ABSTRACT
Formation of the strong binding interaction between actin and myosin is essential for force generation in muscle and in cytoskeletal motor systems. To clarify the role of the closure of myosin's actin-binding cleft in the actomyosin interaction, we performed rapid kinetic, spectroscopic, and calorimetric experiments and atomic-level energetic calculations on a variety of myosin isoforms for which atomic structures are available. Surprisingly, we found that the endothermic actin-binding profile of vertebrate skeletal muscle myosin subfragment-1 is unique among studied myosins. We show that the diverse propensity of myosins for cleft closure determines different energetic profiles as well as structural and kinetic pathways of actin binding. Depending on the type of myosin, strong actin binding may occur via induced-fit or conformational preselection mechanisms. However, cleft closure does not directly determine the kinetics and affinity of actin binding. We also show that cleft closure is enthalpically unfavorable, reflecting the development of an internal strain within myosin in order to adopt precise steric complementarity to the actin filament. We propose that cleft closure leads to an increase in the torsional strain of myosin's central ß-sheet that has been proposed to serve as an allosteric energy-transducing spring during force generation.
Subject(s)
Actomyosin/chemistry , Muscle, Skeletal/metabolism , Myosin Subfragments/chemistry , Myosins/chemistry , Actins/chemistry , Actins/metabolism , Actomyosin/metabolism , Animals , Binding Sites , Calorimetry , Kinetics , Myosin Subfragments/metabolism , Myosins/metabolism , Protein Binding , Rabbits , Spectrometry, Fluorescence , TemperatureABSTRACT
Active site loops that are conserved across superfamilies of myosins, kinesins, and G proteins play key roles in allosteric coupling of NTP hydrolysis to interaction with track filaments or effector proteins. In this study, we investigated how the class-specific natural variation in the switch-2 active site loop contributes to the motor function of the intracellular transporter myosin-5. We used single-molecule, rapid kinetic and spectroscopic experiments and semiempirical quantum chemical simulations to show that the class-specific switch-2 structure including a tyrosine (Y439) in myosin-5 enables rapid processive translocation along actin filaments by facilitating Mg(2+)-dependent ADP release. Using wild-type control and Y439 point mutant myosin-5 proteins, we demonstrate that the translocation speed precisely correlates with the kinetics of nucleotide exchange. Switch-2 variants can thus be used to fine-tune translocation speed while maintaining high processivity. The class-specific variation of switch-2 in various NTPase superfamilies indicates its general role in the kinetic tuning of Mg(2+)-dependent nucleotide exchange.
Subject(s)
Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Myosin Type V/genetics , Myosin Type V/metabolism , Protein Transport , Actins/metabolism , Animals , Computer Simulation , Magnesium/metabolism , Mice , Models, Molecular , Mutation/genetics , Nucleotides/metabolism , Phosphates/metabolism , Protein Binding , Protein Structure, Tertiary , Protein Transport/geneticsABSTRACT
The powerstroke of the myosin motor is the basis of cell division and bodily movement, but has eluded empirical description due to the short lifetime and low abundance of intermediates during force generation. To gain insight into this process, we used well-established single-tryptophan and pyrene fluorescent sensors and electron microscopy to characterize the structural and kinetic properties of myosin complexed with ADP and blebbistatin, a widely used inhibitor. We found that blebbistatin does not weaken the tight actin binding of myosin.ADP, but unexpectedly it induces lever priming, a process for which the gamma-phosphate of ATP (or its analog) had been thought necessary. The results indicate that a significant fraction of the myosin.ADP.blebbistatin complex populates a previously inaccessible conformation of myosin resembling the start of the powerstroke.
Subject(s)
Adenosine Diphosphate/chemistry , Dictyostelium/metabolism , Heterocyclic Compounds, 4 or More Rings/chemistry , Myosins/chemistry , Adenosine Triphosphate/chemistry , Animals , Binding Sites , Fluorescent Dyes/chemistry , Kinetics , Microscopy, Electron/methods , Microscopy, Fluorescence/methods , Models, Biological , Molecular Conformation , Protein Conformation , RabbitsABSTRACT
A cell membrane permeable phosphopeptide corresponding to the SHP-2 binding motif of Grb2-associated binder 1 (Gab1) interferes with the Gab1 adaptor-dependent functions and modulates B cell receptor-triggered intracellular signaling in B cell tumors.
