Browsing by Subject "Myosin"

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Dissecting Transient Protein Interactions Implicated in Cardiovascular Disease: G Protein-Coupled Receptors and Cardiac Myosin-Binding Protein C
(2021-10) Touma, Anja
Weak, transient protein-protein interactions in the cell are being increasingly appreciated, yet characterization of these interactions presents a unique challenge. We have used protein engineering techniques, including ER/K α-helical linkers and DNA nanotechnology, to characterize G protein-coupled receptor (GPCR) and cardiac myosin-binding protein C (cMyBP-C) interactions.The cellular environment can have a significant impact on GPCR signaling and functional selectivity. Our lab has found that GPCR interactions with non-cognate G-proteins can enhance, or ‘prime’, signaling through the canonical pathway. To investigate the impact of non-cognate interactions on signaling in two promiscuous Gi-coupled receptors, adenosine type 1 (A1R) and cannabinoid type 1 (CB1), we utilized a variation of the Systematic Protein Affinity Strength Modulation (SPASM) approach to observe the impact on downstream signaling in live cells. To the C-terminus of intact A1R or CB1, we tethered native G-peptides (s-pep, i-pep, and q-pep) derived from the Gα subunit of G-proteins. We found that i-pep and q-pep enhanced Gi signaling while suppressing Gq signaling. This study provides an initial model for the impact of G-peptide interactions in Gi-coupled receptors, and highlights the potential of G-peptide interactions to enhance receptor specificity. CMyBP-C is an important regulator of cardiac muscle contraction and is commonly implicated in hypertrophic cardiomyopathy (HCM). However, the mechanism of regulation by cMyBP-C remains unclear due to experimental challenges in dissecting these weak, transient interactions. In this study we utilized a nanosurf assay, containing a synthetic β-cardiac myosin thick filament, to systematically probe cMyBP-C interactions with actin and myosin. We recapitulated inhibition of β-cardiac myosin HMM nanotube motility by C0-C2 and C1-C2 N-terminal fragments. Equivalent inhibition of an β-cardiac myosin S1 construct suggests the actin-cMyBP-C interaction dominates this inhibitory mechanism. We found that a C0-C1f fragment lacking the majority of the M-domain did not inhibit β-cardiac myosin nanotube motility, confirming the importance of the M-domain in regulatory interactions. Release of inhibition by phosphomimetic fragments further highlights the importance of the phosphorylatable serines in the regulatory M-domain. These results shed light on the mechanism of cMyBP-C and highlight the utility of the nanosurf assay for precisely manipulating and defining transient protein interactions.
Elucidating the Structural Dynamics of Muscle Myosin Using Novel Methods in Electron Paramagnetic Resonance
(2016-11) Binder, Benjamin
Muscle contraction is fundamentally driven by an interaction between two proteins: actin and myosin. Myosin is a molecular motor, and assumes the active role in this relationship, coupling energy from hydrolysis of ATP with conformational changes to generate force on actin. In the context of a muscle fiber, this force causes filaments of myosin and actin to slide past one another in an ordered lattice, drawing the ends of individual contractile units (called sarcomeres) together. Concerted shortening of sarcomeres along the length of a fiber results in large-scale shortening of the entire fiber. Although muscle myosin has been the focus of intense study for many years, crucial details regarding its mechanism remain unknown. In particular, few structures of actin and myosin together have been reported—this is largely due to the inherent difficulties of handling large, filamentous protein complexes in traditional methods for structure determination. Myosin's interactions with actin are absolutely essential for macroscopic function, and this lack of structural information has created a knowledge gap: there is an abundance of functional and kinetic data for myosin in both normal and pathological states, but often no direct insight into the underlying structural causes for the observed behavior. In the present work, I seek to address this knowledge gap by providing high-resolution insight into the structural states of actin-bound myosin. My work is based on the hypothesis that allosteric coupling in myosin's catalytic domain (the domain responsible for actin binding, ATP hydrolysis, and initiation of force-generating conformational change) is accomplished via subtle internal rearrangements of individual structural elements. Furthermore, I hypothesize that these changes can be detected and quantified by innovative applications of site-directed spectroscopy. In Chapter 4, I establish a method using electron paramagnetic resonance (EPR) of a bifunctional spin label to probe nucleotide-dependent changes in the actomyosin complex. In Chapter 5, this method is expanded to include two complementary EPR techniques, ultimately providing sufficient constraints for direct modeling of nucleotide-dependent changes. Following these results, Chapter 6 addresses the ongoing development and further application of these methods within myosin and other protein systems.
