Rigor Complex Research Articles

Complementary Use of Electron Cryomicroscopy and X-Ray Crystallography: Structural Studies of Actin and Actomyosin Filaments.

Visualization of macromolecular structures is essential for understanding the mechanisms of biological functions because they are all determined by the structure and dynamics of macromolecular complexes. Electron cryomicroscopy (cryoEM) and image analysis has become a powerful tool for structural studies because of recent technical developments in microscope optics, cryostage control, image detection and the methods of sample preparation. In particular, the recent development of CMOS-based direct electron detectors with high sensitivity, high resolution and high frame rate has revolutionized the field of structural biology by making near-atomic resolution structural analysis possible from small amounts of solution samples. However, for some biological systems, it is still difficult to reach high resolution due to somewhat flexible nature of the structure, and a complementary use of cryoEM with X-ray crystallography is essential and useful to gain mechanistic understanding of the biological functions and mechanisms. We will describe our strategy for the structural analyses of actin filament and actomyosin rigor complex and the biological insights we gained from these structures.

Cryo-EM structures reveal specialization at the myosin VI-actin interface and a mechanism of force sensitivity.

Despite extensive scrutiny of the myosin superfamily, the lack of high-resolution structures of actin-bound states has prevented a complete description of its mechanochemical cycle and limited insight into how sequence and structural diversification of the motor domain gives rise to specialized functional properties. Here we present cryo-EM structures of the unique minus-end directed myosin VI motor domain in rigor (4.6 Å) and Mg-ADP (5.5 Å) states bound to F-actin. Comparison to the myosin IIC-F-actin rigor complex reveals an almost complete lack of conservation of residues at the actin-myosin interface despite preservation of the primary sequence regions composing it, suggesting an evolutionary path for motor specialization. Additionally, analysis of the transition from ADP to rigor provides a structural rationale for force sensitivity in this step of the mechanochemical cycle. Finally, we observe reciprocal rearrangements in actin and myosin accompanying the transition between these states, supporting a role for actin structural plasticity during force generation by myosin VI.

Reconstruction of Functional Insect Flight Muscle Fibers with Rabbit Skeletal Muscle Actin

Stretch activation (SA, delayed rise of force after stretch) is a mechanism essential for the fast wing-beat of insects with asynchronous flight muscles. To determine which constituent protein(s) is (are) indispensable for SA, we have been conducting experiments to replace the endogenous actin of bumblebee flight muscle with rabbit skeletal muscle actin, and we have demonstrated that actin filaments can be regenerated as judged from X-ray diffraction patterns (2016 annual meeting). In this experiment, the endogenous actin is removed by gelsolin, and after this, rabbit G-actin is polymerized in situ. To prevent spontaneous contraction, a myosin inhibitor must be added to the solutions. For vertebrate skeletal and cardiac muscles, the preferred inhibitor is BDM (butanedione monoxime, Fujita et al., 1996). However, BDM is not an effective inhibitor for insect flight muscle. For this reason, we used blebbistatin instead, and the actin filaments were successfully restored. The problem is that the inhibition by blebbistatin is irreversible, and it is not reversed by extensive washout or irradiation with blue light. Here we used a high concentration of BDM (100 mM) and repeated the experiments. The actin filaments were restored, and unlike in the case of blebbistatin, actin-based layer line reflections were intensified after washout of BDM and ATP. This indicates that the ability of the endogenous myosin to form rigor complexes is not compromised by the use of BDM. We are currently trying to activate the flight muscle fibers prepared in this way.

Functional Analysis of Freeman-Sheldon Syndrome Causing Mutations on Embryonic Myosin

Freeman-Sheldon Syndrome (FSS) is a rare genetic disorder characterized primarily by multiple congenital contractures of facial muscles and distal joints, craniofacial abnormalities among other phenotypes. Mutations in embryonic skeletal myosin (MYH3) are the only known cause of FSS, and the vast majority of patients have one of three mutations: R672C, R672H, and T178I. The goal of this study was to determine the functional effects of these FSS- causing mutations on the human embryonic myosin motor domain. Preliminary data show a 10-fold reduction in ATP binding for both S1 (k2+) and actin-S1 (k′2+) for the R672C mutation. ADP affinity appears unaffected for this mutant. In the case of R672H, measurements of ATP binding were difficult because there was such a small change in tryptophan fluorescence (1.5% compared to 7-8% for Emb-WT). There was a 30 fold reduction in ATP binding to S1. Taken together with the reduction in tryptophan fluorescence change, we speculate a disruption in the conformational change in the recovery stroke. However when measured in the presence of actin we see values similar to Emb-WT. Interestingly, the affinity of ADP for actin-S1 is >3 fold tighter in this mutant with the rate constant for ADP release being reduced >3 fold. The affinity for actin is ∼20 fold weaker in the rigor complex and 10 fold tighter in the presence of ADP. For the T178I mutant which in the myosin structure forms a hydrogen bond with the R672 residue, we expect a similar effect in nucleotide binding. Our preliminary data shows a 5 fold reduction in ATP binding to S1 and 3 fold to actin-S1. When comparing to Emb-WT, the functional characteristics of these mutants are severely disrupted specifically in ATP induction of cross-bridges, which can explain the disease phenotype.

