A strain-dependent ADP release mechanism. The cartoon depicts a myosin motor (red) attached to an actin filament (blue). Following an isometric power stroke (coupled to Pi release; A-M.D.P to A.M.D), the ADP (D) remains trapped in the nucleotide pocket of the strained myosin head. A further movement (A.M.D to A.M'.D) of the converter domain/LCBD (yellow) is required to open the nucleotide pocket and allow ADP dissociation. If such a movement of the converter domain is in the same direction as the power stroke, then the strain on the isometric motor will oppose the additional movement required. If the strain is dissipated by movement of the tail of the myosin relative to actin then the movement is not inhibited. For effective inhibition of ADP release, the free energy associated with the conformational change and subsequent ADP release must be small compared to the energy required to stretch the elastic element. In addition, the affinity of the motor for actin must remain high in the A.M.D state to prevent strain-induced detachment of the motor. Thus, a strain-sensitive ADP mechanism requires: (i) ADP-coupled movement of the converter; (ii) high affinity (i.e., low delta G) of ADP; and (iii) low coupling between ADP and actin affinities such that both remain tightly bound in the ternary complex.

3D Maps of the ADP and rigour states of Myo1c calculated by cryo-EM and image analysis. In each panel, the molecular envelope of the bound myosin head is shown in chicken wire representation. The actin filament is at right running vertically, with the pointed end of the filament towards the bottom (as in Figure 6). The alpha-carbon backbone of a motor domain X-ray structure (white) has been docked into the molecular envelope. The LCBD model consists of a C-terminal heavy-chain helix (white). The calmodulin light chains are represented by three chicken skeletal muscle myosin essential light chains (yellow, green, yellow) whose positions and orientations are dictated by the spacing of the IQ motifs in the sequence. Shown are the ADP (top) and rigour (middle) states. In the bottom panel, these two 3D maps have been superimposed to show the 33° swing of the LCBD clearly. The Myo1c tail domain (235 amino acids) is not visible either because it is flexibly linked to the molecule or because of insufficient resolution in the images.

Powerstroke size measured using an optical-tweezers transducer. (A) Cartoon showing two optically trapped beads, positioned over the four-quadrant photodiode detectors. The trapped beads hold a single actin filament in the vicinity of a third surface bead coated with Myo1c. (B) Top trace shows the position of the optically trapped beads measured in the absence of binding interactions. Such data were used to find the distribution of bead positions caused by thermal vibration and the applied optical-tweezer oscillation. Middle trace shows data recorded parallel to the actin filament. When myosin bound to actin, the system stiffness increased about 10-fold from 0.04 pN nm⁻¹ (equal trap stiffness 2κ_trap) to 0.5 pN nm⁻¹ and system noise was dramatically reduced. The arrow indicates the two phases of the actomyosin binding interaction. Reduction in the amplitude of the 100 Hz carrier signal (measured from a discrete Fourier transform of the data) is shown in the lower trace and this was used to identify the start and end of individual binding events so that event lifetimes could be accurately determined. From the distribution of bound lifetimes we found the off-rate to be 0.06 μM⁻¹ s⁻¹, giving an average bound lifetime, at 20 μM ATP of 0.83 s. (C) The upper record shows raw data for an individual binding event (from trace B) and indicates how sections of data were extracted from the start and end of the binding event. Events were synchronised in this way and then averaged to give the ensemble response shown in the lower section (amplitude of phase 1=3.1 nm, phase 2=1.1 nm; overall displacement 4.2 nm, s.d. 1.4 nm, n=85). Data shown here were recorded at 22°C, pH 7.4 and 20 μM ATP.