Myosin structural dynamics: mechanistic insights and therapeutic technology developments
(2019-03) Rohde, John
A major focus in molecular biophysics is to understand how protein structural isomerizations correspond to cellular and organismal physiology. The heart generates force to perfuse the body with oxygenated blood through contractile units in myocytes called sarcomeres. The primary force-generating protein in this contractile apparatus is myosin. Our lab has developed a strategic tool called transient time-resolved FRET, (TR)2FRET, to measure directly, with sub-nanometer and sub-millisecond resolution, the structural and biochemical kinetics of muscle myosin. This tool allows us to directly determine how myosin’s power stroke is coupled to the thermodynamic drive for force generation—the entropically-favored dissociation of inorganic phosphate. My research revealed that actin initiates the force-generating power stroke before phosphate dissociation, revealing how power output and efficiency are regulated by the distribution of myosin’s structural states. (TR)2FRET is also a powerful tool to examine small-molecule perturbations of structural transitions within myosin’s kinetic cycle. Omecamtiv mecarbil (OM), a putative heart failure therapeutic, increases cardiac contractility. My results demonstrate that OM stabilizes myosin’s pre-powerstroke structural state and significantly slows the actin-induced powerstroke. I also used transient biochemical and structural kinetics to elucidate the molecular mechanism of mavacamten, an allosteric cardiac myosin inhibitor and prospective therapeutic for hypertrophic cardiomyopathy. I found that mavacamten stabilizes an auto-inhibited state of two-headed cardiac myosin, not present in the single-headed myosin motor fragment. From these results, we predicted that cardiac myosin is regulated by an interaction between its two heads and the thick filament, and proposed that mavacamten stabilizes this state. I also investigated two mutations in the converter domain of myosin V to examine how point mutations alter specific structural transitions in the myosin motor’s ATPase cycle. Transient kinetics analyses and FRET-based experiments demonstrated that one mutation slowed the recovery-stroke rate constants, while a second mutation enhanced these steps. These mutations correspond to human mutations that give rise to dilated or hypertrophic cardiomyopathies, respectively. Together these experiments reveal new and important mechanistic insights into myosin’s structural dynamics and provide proof-of-concept results for developing therapeutic technology.
Phosphorylation-induced structural changes in smooth muscle myosin.
(2010-07) Kast, David John Edward
Activation of smooth muscle myosin (SMM) requires phosphorylation of the myosin's regulatory light chain (RLC) to relieve autoinhibitory head-head interactions, but the structural basis of this mechanism in unknown. There are no crystal structures of any fragment of SMM, and there are no crystal structures of any RLC that contains the N-terminal 24 amino acids required for phosphorylation. Site-directed spin labeling of this N-terminal segment, referred to as the phosphorylation domain (PD), showed that phosphorylation increases α-helicity, mobility and solvent accessibility of the PD. A model emerged, where the unphosphorylated RLC is compact with a disordered PD, and phosphorylation causes the PD extend away from the RLC while inducing helical ordering. The goal of this research is to test the hypothesis that the PD functions as a structural switch that changes the structure of RLC upon phosphorylation, and to define these structural changes in atomic detail. Complementary fluorescence resonance energy transfer (FRET) experiments and molecular dynamics (MD) simulations were performed to elucidate structural changes in the phosphorylation domain (PD) of smooth muscle regulatory light chain (RLC). MD simulations on the isolated PD reveal disorder-to-order transition, where residues K11-Q15 are disordered in the unphosphorylated PD but completely α-helical in the phosphorylated PD. A salt bridge formed between R16 and the phosphorylated S19 promotes ordering by stabilizing α-helicity and reducing conformational fluctuations. Consequently, this disorder-to-order transition is regulated by delicate balance between enthalpy and entropy. To elucidate the structural changes of the PD in context with the RLC bound to smooth muscle myosin, donor-acceptor pairs of probes were attached to three site-directed di-Cys mutants of RLC, each having one