Kinetic Characterization of the ATPase and Actin-activated ATPase Activities of Acanthamoeba castellanii Myosin-2

Phosphorylation of Ser-639 in loop-2 of the catalytic motor domain of the heavy chain of Acanthamoeba castellanii myosin-2 and the phosphomimetic mutation S639D have been shown previously to down-regulate the actin-activated ATPase activity of both the full-length myosin and single-headed subfragment-1 (Liu, X., Lee, D. Y., Cai, S., Yu, S., Shu, S., Levine, R. L., and Korn, E. D. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, E23-E32). In the present study we determined the kinetic constants for each step in the myosin and actomyosin ATPase cycles of recombinant wild-type S1 and S1-S639D. The kinetic parameter predominantly affected by the S639D mutation is the actin-activated release of inorganic phosphate from the acto myosin·ADP·Pi complex, which is the rate-limiting step in the steady-state actomyosin ATPase cycle. As consequence of this change, the duty ratio of this conventional myosin decreases. We speculate on the effect of Ser-639 phosphorylation on the processive behavior of myosin-2 filaments.

3P147 クライオ電子顕微鏡法を用いたアクチンミオシン硬直複合体の高分解能密度マップ取得への試み(11.分子モーター,ポスター,日本生物物理学会年会第51回(2013年度))

3P147 クライオ電子顕微鏡法を用いたアクチンミオシン硬直複合体の高分解能密度マップ取得への試み(11.分子モーター,ポスター,日本生物物理学会年会第51回(2013年度))

Structural Model of the Pre-Powerstroke State of the Actomyosin Complex

We created a structural model of the ADP.Pi pre-powerstroke state of Dictyostelium myosin motor domain complexed with actin trimer by an in silico protein docking procedure followed by a long timescale molecular dynamics relaxation. Furthermore, we also modeled the ADP.Pi down lever state of myosin motor domain and the rigor complexes using 1q5q (apo structure of Dictyostelium myosin motor domain) or squid myosin S1 (3I5G) atomic structures and actin trimer model. During the molecular dynamics of the apo myosin (1q5q ) complexed with actin trimer the actin binding cleft of myosin motor domain spontaneously closed and the relaxed actomyosin rigor structure fits well with the structural rigor model determined by EM. The analysis of the ADP.Pi pre-powerstroke actomyosin complex showed that actin spontaneously induces significant conformational changes in the myosin motor domain. Most strikingly, actin further closes the closed switch 2 loop coupled with a further up movement of the lever. Interestingly, if the interaction between the N-terminal region of actin and myosin activation loop is interrupted by a single mutation (K520N) this conformational change does not occur upon actin binding. Furthermore, actin binding rearranges correlating movements of different myosin motor regions which effect was significantly reduced in the mutant. Recently we shoved experimentally that the interaction between actin and the activation loop is responsible for channeling the enzymatic pathway of actomyosin into the effective powerstroke path. The further closure of switch 2 induced by actin indicates that cocking is induced by actin mainly through the actin binding of activation loop. In order to test the indicated conformational change we have produced a myosin motor domain containg a FLASH and REASH probes located at the N and C-terminus, respectively, to sensitively follow the lever movement upon actin binding by FRET.

1PS008 クライオ電子顕微鏡法を用いたアクチン-ミオシン硬直複合体の高分解能構造への試み(日本生物物理学会第50回年会(2012年度))

1PS008 クライオ電子顕微鏡法を用いたアクチン-ミオシン硬直複合体の高分解能構造への試み(日本生物物理学会第50回年会(2012年度))

Single actomyosin motor interactions in skeletal muscle

We present a study of intramuscular motion during contraction of skeletal muscle myofibrils. Myofibrillar actin was labeled with fluorescent dye so that the ratio of fluorescently labeled to unlabeled protein was 1:105. Such sparse labeling assured that there was on average only one actin-marker present in the focus at a given time. From the intensity signal in the two orthogonal detection channels, significant fluctuations, similar to fluorescent burst in diffusion-based single-molecule detection schemes, were identified via a threshold algorithm and analyzed with respect to their intensity and polarization. When only rigor complexes were formed, the fluctuations of polarized intensity were characterized by unimodal Gaussian photon distributions. During contraction, in contrast, bimodal Gaussian photon distributions were observed above the rigor background threshold. This suggests that the bimodal Gaussian photon distributions represent pre- and post-power stroke conformations. Clusters of polarized photons indicated an anisotropy decay of single actomyosin motors of ~9s during muscle contraction.

