The ATPase mechanism of myosin 15, the molecular motor mutated in DFNB3 human deafness

Figures

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  Figure 1. Purification and steady-state activity of the unconventional myosin 15 (MYO15) motor domain.A, drawing of alternative splice isoforms of MYO15 expressed in the inner ear. Each isoform shares a common ATPase domain (red) and three light-chain binding domains (LCBDs), in addition to MyTH4 (green), SH3 (yellow), and FERM (blue) domains. Both isoforms are identical, except for the addition of a 133-kDa N-terminal domain in MYO15-1. Baculovirus expression constructs used in this study encode the truncated MYO15 motor domain and two consensus LCBDs fused to enhanced green fluorescent protein (EGFP) and a FLAG tag. B, the effect of UNC45 expression upon solubility of the MYO15 motor domain. Sf9 cells were infected with baculovirus to express EGFP-M15-2IQ, ELC (MYL6), and RLC (MYL12), plus optionally UNC45A or UNC45B. Representative Western blots (anti-FLAG) of EGFP-M15-2IQ in Sf9 cell total lysate (T) versus supernatant (S) fractions after sedimentation at 48 kG. Blots were probed for alpha-tubulin as loading controls. A solubility ratio (= S/T) was calculated using densitometry from three independent experimental determinations (data are mean ± SD). Coexpression of either UNC45A (p =…

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  Figure 2. ATPase mechanism of the MYO15 motor domain. Species are abbreviated as follows: actin (A), myosin (M), ATP (T), ADP (D) and inorganic phosphate (P). Strongly actin-bound species (dark gray) and weakly actin-bound species (light gray). Equilibrium constants are defined as Ki = k+i/k−i, where the k+i reaction proceeds up or to the right. Actin-bound state transitions are denoted with an apostrophe (e.g., k′+2).

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  Figure 3. Transient kinetic analysis of ATP binding to EGFP-M15-2IQ using stopped-flow fluorescence spectroscopy.A, intrinsic fluorescence enhancement as 0.25-μM EGFP-M15-2IQ was rapidly mixed with 25-μM ATP. The transient was fit to a single exponential equation, I(t) = −9.2·e−4.52t + C (dotted line, residuals above). B, dependence of intrinsic fluorescence observed rate constants (kobs) upon ATP concentration. The hyperbola kobs = (K1·k+2·[ATP])/(1 + K1·[ATP]) is shown (dotted line), where 1/K1 = 1898 ± 145 μM, k+2 = 322 ± 10 s−1. Inset: kobs is shown at lower [ATP]. ATP binding was irreversible. C, fluorescence enhancement of 10-μM mant-ATP after rapid mixing with 0.25-μM EGFP-M15-2IQ. The mant fluorophore was excited by FRET from vicinal tryptophan residues. The transient was fit to a single exponential equation I(t) = −5.9 e−1.71t + C (dotted line, residuals above). D, dependence of kobs upon mant-ATP concentration reveals the apparent association rate constant K1·k+2 = 0.15 ± 0.002 μM−1 s−1 with an intercept of ∼0, showing irreversible binding of mant-ATP to EGFP-M15-2IQ. For all experiments, conditions in the observation cell were 20-mM Mops (pH 7.0), 100-mM KCl, 5-mM MgCl2, and…

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  Figure 4. Transient kinetic analysis of ATP binding to actomyosin using stopped-flow spectroscopy.A, reduction in orthogonal light scattering (measured at 340 nm) as 50-, 250-, or 2500-μM MgATP bound to a preincubated mix of 0.25-μM EGFP-M15-2IQ plus 0.5-μM actin in the stopped flow. Transients were fit to a biphasic exponential (dotted lines) with I(t) = 36.2·e−8.7t + 13.3·e−1.7t + C, I(t) = 42.2·e−28.7t + 10.1·e−5.3t + C, I(t) = 37.5·e−150.6t + 8.4·e−14.7t + C, for 50-, 250-, and 2500-μM MgATP, respectively. B, dependence of fast and slow observed rate constants upon [MgATP]. Hyperbolae were fit (dotted lines) to both fast (circle) and slow (square) phases to yield maximum values of 307 ± 15 s−1 and 30.9 ± 1.9 s−1, with half maximal activation at 2749 ± 268 μM and 1428 ± 225 μM, respectively. Inset: normalized amplitudes of fast (circle) and slow (square) phases at different [ATP]. C, fluorescence enhancement of 10-μM mant-ATP after rapid mixing with 0.25-μM EGFP-M15-2IQ + 0.5 μM actin. The mant fluorophore was excited by FRET from vicinal tryptophan residues in EGFP-M15-2IQ. The transient was fit to a biphasic exponential I(t) = −5.0·e−1.2t – 0.9·e−0.4t + C (dotted line, fit residuals…

