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Mechanochemical properties of human myosin-1C are modulated by isoform-specific differences in the N-terminal extension

2020; Elsevier BV; Volume: 296; Linguagem: Inglês

10.1074/jbc.ra120.015187

ISSN

1083-351X

Autores

Sven Giese, Theresia Reindl, P. Reinke, Lilach Zattelman, Roman Fedorov, Arnon Henn, Manuel H. Taft, Dietmar J. Manstein,

Tópico(s)

Cellular Mechanics and Interactions

Resumo

Myosin-1C is a single-headed, short-tailed member of the myosin class I subfamily that supports a variety of actin-based functions in the cytosol and nucleus. In vertebrates, alternative splicing of the MYO1C gene leads to the production of three isoforms, myosin-1C0, myosin-1C16, and myosin-1C35, that carry N-terminal extensions of different lengths. However, it is not clear how these extensions affect the chemomechanical coupling of human myosin-1C isoforms. Here, we report on the motor activity of the different myosin-1C isoforms measuring the unloaded velocities of constructs lacking the C-terminal lipid-binding domain on nitrocellulose-coated glass surfaces and full-length constructs on reconstituted, supported lipid bilayers. The higher yields of purified proteins obtained with constructs lacking the lipid-binding domain allowed a detailed characterization of the individual kinetic steps of human myosin-1C isoforms in their productive interaction with nucleotides and filamentous actin. Isoform-specific differences include 18-fold changes in the maximum power output per myosin-1C motor and 4-fold changes in the velocity and the resistive force at which maximum power output occurs. Our results support a model in which the isoform-specific N-terminal extensions affect chemomechanical coupling by combined steric and allosteric effects, thereby reducing both the length of the working stroke and the rate of ADP release in the absence of external loads by a factor of 2 for myosin-1C35. As the large change in maximum power output shows, the functional differences between the isoforms are further amplified by the presence of external loads. Myosin-1C is a single-headed, short-tailed member of the myosin class I subfamily that supports a variety of actin-based functions in the cytosol and nucleus. In vertebrates, alternative splicing of the MYO1C gene leads to the production of three isoforms, myosin-1C0, myosin-1C16, and myosin-1C35, that carry N-terminal extensions of different lengths. However, it is not clear how these extensions affect the chemomechanical coupling of human myosin-1C isoforms. Here, we report on the motor activity of the different myosin-1C isoforms measuring the unloaded velocities of constructs lacking the C-terminal lipid-binding domain on nitrocellulose-coated glass surfaces and full-length constructs on reconstituted, supported lipid bilayers. The higher yields of purified proteins obtained with constructs lacking the lipid-binding domain allowed a detailed characterization of the individual kinetic steps of human myosin-1C isoforms in their productive interaction with nucleotides and filamentous actin. Isoform-specific differences include 18-fold changes in the maximum power output per myosin-1C motor and 4-fold changes in the velocity and the resistive force at which maximum power output occurs. Our results support a model in which the isoform-specific N-terminal extensions affect chemomechanical coupling by combined steric and allosteric effects, thereby reducing both the length of the working stroke and the rate of ADP release in the absence of external loads by a factor of 2 for myosin-1C35. As the large change in maximum power output shows, the functional differences between the isoforms are further amplified by the presence of external loads. Myosin-1C connects cell and vesicle membranes with actin-rich structures of the cytoskeleton to support critical cellular processes at multiple intracellular locations. Myosin-1C has been shown to contribute to the adaptation response in sensory hair cells (1Gillespie P.G. Cyr J.L. Myosin-1c, the hair Cell's adaptation motor.Annu. Rev. Physiol. 2004; 66: 521-545Crossref PubMed Scopus (129) Google Scholar), to act as a cofactor of the transcriptional machinery by interacting with RNA polymerase I and II in the nucleus (2Pestic-Dragovich L. Stojiljkovic L. Philimonenko A.A. Nowak G. Ke Y. Settlage R.E. Shabanowitz J. Hunt D.F. Hozak P. Lanerolle P. de A myosin I isoform in the nucleus.Science. 