3E1048 P07 1YE1030 低温電子顕微鏡によるアクチン繊維-ミオシンII頭部複合体の高分解能構造解析(3E 筋肉1,日本生物物理学会第49回年会)

3E1048 P07 1YE1030 低温電子顕微鏡によるアクチン繊維-ミオシンII頭部複合体の高分解能構造解析(3E 筋肉1,日本生物物理学会第49回年会)

2P156 Fアクチンのアロステリシティ : Rigor Complex形成の場合(筋肉(筋蛋白質・収縮),第48回日本生物物理学会年会)

2P156 Fアクチンのアロステリシティ : Rigor Complex形成の場合(筋肉(筋蛋白質・収縮),第48回日本生物物理学会年会)

Single Molecule Investigation of the Acto-Myosin-10 Complex Using Optical Tweezers

Recent cell biological studies of myosin-10 have revealed that myosin-10 is essential to cellular processes such as filopodia extensions and phagocytosis. Steady-state and transient kinetic biochemical studies of the ATPase cycle of a single-headed, subfragment-1 like (S1), construct of myosin-10 show that it has an intermediate duty-cycle ratio. It remains tightly bound to actin for about 16% of its total ATPase cycle time but spends around 90% associated with actin in both weak and strongly bound states. Furthermore, the acto-myosin-10-S1 complex has two ADP bound states and a surprisingly low affinity for actin, comparable to that of the rigor complex between actin and skeletal muscle myosin II. To study the mechano-chemical coupling of myosin-10, we used the actin filament sliding assay and a dual-beam, optical tweezers apparatus to perform single molecule mechanical studies. For these studies we used a two-headed, heavy meromyosin-like (HMM) construct of myosin-10 that contained a leucine-zipper at the C-terminal end to force dimerization. The actin filament gliding assay showed that myosin-10-HMM moves filaments at a velocity of ∼125 nm s-1. This is similar to the speed of intact, GFP-tagged, myosin-10 moving within filopodia of live mammalian cells (∼140 nm s-1) measured by TIRF microscopy. Optical trapping results showed that the average powerstroke size was ∼10 nm, with the rate of ATP binding of ∼1.3 μM-1 s-1. In most of the raw data traces, we observed displacements of unitary size, however at low ATP concentrations (2 μM) we also observed a number of interactions that exhibited multiple, staircase-like movements consisting of up to 3 steps per binding interaction. This behaviour is characteristic of a processive molecular motor. We will discuss these measurements in the context of mechano-chemical coupling and the functional significance in the living cell.

1P-114 アクチン-ミオシン硬直複合体の高分解能3次元像の獲得(筋肉(筋蛋白質・収縮),第47回日本生物物理学会年会)

1P-114 アクチン-ミオシン硬直複合体の高分解能3次元像の獲得(筋肉(筋蛋白質・収縮),第47回日本生物物理学会年会)

The Structural Basis for the Large Powerstroke of Myosin VI

Due to a unique addition to the lever arm-positioning region (converter), class VI myosins move in the opposite direction (toward the minus-end of actin filaments) compared to other characterized myosin classes. However, the large size of the myosin VI lever arm swing (powerstroke) cannot be explained by our current view of the structural transitions that occur within the myosin motor. We have solved the crystal structure of a fragment of the myosin VI motor in the structural state that represents the starting point for movement on actin; the pre-powerstroke state. Unexpectedly, the converter itself rearranges to achieve a conformation that has not been seen for other myosins. This results in a much larger powerstroke than is achievable without the converter rearrangement. Moreover, it provides a new mechanism that could be exploited to increase the powerstroke of yet to be characterized plus-end-directed myosin classes.

Identification and Characterization of an 8-kDa Light Chain Associated with Dictyostelium discoideum MyoB, a Class I Myosin

Dictyostelium discoideum MyoB is a single-headed class I myosin. Analysis of purified MyoB by SDS-PAGE indicated the presence of an approximately 9-kDa light chain. A tryptic digest of MyoB yielded a partial sequence for the light chain that exactly matched a sequence in a 73-amino acid, 8,296-Da protein (dictyBase number DDB0188713). This protein, termed MlcB, contains two EF-hand motifs and shares approximately 30% sequence identity with the N- and C-terminal lobes of calmodulin. FLAG-MlcB expressed in Dictyostelium co-immunoprecipitated with MyoB but not with the related class myosins and MyoD. Recombinant MlcB bound Ca2+ with a Kd value of 0.2 microm and underwent a Ca2+-induced change in conformation that increased alpha-helical content and surface hydrophobicity. Mutational analysis showed that the first EF-hand was responsible for Ca2+ binding. In the presence and absence of Ca2+ MlcB was a monomer in solution and bound to a MyoB IQ motif peptide with a Kd value of approximately 0.5 microm. A MyoB head-neck construct with a Ser to Glu mutation at the TEDS site bound MlcB and displayed an actin-activated Mg2+ ATPase activity that was insensitive to Ca2+. We conclude that MlcB represents a novel type of small myosin light chain that binds to IQ motifs in a manner comparable with a single lobe of a typical four-EF-hand protein.

