Insights into the Importance of Hydrogen Bonding in the γ-Phosphate Binding Pocket of Myosin: Structural and Functional Studies of Serine 236†,‡

The active site of myosin contains a group of highly conserved amino acid residues whose roles in nucleotide hydrolysis and energy transduction might appear to be obvious from the initial structural and kinetic analyses but become less clear on deeper investigation. One such residue is Ser236 (Dictyostelium discoideum myosin II numbering) which was proposed to be involved in a hydrogen transfer network during γ-phosphate hydrolysis of ATP, which would imply a critical function in ATP hydrolysis and motility. The S236A mutant protein shows a comparatively small decrease in hydrolytic activity and motility, and thus this residue does not appear to be essential. To understand better the contribution of Ser236 to the function of myosin, structural and kinetic studies have been performed on the S236A mutant protein. The structures of the D. discoideum motor domain (S1dC) S236A mutant protein in complex with magnesium pyrophosphate, MgAMPPNP, and MgADP·vanadate have been determined. In contrast to the previous structure of wild-type S1dC, the S236A·MgAMPPNP complex crystallized in the closed state. Furthermore, transient-state kinetics showed a 4-fold reduction of the nucleotide release step, suggesting that the mutation stabilizes a closed active site. The structures show that a water molecule approximately adopts the location of the missing hydroxyl of Ser236 in the magnesium pyrophosphate and MgAMPPNP structures. This study suggests that the S236A mutant myosin proceeds via a different structural mechanism than wild-type myosin, where the alternate mechanism is able to maintain near normal transient-state kinetic values

High-field high-repetition-rate sources for the coherent THz control of matter

Ultrashort flashes of THz light with low photon energies of a few meV, but strong electric or magnetic field transients have recently been employed to prepare various fascinating nonequilibrium states in matter. Here we present a new class of sources based on superradiant enhancement of radiation from relativistic electron bunches in a compact electron accelerator that we believe will revolutionize experiments in this field. Our prototype source generates high-field THz pulses at unprecedented quasicontinuous-wave repetition rates up to the MHz regime. We demonstrate parameters that exceed state-of-the-art laser-based sources by more than 2 orders of magnitude. The peak fields and the repetition rates are highly scalable and once fully operational this type of sources will routinely provide 1 MV/cm electric fields and 0.3 T magnetic fields at repetition rates of few 100 kHz. We benchmark the unique properties by performing a resonant coherent THz control experiment with few 10 fs resolution

Molecular features of the UNC-45 chaperone critical for binding and folding muscle myosin

Myosin is a motor protein that is essential for a variety of processes ranging from intracellular transport to muscle contraction. Folding and assembly of myosin relies on a specific chaperone, UNC-45. To address its substrate-targeting mechanism, we reconstitute the interplay between Caenorhabditis elegans UNC-45 and muscle myosin MHC-B in insect cells. In addition to providing a cellular chaperone assay, the established system enabled us to produce large amounts of functional muscle myosin, as evidenced by a biochemical and structural characterization, and to directly monitor substrate binding to UNC-45. Data from in vitro and cellular chaperone assays, together with crystal structures of binding-deficient UNC-45 mutants, highlight the importance of utilizing a flexible myosin-binding domain. This so-called UCS domain can adopt discrete conformations to efficiently bind and fold substrate. Moreover, our data uncover the molecular basis of temperature-sensitive UNC-45 mutations underlying one of the most prominent motility defects in C. elegans

Kinetic resolution of a conformational transition and the ATP hydrolysis step using relaxation methods with a Dictyostelium myosin II mutant containing a single tryptophan residue