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  Figure 5. Hydrolysis and actin-activated phosphate release.A, ATP hydrolysis measured by quenched-flow. EGFP-M15-2IQ (3 μM) was reacted under multiple turnover conditions with 0.5-mM [γ-32P]ATP and aged for varying intervals before acid quench. The observed phosphate burst was fit to a single exponential, I(t) = (K3/1 + K3)·(1 − e−kobs·t) to yield kobs = 50.2 ± 5.7 s−1 and K3 = 1.34 ± 0.07. Data are from one myosin preparation. B, double-mixing stopped flow experiment to measure actin-activated phosphate release during a single turnover. EGFP-M15-2IQ (2 μM) was 1:1 mixed with 1-μM ATP and aged for 5 s (postmix concentrations), followed by a subsequent 1:1 mix with actin (final condition in reaction cell, 1-μM myosin, 0.5-μM ATP, 50-μM actin). MDCC-PbP was included in all solutions at 5 μM. Transient is shown overlaid with I(t) = −1.39e−6.59t – 1.06e−0.17t + C (dotted line, fit residuals above). C, dependence of observed rate constants upon [actin]. A linear fit to the fast phase (circles) yields K9·k′+4 = 0.13 ± 0.004 μM−1 s−1. The slow observed phase (triangles) did not vary systematically with [actin]. Data are from two independent determinations, from one myosin preparation. Conditions…

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  Figure 6. The interaction of EGFP-M15-2IQ with actin filaments.A, reduction in fluorescence measured as 0.15-μM EGFP-M15-2IQ binds and quenches 1.5-μM pyrene-labeled actin in a stopped-flow spectrophotometer in the absence of nucleotide (apyrase-treated). The transient was fit to a single exponential function I(t) = 5.6e−5.4t + C (dotted line, fit residuals above). Similar experiments were repeated in the presence of 1-mM ADP (not shown). B, dependence of kobs upon [actin] is shown for nucleotide-free conditions (circles) and with 1-mM ADP present (squares). Linear regression to kobs = k+A[actin] + k−A yields the association rate constant for actin binding k+6 = 3.18 ± 0.15 μM−1s−1 and off-rate k−6 = 0.65 ± 0.3 s−1. In the presence of ADP, k+10 = 1.75 ± 0.09 μM−1s−1 and k−10 = 0.55 ± 0.17 s−1. Experimental conditions in observation cell: 0.05- to 0.3-μM EGFP-M15-2IQ, 0.5- to 3-μM pyrene-actin, 1-mM ADP (optional), 20-mM Mops (pH 7.0), 100-mM KCl, 5-mM MgCl2, 0.1-mM EGTA at 20 °C. Experimental data were measured from three independent myosin preparations.

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  Figure 7. Kinetics of ADP binding to EGFP-M15-2IQ.A, enhancement of intrinsic protein fluorescence as 200-μM MgADP bound to 0.25-μM EGFP-M15-2IQ in the stopped-flow. After an initial lag phase (∼10 ms), the recorded transient follows a single exponential time course with kobs = 23.8 ± 0.2 s−1 (dotted line, fit residuals above). B, observed rate constants vary hyperbolically (dotted line) with respect to [MgADP], indicating that ADP binding is at least a two-step process. Referring to Equation 3, the data points were fit to kobs = k−2D + (K1D·k+2D·[ADP])/(1 + K1D·[ADP]), yielding k+2 = 25.2 ± 0.4 s−1, k−2 = 3.5 ± 0.3 s−1, and 1/K1D = 60.6 ± 4.7 μM. C, fluorescence enhancement after rapid mixing of 10-μM mant-ADP with 0.25-μM EGFP-M15-2IQ in the stopped flow. The mant fluorophore was excited by FRET from vicinal tryptophan residues. The transient followed a monophasic exponential increase, I(t) = −1.7e−28.8t + C (dotted line, fit residuals above). D, observed rate constants varied linearly with [mant-ADP]. Linear regression to kobs = k+5 + k−5 [ADP] yields k−5 = 0.11 ± 0.01 μM s−1 and k+5 = 1.6 ± 0.07 s−1. E, displacement reaction where 0.25-μM EGFP-M15-2IQ was pre-equilibrated with 25-μM…

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  Figure 8. ADP binding to actomyosin.A, the actin-attached dissociation constant (K′5) of ADP from EGFP-M15-2IQ was measured using a displacement reaction. EGFP-M15-2IQ (0.25 μM), actin (0.5 μM), and ADP (0–0.5 mM) were pre-equilibrated before rapid mixing with ATP (50 μM) in a stopped-flow spectrophotometer. ATP induced dissociation of the actomyosin complex was monitored using orthogonally scattered light at 340 nm. The ATP concentration was chosen to allow ATP and ADP to compete for binding to EGFP-M15-2IQ. Example traces are shown for 0 μM (blue), 50 μM (green), and 250 μM ADP (red) and are shown with biphasic exponential fits overlaid (dotted line, residuals above). B, dependence of fast (circles) and slow (square) observed rate constants upon [ADP]. The fast phase was interpreted as direct binding of ATP to actomyosin. Fitting of the fast phase to an inverse-hyperbola reveals half-maximal inhibition at 36.1 ± 5.7 μM ADP. C, ADP release from actomyosin was measured using a displacement reaction. EGFP-M15-2IQ (0.25 μM) was pre-equilibrated with actin (0.5 μM) and saturating ADP (0.5 mM), before rapid mixing with 5-mM MgATP in the stopped flow. The high concentration of ATP prevents ADP…