Cys at position 129 in the C-terminal lobe and the other at position 2, 3, or 7 in the N-terminal PD. Labeled RLC was reconstituted onto myosin S1. Time-resolved FRET demonstrated two simultaneously resolved structural states of the RLC, closed and open, which are present in both unphosphorylated and phosphorylated biochemical states. All three FRET pairs show that phosphorylation shifts the equilibrium toward the open state, increasing its mole fraction by 23%. Molecular dynamics simulations agree with FRET data in remarkable detail, supporting the coexistence of two structural states, with phosphorylation shifting the system toward a more open and mobile structure. This agreement between experiment and simulation validates the additional structural details provided by the MD simulations: In the closed state, the PD is bent onto the surface of the C-terminal lobe, stabilized by two specific interdomain salt bridges. In the open state, the PD is more helical and straight, resides farther from the C-terminal lobe, and is stabilized by a specific intradomain salt bridge. The closed and open states are also present in phosphorylated HMM, while unphosphorylated HMM possess the closed state an intermediate distance distribution. Phosphorylation forces the PD to adopt S1-like states without increasing the mean separation of the two myosin heads. The result is a vivid atomic-resolution model of the molecular mechanism by which phosphorylation activates smooth muscle.
Site-directed modifications of myosin
(2013-06) Moen, Rebecca Jane
Myosins are a diverse class of molecular motors responsible for movement in all eukaryotic cells. The conversion of chemical energy from ATP hydrolysis into mechanical force produces movement along an actin filament. The mechanism of movement is involved in muscle contraction as well as various cellular processes including cytokinesis, adhesion, and vesicle transport. All myosins contain three functionally important domains: the catalytic head domain (CD), the light chain or lever arm domain (LCD), and the tail. The catalytic head domain is very similar between all classes of myosins, containing the site of ATP binding and hydrolysis and the actin-binding interface. The tail domain of myosins are highly divergent, containing either coiled-coil domains, individual subdomains, or both, that confer each myosin's specific function and cellular localization. The biochemical steps of ATP hydrolysis in myosins are accompanied by a sequence of structural transitions. A large-scale conformational change within the myosin molecule occurs where the LCD, functioning as a lever arm, rotates relative to CD. In muscle myosin, this large-scale conformation change is associated with a transition of the actomyosin complex from a state of disordered, weak actin binding to a state of ordered, strong actin binding. This research focuses on two functionally important domains within the myosin molecule: the catalytic domain and the tail domain. First, the structural transitions that occur within the myosin II catalytic domain during the actomyosin ATPase cycle are investigated using a combination of biochemical and spectroscopic approaches, specifically studying how various chemical modifications (chemical crosslinking, oxidative modifications including methionine oxidation and glutathionylation) produce functional and structural changes. Chemical crosslinking is used to capture a dynamic intermediate in the myosin ATPase cycle, resembling a weak binding state, which is defective in actomyosin functional interaction and is dynamically disordered when bound to oriented actin. In vitro oxidative modification of the myosin catalytic domain, as a model for aging and oxidative stress in muscle shows chemical, functional, and structural perturbations are predominantly caused by a specific methionine residue in the actomyosin binding interface. These combined results illustrate a crucial role in proper actin binding cleft structural dynamics in myosin function. Modification of dynamics in this region, either by crosslinking or oxidation at critical residues in cleft, affect muscle function by interfering with the critical structural transitions necessary for actomyosin functional interaction. The focus then shifts to the tail domain of myosin VII, again using biochemical and spectroscopic approaches to elucidate the functional and structural properties of a myosin tail subdomain, the MyTH/FERM domain.
The structural dynamics of force generation in muscle, probed by electron paramagnetic resonance of bifunctionally labeled myosin.