The fluorescence emission intensity from a conserved tryptophan residue (W501) located in the relay loop (F466 to L516) of the Dicytostelium discoideum myosin II motor domain is sensitive to ATP binding and hydrolysis. The initial binding process is accompanied by a small quench in fluorescence, and this is followed by a large enhancement that appears coincident with the hydrolysis step. Using temperature and pressure jump methods, we show that the enhancement process is kinetically distinct from but coupled to the hydrolysis step. The fluorescence enhancement corresponds to the open-closed transition (k(obs) approximately 1000 s(-1) at 20 degrees C). From the overall steady-state fluorescence signal and the presence or absence of a relaxation transient, we conclude that the ADP state is largely in the open state, while the ADP.AlF(4) state is largely closed. At 20 degrees C the open-closed equilibria for the AMP.PNP and ADP.BeF(x) complexes are close to unity and are readily perturbed by temperature and pressure. In the case of ATP, the equilibrium of this step slightly favors the open state, but coupling to the subsequent hydrolysis step gives rise to a predominantly closed state in the steady state. Pressure jump during steady-state ATP turnover reveals the distinct transients for the rapid open-closed transition and the slower hydrolysis step

Reversible movement of switch 1 loop of myosin determines actin interaction

The conserved switch 1 loop of P-loop NTPases is implicated as a central element that transmits information between the nucleotide-binding pocket and the binding site of the partner proteins. Recent structural studies have identified two states of switch 1 in G-proteins and myosin, but their role in the transduction mechanism has yet to be clarified. Single tryptophan residues were introduced into the switch 1 region of myosin II motor domain and studied by rapid reaction methods. We found that in the presence of MgADP, two states of switch 1 exist in dynamic equilibrium. Actin binding shifts the equilibrium towards one of the MgADP states, whereas ATP strongly favors the other. In the light of electron cryo-microscopic and X-ray crystallographic results, these findings lead to a specific structural model in which the equilibrium constant between the two states of switch 1 is coupled to the strength of the actin-myosin interaction. This has implications for the enzymatic mechanism of G-proteins and possibly P-loop NTPases in genera

Myosin cleft movement and its coupling to actomyosin dissociation

It has long been known that binding of actin and nucleotides to myosin are antagonistic, an observation that led to the biochemical basis for the crossbridge cycle of muscle contraction. Thus ATP binding to actomyosin causes actin dissociation, while actin binding to the myosin accelerates ADP and phosphate release. Structural studies have indicated that communication between the actin and nucleotide binding sites involves the opening and closing of the cleft between the upper and lower 50K domains of the myosin he ad. Here we test the proposal that the cleft responds to actin and nucleotide binding in a reciprocal manner and show that cleft movement is coupled to actin binding and dissociation. We monitored cleft movement using pyrene excimer fluorescence from probes engineered across the cleft

Cycling Cross-Bridges Contribute to Thin Filament Activation in Human Slow-Twitch Fibers

It has been shown that not only calcium but also strong binding myosin heads contribute to thin filament activation in isometrically contracting animal fast-twitch and cardiac muscle preparations. This behavior has not been studied in human muscle fibers or animal slow-twitch fibers. Human slow-twitch fibers are interesting since they contain the same myosin heavy chain isoform as the human heart. To explore myosin-induced activation of the thin filament in isometrically contracting human slow-twitch fibers, the endogenous troponin complex was exchanged for a well-characterized fast-twitch skeletal troponin complex labeled with the fluorescent dye N-((2-(Iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (fsTn-IANBD). The exchange was approximate to 70% complete (n = 8). The relative contributions of calcium and strong binding cross-bridges to thin filament activation were dissected by increasing the concentration of calcium from relaxing (pCa 7.5) to saturating levels (pCa 4.5) before and after incubating the exchanged fibers in the myosin inhibitor para-aminoblebbistatin (AmBleb). At pCa 4.5, the relative contributions of calcium and strong binding cross-bridges to thin filament activation were approximate to 69 and approximate to 31%, respectively. Additionally, switching from isometric to isotonic contraction at pCa 4.5 revealed that strong binding cross-bridges contributed approximate to 29% to thin filament activation (i.e., virtually the same magnitude obtained with AmBleb). Thus, we showed through two different approaches that lowering the number of strong binding cross-bridges, at saturating calcium, significantly reduced the activation of the thin filament in human slow-twitch fibers. The contribution of myosin to activation resembled that which was previously reported in rat cardiac and rabbit fast-twitch muscle preparations. This method could be applied to slow-twitch human fibers obtained from the soleus muscle of cardiomyopathy patients. Such studies could lead to a better understanding of the effect of point mutations of the cardiac myosin head on the regulation of muscle contraction and could lead to better management by pharmacological approaches