiled-coil rod responsible for filament formation. The motor domain confers the mechanoenzymatic function of

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Section: Futile Lever Swing Is Kinetically Blockedmentioning

confidence: 99%

Emerging complex pathways of the actomyosin powerstroke

Málnási‐Csizmadia

Kovács

2010

Trends in Biochemical Sciences

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“…We showed that in Dictyostelium myosin II, actin binding in the presence of ATP straightens the relay helix before dissociation of hydrolyzed phosphate (5). If the straight structural state of the relay helix is tightly coupled to the postpower stroke orientation of the LCD as proposed (14,15), then actin should induce LCD rotation before phosphate release. This hypothesis disagrees with several existing proposals (2, 4) but agrees with recent studies performed in isolated muscle fibers (6).…”

Section: Significancementioning

confidence: 99%

“…The structure of this helix is allosterically coupled to interactions between switch-2 and bound ATP, ADP, and hydrolyzed phosphate (14,15). Movement of the relay helix is hypothesized to be tightly coupled to LCD rotation (14)(15)(16).…”

Section: Significancementioning

confidence: 99%

Direct real-time detection of the structural and biochemical events in the myosin power stroke

Muretta

Rohde

Johnsrud

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

156

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A principal goal of molecular biophysics is to show how protein structural transitions explain physiology. We have developed a strategic tool, transient time-resolved FRET [(TR) 2 FRET], for this purpose and use it here to measure directly, with millisecond resolution, the structural and biochemical kinetics of muscle myosin and to determine directly how myosin's power stroke is coupled to the thermodynamic drive for force generation, actin-activated phosphate release, and the weak-to-strong actin-binding transition. We find that actin initiates the power stroke before phosphate dissociation and not after, as many models propose. This result supports a model for muscle contraction in which power output and efficiency are tuned by the distribution of myosin structural states. This technology should have wide application to other systems in which questions about the temporal coupling of allosteric structural and biochemical transitions remain unanswered.FRET | myosin | power stroke | phosphate release | structural kinetics M yosin family proteins use ATP hydrolysis to generate force and movement required for normal physiology. They drive muscle contraction, help control cell division and cellular motility, move organelles through the cytoplasm, and are important elements of the cellular mechanical-sensing machinery (1, 2). The key to understanding how myosin and related enzymes function in cells, and how to modulate their activity to treat disease, is to determine how the protein's structural dynamics and biochemical kinetics are coupled. Although high-resolution crystal structures provide best-guess snapshots of protein structure over a range of biochemical states, determining the physiological relevance of these snapshots remains one of the central challenges of structural biophysics.How myosin generates force remains debated despite more than 50 y of intense research (1-3). The most popular current model (2, 4) proposes that after ATP hydrolysis, myosin interacts weakly with actin and this interaction initiates an ordered series of structural and biochemical transitions that culminate in the dissociation of hydrolyzed phosphate, followed by the isomerization of the actin-binding interface to a state that binds actin with nanomolar affinity and then the rotation of the myosin lightchain domain (LCD) toward the plus end of the actin filament. This rotation converts the thermodynamic energy of phosphate release and actin binding into mechanical energy that performs work. A number of results question this model, however, including spectroscopic data showing that a structural transition in the myosin relay helix, hypothesized to be coupled to LCD rotation, precedes P i release (5) and force development precedes P i release in muscle fibers (6).Determining how these events take place in solution and in cells is an important question, because (i) differences in the mechanics of different myosins likely reflect differences in how the biochemical and structural transitions described above are coordinated (4); (ii) disea...

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“…S1 shows a simple illustration of the method. Examples of applications include a bacterial pore (21) and myosin (5), for which the transition mechanism found by CPR was subsequently verified by experiment (22).…”

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confidence: 99%

Unsuspected pathway of the allosteric transition in hemoglobin

Fischer

Olsen

Nam

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Large conformational transitions play an essential role in the function of many proteins, but experiments do not provide the atomic details of the path followed in going from one end structure to the other. For the hemoglobin tetramer, the transition path between the unliganded (T) and tetraoxygenated (R) structures is not known, which limits our understanding of the cooperative mechanism in this classic allosteric system, where both tertiary and quaternary changes are involved. The conjugate peak refinement algorithm is used to compute an unbiased minimum energy path at atomic detail between the two end states. Although the results confirm some of the proposals of Perutz [Perutz MF (1970) Stereochemistry of cooperative effects in haemoglobin. Nature 228:726-734], the subunit motions do not follow the textbook description of a simple rotation of one αβ-dimer relative to the other. Instead, the path consists of two sequential quaternary rotations, each involving different subdomains and axes. The quaternary transitions are preceded and followed by phases of tertiary structural changes. The results explain the recent photodissociation measurements, which suggest that the quaternary transition has a fast (2 μs) as well as a slow (20 μs) component and provide a testable model for single molecule FRET experiments.conformational change | cooperativity | domain motion | protein hinges M any proteins undergo large conformational transitions that are essential for their functions (1-3). The transitions can occur in monomers such as the myosin molecular motor (4-6), in multisubunit complexes such as the chaperone GroEL (7), and in systems such as the flagellar motor of bacteria, which is composed of several hundred proteins (8). Probably the most studied example is the T (tense) to R (relaxed) transition in the vertebrate hemoglobin tetramer (Hb) (9-11), for which the conformational transition increases the efficiency of oxygen transport. Hemoglobin, which is composed of two identical α-and two identical β-subunits, is the paradigm for the development of models of cooperativity and allosteric regulation in proteins (9-13). Phenomenologically, the positive cooperativity involves the increase in the affinity for oxygen of unliganded subunits upon the successive binding of oxygen to other subunits. This is achieved by structural changes within subunits (tertiary changes) and between subunits (quaternary changes). Based on the superposition of the crystallographic structures for the deoxy unliganded T state and the fully liganded R state of the protein, the quaternary T → R transition has been described as involving primarily an approximately 15°rotation of one "dimer" (α 1 β 1 ) relative to the other (α 2 β 2 ) around a virtual axis (Fig. 1A) (9, 14). Together with a small relative translation of the αβ-dimers, this reduces the central cavity between the dimers (a channel along the C2 axis of symmetry), where the heterotropic effector 2,3-bisphosphoglycerate is bound in the T state. An important aspect of these structural ch...

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Experimental Investigation of the Seesaw Mechanism of the Relay Region That Moves the Myosin Lever Arm

Cited by 20 publications

References 27 publications

Emerging complex pathways of the actomyosin powerstroke

Emerging complex pathways of the actomyosin powerstroke

Direct real-time detection of the structural and biochemical events in the myosin power stroke

Unsuspected pathway of the allosteric transition in hemoglobin

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