(2009-05) Thompson, Andrew Russell
Two proteins in muscle, actin and myosin, are the key structural components that interact in order to produce muscle contraction. Myosin is a molecular motor that utilizes the chemical energy of ATP to undergo conformational changes that translate actin linearly, resulting in mechanical work. While previous studies have provided high-resolution measurement of these structural changes, many are unable to do so in intact muscle or in systems where myosin and actin can interact. This project seeks to make high-resolution structural measurements of myosin in actomyosin complexes during the different biochemical states associated with contraction. These measurements are being made using electron paramagnetic resonance (EPR), a spectroscopic technique sensitive to protein dynamics and orientation. In order to study myosin with EPR, a spin label is chemically attached to cysteine within the protein structure. In certain cases, native cysteines are used for spin labeling whereas in others, mutant protein is created with cysteines engineered in desired locations, a process known as site-directed spin labeling.Traditional spin probes attach via a single, flexible bond. This monofunctional attachment limits the sensitivity of EPR to protein orientation and dynamics because the resultant spectra are a mixture of probe and protein states. This project, on the other hand, uses a novel bifunctional spin label that is rigidly coupled to the protein via attachment to two engineered cysteines. Due to this rigid coupling, high-resolution structural measurements can be made with a degree of sensitivity not available to other techniques.
Structural dynamics of the myosin force-generating region.
(2009-12) Agafonov, Roman
Myosin is a molecular motor that generates force on actin using energy from ATP hydrolysis. Myosin plays a key role in muscle contraction and is responsible for a variety of motility processes at the cellular level. It works cyclically, changing its conformation during the power stroke and the recovery stroke. X-ray crystallography has provided information about the structural organization of myosin in different biochemical states (as defined by bound nucleotide), inspiring several structural models that could explain the molecular mechanism of myosin's function. Spectroscopy, in combination with site-directed labeling and transient experiments, can test and refine these models and provide information about myosin's dynamic properties. The goal of this project was to determine the structural dynamics of the myosin force-generating domain and study coupling mechanisms between this domain and the myosin active site. We have chosen Dictyostelium discoideum (Dicty) as our experimental system since it provides multiple advantages in comparison with the muscle myosin. In particular, it is possible to manipulate the Dicty DNA sequence, engineering labeling sites at desired locations and introducing functional mutations at the points of interest. As a first part of the project, we have tested Dicty myosin in comparison with myosin purified from rabbit skeletal muscle, and have shown that structural changes in the force-generating domain of Dicty and rabbit myosin are identical. We then focused on specific elements within the force-generating domain, relay helix and relay loop, as these elements appeared to be crucial for interdomain coupling and force generation. Using time-resolved EPR and FRET, we have developed a spectroscopic approach to determine the conformation of the relay helix. We have also developed a novel technique that we called transient time-resolved FRET [(TR)2FRET], which allowed us to monitor structural changes within the relay helix in real time. We then studied the relationship between the state of the myosin active site (which is determined by the bound ligand) and the structure of the relay helix. To obtain insights about regulatory mechanisms, we have investigated the effect of a mutation that is known to abolish myosin motor function, despite leaving enzymatic activity intact. These experiments revealed important coupling mechanisms between the relay loop and relay helix, providing a structural explanation for the previously observed functional effects and a model for power stroke activation in myosin.
Structural transitions of myosin associated with force generation in spin-labeled muscle fibers.
