Thermodynamics of Nucleotide Binding to Actomyosin V and VI: A Positive Heat Capacity Change Accompanies Strong ADP Binding†

James P. Robblee, Wenxiang Cao, Arnon Henn, Diane E. Hannemann, Enrique M. De La Cruz
2005 Biochemistry  
We have measured the energetics of ATP and ADP binding to single-headed actomyosin V and VI from the temperature dependence of the rate and equilibrium binding constants. Nucleotide binding to actomyosin V and VI can be modeled as two-step binding mechanisms involving the formation of collision complexes followed by isomerization to states with high nucleotide affinity. Formation of the actomyosin VI-ATP collision complex is much weaker and slower than for actomyosin V. A three-step binding
more » ... ee-step binding mechanism where actomyosin VI isomerizes between two conformations, one competent to bind ATP and one not, followed by rapid ATP binding best accounts for the data. ADP binds to actomyosin V more tightly than actomyosin VI. At 25°C, the strong ADP-binding equilibria are comparable for actomyosin V and VI, and the different overall ADP affinities arise from differences in the ADP collision complex affinity. The actomyosin-ADP isomerization leading to strong ADP binding is entropy driven at >15°C and occurs with a large, positive change in heat capacity (∆C P°) for both actomyosin V and VI. Sucrose slows ADP binding and dissociation from actomyosin V and VI but not the overall equilibrium constants for strong ADP binding, indicating that solvent viscosity dampens ADP-dependent kinetic transitions, presumably a tail swing that occurs with ADP binding and release. We favor a mechanism where strong ADP binding increases the dynamics and flexibility of the actomyosin complex. The heat capacity (∆C P°) and entropy (∆S°) changes are greater for actomyosin VI than actomyosin V, suggesting different extents of ADP-induced structural rearrangement. regarding the thermodynamic basis of the high ADP affinities that help dictate the duty ratios is available. In this study, we have investigated the energetics of nucleotide binding to myosins V and VI from the physical linkage of the rate and equilibrium constants to temperature. Our results indicate that strong ADP binding to myosins V and VI occurs with a positive heat capacity change, consistent with significant nucleotide-dependent conformational rearrangement and population of multiple conformational states contributing to the stability of the actomyosin-ADP complex. In addition, the kinetic data favor a mechanism where a conformational change precedes ATP binding to actomyosin VI. EXPERIMENTAL PROCEDURES Reagents. All chemicals were the highest purity commercially available. ATP (99+% purity as assayed by HPLC, data not shown) was purchased from Roche Molecular Biochemicals (Indianapolis, IN), and ADP (Sigma A-5285, 99+% purity as assayed by HPLC, data not shown) was purchased from Sigma (St. Louis, MO). ATP and ADP concentrations were determined by absorbance at 259 nm using a 259 of 15 400 M -1 cm -1 . A molar equivalent of MgCl 2 was added to nucleotides immediately before use. Pyrenyl-iodoacetamide came from Molecular Probes (Eugene, OR). Fluorescence-grade (99+%, titration) imidazole and phalloidin were purchased from Sigma. Ultrapure sucrose was from American Bioanalytical. Protein Expression and Purification. Single-headed chicken myosin V-1IQ with bound LC-1sa light chain (13, 14) and single-headed porcine myosin VI (T406A mutant) with bound calmodulin light chain (5, 10) were purified from Sf9 cells by Flag affinity chromatography (16). Purity was >98% for all preparations, with actin being the only detectible contaminant. Actin was purified from rabbit skeletal muscle (17) , labeled with pyrene (18), and gel-filtered over Sephacryl S-300HR. Ca 2+ -actin monomers were converted to Mg 2+ -actin monomers with 0.2 mM EGTA and 50 µM MgCl 2 (excess over [actin]) immediately prior to polymerization by dialysis into KMg50 buffer (3 × 500 mL). Phalloidin (1.1 M equiv) was used to stabilize actin filaments. Stopped-Flow Measurements and Kinetic Modeling. All experiments were performed in KMg50 buffer (50 mM KCl, 2 mM MgCl 2 , 1 mM EGTA, 1 mM DTT, and 10 mM imidazole at pH 7.0) with an Applied Photophysics (Surrey, U.K.) SX.18MV-R stopped-flow apparatus thermostated at the indicated temperatures ((0.1°C). The buffer pH was adjusted at the experimental temperatures. Pyrene (λ ex ) 366 nm) fluorescence were monitored through a 400 nm long pass colored glass filter. Long time courses of pyrene fluorescence change were corrected for photobleaching by subtracting the time courses of fluorescence acquired after mixing pyrene actin filaments with nucleotides. Essentially identical results were obtained when the time courses after mixing actomyosin and buffer were subtracted. Most of the time courses shown are of individual, unaveraged, 1000-point transients collected with the instrument in oversampling mode. The intrinsic time constant for data acquisition is ∼30 µs. Time courses that display fast and slow phases were collected on a logarithmic time scale. Typically, multiple (3-8) time courses were averaged before Thermodynamics of Nucleotide Binding to Actomyosin V and VI
doi:10.1021/bi050232g pmid:16042401 fatcat:tyh434aehja3zczpt2ykcv424q