Lifetime measurements as a function of ATP concentration; effect of Ca²⁺. (A) Scatter plot showing the lifetimes of Myo1c at 5, 20 and 100 μM ATP (n=127, 427 and 97, respectively). Data are plotted as a cumulative distribution (e.g., data values that are less than each time point are scored and plotted at that time point) and normalised to 100%. Myo1c bound lifetime distribution exhibits a double exponential decay at all three ATP concentrations, suggesting that there are two exit routes from the attached state. Rate constants obtained by fitting a dual exponential decay indicate that there is a fast, ATP-independent phase and a slow, ATP-dependent phase (Table I). (B) Lifetime distribution for data obtained at pCa 4.11 with 5 and 20 μM ATP plotted as before. The data for 20 μM ATP pCa 8 are replotted from (A) for comparison. The slow phase is accelerated by about 10-fold. (C) The relationship between step size estimate and duration of attachment is plotted by first ranking all event amplitudes on the basis of their attached lifetime and then calculating the running average of the lifetime and step size over a window of 80 consecutive data points (each estimate of mean step size for a given time window has a s.e.m. given by s.d./√n=14/8.9=1.5 nm). The data are fitted to an equation which assumes that the fast population produces no work stroke, whereas the slow population gives a work stroke (d_max) of 4.2 nm in the absence of calcium and 7.2 nm in the presence of calcium. The fitted line: Work stroke_t= d_max × { Ae^{- r_st} /(Ae^{- r_st}+ (1-A)e^{- r_ft})}, where A_slow=relative amplitude of slow phase (%/100), d_max =work stroke produced by slow population, r_s=rate constant of slow phase, and r_f=rate constant of fast phase. See Table I for fitting parameters. Note the similarity of fit parameters used for lifetime data plots (A) and amplitude data plots (C).

ATP-induced dissociation of acto-Myo1c. Pyrene-labelled acto-Myo1c (25 nM) was mixed with 50 μM–5 mM ATP. (A) At low ATP (50 μM) the change in fluorescence signal (ΔF=F_t−F_∞) is fit with a single exponential (ΔF=ΔF_max exp^{- k_obst}). (B) At high ATP concentration, the change in fluorescence is biphasic and exhibits a fast (1) and a slow (2) phase (ΔF=ΔF_max1 exp ^{- k_obs1t}+ΔF_max2 exp ^{- k_obs2t}). The magnitude of the fast phase is variable representing 30% (maximum) of the total signal change and is linearly dependent upon ATP concentration up to 1 mM (not shown; second-order rate constant=0.014 × 10⁶ M⁻¹ s⁻¹). The fast phase is difficult to detect in the presence of Ca²⁺. (C) The slow phase shows a hyperbolic dependence on ATP concentration (k_obs=k_max[ATP]/(K_0.5+[ATP]). The slow phase saturates at about 2.5 s⁻¹ (k_max) and requires 680 μM ATP for half-saturation (K_0.5) and shows no strong dependence on Ca²⁺. Treatment of the sample with apyrase does not affect the results.

Schematic model for the role of Myo1c in adaptation in the hair cell. This model is adapted from Holt et al (2002) to depict the nucleotide state of the Myo1c molecules in the adaptation motor complex at rest (left), under positive deflection (middle) and under negative deflection (right). Red signifies Myo1c in rigour (nucleotide-free); blue, Myo1c with ADP bound; yellow, Myo1c with ADP.Pi; and green, Myo1c undergoing Pi release. The pointed ends of actin lie towards the bottom of the illustration. At rest, P_o is 0.1. The tension in the tip links is balanced by force produced by the motors. Strain is maintained by Myo1c in the ADP state. At positive deflection, P_o approaches 1.0, the channels open and the tension in the tip link causes detachment of the motor complex from the actin filament. The motor complex, still attached to the deflecting membrane, ‘slips' relative to its starting position on the actin filament toward the minus end of the filament. During negative deflection, P_o approaches 0. A reduction in tip link tension reduces the strain on Myo1c allowing the motor molecules, some of which are expected to be in the strained ADP state and some at the beginning of the powerstroke, to complete the powerstroke and move up the filament to re-establish the resting state.