(2012-06) Mello, Ryan Nicholas
Muscle contraction is driven by the actin-activated hydrolysis of ATP by myosin, resulting in the relative sliding of actin and myosin filaments. Current models propose that filament sliding is driven by a structural transition of myosin’s catalytic domain (CD) and light chain domain (LCD). The goal of this research is to measure structural transitions of myosin II (muscle and nonmuscle) that are associated for force generation. Structural measurements were made using electron paramagnetic resonance (EPR) spectroscopy. This work is comprised of two separate, but related, projects. In the first project (Chapter 3), thiol crosslinking and EPR were used to resolve structural transitions of myosin’s LCD and CD that are associated with force generation. Spin labels were incorporated into the LCD of muscle fibers by exchanging spin-labeled regulatory light chain (RLC) for endogenous RLC, with full retention of function. LCD orientation and dynamics were measured in three biochemical states: relaxation (A.M.T), post-hydrolysis intermediate (A.M.D.P), and rigor (A.M.D). To trap myosin in a structural state analogous to the elusive post-hydrolysis ternary complex A.M.D.P, we used pPDM to crosslink SH1 (Cys707) to SH2 (Cys697) on the CD. EPR showed that the LCD of crosslinked fibers has an orientational distribution intermediate between relaxation and rigor, and saturation transfer EPR revealed slow rotational dynamics indistinguishable from that of rigor. Similar results were obtained for the CD using a bifunctional spin label to crosslink SH1 to SH2, but the CD was more disordered than the LCD. We conclude that SH1-SH2 crosslinking traps a state in which both the LCD and CD are in a structural state intermediate between relaxation (highly disordered and microsecond dynamics) and rigor (highly ordered and rigid), supporting the hypothesis that the crosslinked state is an A.M.D.P analog on the force generation pathway. In the second project, we present a method for obtaining high-resolution structural information of proteins using EPR of a bifunctional spin label (BSL). Two complimentary EPR techniques were employed to measure dynamics and orientation (conventional EPR) and intraprotein distances (dipolar electron-electron resonance). The exploitation of BSL is a key feature of this work. BSL attaches at residue positions i and i+4, which drastically restricts probe motion compared to monofunctional probes. For comparison, measurements were also made with the monofunctional spin label MSL. Subfragment 1 of Dictyostelium myosin II (S1dC) was used to exemplify the increased resolution provided by BSL. Using this approach, we demonstrate with experiments that BSL significantly increases resolution when measuring distance and orientation compared to MSL. And while this work does focus on the methodology, there is significant biological insight into myosin’s nucleotide-dependent structural transitions.
An unconventional myosin is necessary for chemotaxis in Dictyostellium discoideum.
(2009-08) Breshears, Laura Marie
Directed cell migration (chemotaxis) is a fundamental biological process necessary for embryonic development, wound healing, and proper function of the immune system. Chemotaxis also plays a significant role in many developmental disorders and post-embryonic diseases in humans, such as cancer. Chemotaxis is driven by extracellular cues that act, in large part, to induce changes in the actin cytoskeleton, such as actin polymerization, that facilitate directed cell migration. Myosins are actin-associated motors that have a variety of functions in different cellular contexts. Myosins can effect cortical tension, pseudopod and filopodia formation, phagocytosis, the function of sensory structures, and the basic mechanics of cell motility. Members of the MyTH/FERM family of unconventional myosins all have roles in actin-based processes and one member, vertebrate myosin X, has recently been shown to play a role in actin dynamics in response to extracellular migration cues. The social amoeba Dictyostelium discoideum is a powerful model system for dissecting chemoattractant signaling pathways and identifying the cytoskeletal components necessary for directed cell migration. MyoG is a novel unconventional myosin characterized by two MyTH/FERM domains in its tail region. The potential role of this myosin in Dictyostelium cell migration was investigated by analyzing the phenotype of three independent myoG null mutants. The initial stages of Dictyostelium development, induced by starvation, depend on chemotaxis to cAMP, resulting in the formation of a multi-cellular aggregate. Upon starvation myoG — cells fail to aggregate, arresting as a smooth monolayer of cells. The myoG — cells neither polarize in a cAMP gradient nor do they chemotax toward the cAMP source. Analysis of the ability of myoG — cells to polymerize actin in response to cAMP revealed that the response is dampened in the mutants. myoG — cells are also defective in signaling to PI3K in response to cAMP. These data show that while the mutant cells retain some ability to respond to the gradient, the major pathways regulating polarity and chemotaxis are not functional. The mutant phenotype suggests that MyoG acts in transducing the chemotactic signal from the cAMP receptor to PI3K and the actin cytoskeleton, facilitating the morphological changes that lead to polarization and directional migration. The role of MyoG in chemotactic signaling represents a novel function for an unconventional myosin. The work presented here clearly demonstrates that MyoG is necessary for signaling from the cAMP receptor to both PI3K and the actin cytoskeleton. Sequence analysis shows that there is no direct homologue of MyoG in other organisms, but the high degree of conservation of the chemotactic signaling pathways indicates that there are likely to be functional homologues in higher eukaryotic cells, such as neutrophils, that rely on chemotaxis for cellular function.

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