The structure of the rigor complex and its implications for the power stroke

Decorated actin provides a model system for studying the strong interaction between actin and myosin. Cryo-energy-filter electron microscopy has recently yielded a 14 A resolution map of rabbit skeletal actin decorated with chicken skeletal S1. The crystal structure of the cross-bridge from skeletal chicken myosin could not be fitted into the three-dimensional electron microscope map without some deformation. However, a newly published structure of the nucleotide-free myosin V cross-bridge, which is apparently already in the strong binding form, can be fitted into the three-dimensional reconstruction without distortion. This supports the notion that nucleotide-free myosin V is an excellent model for strongly bound myosin and allows us to describe the actin-myosin interface. In myosin V the switch 2 element is closed although the lever arm is down (post-power stroke). Therefore, it appears likely that switch 2 does not open very much during the power stroke. The myosin V structure also differs from the chicken skeletal myosin structure in the nucleotide-binding site and the degree of bending of the backbone beta-sheet. These suggest a mechanism for the control of the power stroke by strong actin binding.

Myo1c is designed for the adaptation response in the inner ear.

The molecular motor, Myo1c, a member of the myosin family, is widely expressed in vertebrate tissues. Its presence at strategic places in the stereocilia of the hair cells in the inner ear and studies using transgenic mice expressing a mutant Myo1c that can be selectively inhibited implicate it as the mediator of slow adaptation of mechanoelectrical transduction, which is required for balance. Here, we have studied the structural, mechanical and biochemical properties of Myo1c to gain an insight into how this molecular motor works. Our results support a model in which Myo1c possesses a strain-sensing ADP-release mechanism, which allows it to adapt to mechanical load.

1P180 3次元クライオ電子顕微鏡像からのアクチン・ミオシン硬直複合体原子モデル構築(筋肉 : 筋蛋白・収縮)

1P180 3次元クライオ電子顕微鏡像からのアクチン・ミオシン硬直複合体原子モデル構築(筋肉 : 筋蛋白・収縮)

A structural state of the myosin V motor without bound nucleotide.

The myosin superfamily of molecular motors use ATP hydrolysis and actin-activated product release to produce directed movement and force. Although this is generally thought to involve movement of a mechanical lever arm attached to a motor core, the structural details of the rearrangement in myosin that drive the lever arm motion on actin attachment are unknown. Motivated by kinetic evidence that the processive unconventional myosin, myosin V, populates a unique state in the absence of nucleotide and actin, we obtained a 2.0 A structure of a myosin V fragment. Here we reveal a conformation of myosin without bound nucleotide. The nucleotide-binding site has adopted new conformations of the nucleotide-binding elements that reduce the affinity for the nucleotide. The major cleft in the molecule has closed, and the lever arm has assumed a position consistent with that in an actomyosin rigor complex. These changes have been accomplished by relative movements of the subdomains of the molecule, and reveal elements of the structural communication between the actin-binding interface and nucleotide-binding site of myosin that underlie the mechanism of chemo-mechanical transduction.

Motor domain mutation traps kinesin as a microtubule rigor complex.

Conventional kinesin is a highly processive, microtubule-based motor protein that drives the movement of membranous organelles in neurons. Using in vivo genetics in Drosophila melanogaster, Glu164 was identified as an amino acid critical for kinesin function [Brendza, K. M., Rose, D. J., Gilbert, S. P., and Saxton, W. M. (1999) J. Biol. Chem. 274, 31506-31514]. Glu164 is located at the beta-strand 5a/loop 8b junction of the catalytic core and projects toward the microtubule binding face in close proximity to key residues on beta-tubulin helix alpha12. Substitution of Glu(164) with alanine (E164A) results in a dimeric kinesin with a dramatic reduction in the microtubule-activated steady-state ATPase (5 s(-1) per site versus 22 s(-1) per site for wild-type). Our analysis shows that E164A binds ATP and microtubules with a higher affinity than wild-type kinesin. The rapid quench and stopped-flow results provide evidence that ATP hydrolysis is significantly faster and the precise coordination between the motor domains is disrupted. The data reveal an E164A intermediate that is stalled on the microtubule and cannot bind and hydrolyze ATP at the second head.