2000; 290: 337-341Crossref PubMed Scopus (194) Google Scholar, 3Ihnatovych I. Migocka-Patrzalek M. Dukh M. Hofmann W.A. Identification and characterization of a novel myosin Ic isoform that localizes to the nucleus.Cytoskeleton. 2012; 69: 555-565Crossref Scopus (28) Google Scholar), to support the delivery of organelles to membranes such as the insulin-induced translocation of GLUT4-containing vesicles to plasma membrane (4Bose A. Guilherme A. Robida S.I. Nicoloro S.M.C. Zhou Q.L. Jiang Z.Y. Pomerleau D.P. Czech M.P. Glucose transporter recycling in response to insulin is facilitated by myosin Myo1c.Nature. 2002; 420: 821Crossref PubMed Scopus (210) Google Scholar), and to play a role in the formation of membrane extensions and the regulation of cellular tension (5Diefenbach T.J. Latham V.M. Yimlamai D. Liu C.A. Herman I.M. Jay D.G. Myosin 1c and myosin IIB serve opposing roles in lamellipodial dynamics of the neuronal growth cone.J. Cell Biol. 2002; 158: 1207-1217Crossref PubMed Scopus (98) Google Scholar, 6Venit T. Kalendová A. Petr M. Dzijak R. Pastorek L. Rohožková J. Malohlava J. Hozák P. Nuclear myosin I regulates cell membrane tension.Sci. Rep. 2016; 6: 30864Crossref PubMed Scopus (12) Google Scholar). All myosins share a generic myosin motor domain, which contains an active site and an actin-binding region. Members of different myosin classes have evolved structural modifications to adapt kinetic and mechanical properties to generate force and motion according to their physiological function (7Foth B.J. Goedecke M.C. Soldati D. New insights into myosin evolution and classification.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3681-3686Crossref PubMed Scopus (343) Google Scholar, 8Preller M. Manstein D.J. Comprehensive Biophysics. Elsevier, Amsterdam2017: 118-150Google Scholar) Myosin-1C is a member of the short-tailed class I myosin subfamily (9Gillespie P.G. Albanesi J.P. Bähler M. Bement W.M. Berg J.S. Burgess D.R. Burnside B. Cheney R.E. Corey D.P. Coudrier E. de Lanerolle P. Hammer J.A. Hasson T. Holt J.R. Hudspeth A.J. et al.Myosin-I nomenclature.J. Cell Biol. 2001; 155: 703-704Crossref PubMed Scopus (60) Google Scholar). Its generic motor domain is followed by a neck region that serves as a lever arm and consists of three IQ motifs and a post-IQ domain (Fig. 1A). IQ1 and IQ2 each bind one calmodulin, while a third calmodulin is bound to both IQ3 and the post-IQ domain (10Lu Q. Li J. Ye F. Zhang M. Structure of myosin-1c tail bound to calmodulin provides insights into calcium-mediated conformational coupling.Nat. Struct. Mol. Biol. 2015; 22: 81-88Crossref PubMed Scopus (35) Google Scholar). The C-terminal 176 residues form the rigid globular tail homology region 1 (TH1), which is found in all members of the class I subfamily. The TH1 domain contains a generic 56-residue, lipid membrane–binding pleckstrin homology (PH) domain in its center. In humans, alternative splicing of the MYO1C gene leads to the production of three isoforms, which differ in the length of their N-terminal extension (NTE) (3Ihnatovych I. Migocka-Patrzalek M. Dukh M. Hofmann W.A. Identification and characterization of a novel myosin Ic isoform that localizes to the nucleus.Cytoskeleton. 2012; 69: 555-565Crossref Scopus (28) Google Scholar, 11Nowak G. Pestic-Dragovich L. Hozák P. Philimonenko A. Simerly C. Schatten G. Lanerolle P. de Evidence for the presence of myosin I in the nucleus.J. Biol. Chem. 1997; 272: 17176-17181Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Compared to myosin-1C0, the isoforms myosin-1C16 and myosin-1C35 contain 16 and 35 additional amino acids at their N terminus (Fig. 1B). The three human myosin-1C splice isoforms are otherwise identical in their structural organization, undergo analogous post-translational modifications, and are capable of interacting with the same partner proteins (12Dzijak R. Yildirim S. Kahle M. Novák P. Hnilicová J. Venit T. Hozák P. Specific nuclear Localizing sequence Directs two myosin isoforms to the cell nucleus in calmodulin-sensitive manner.PLoS ONE. 2012; 7: e30529Crossref PubMed Scopus (33) Google Scholar, 13Venit T. Dzijak R. Kalendová A. Kahle M. Rohožková J. Schmidt V. Rülicke T. Rathkolb B. Hans W. Bohla A. Eickelberg O. Stoeger T. Wolf E. Yildirim A.Ö. Gailus-Durner V. et al.Mouse nuclear myosin I Knock-out shows Interchangeability and Redundancy of myosin isoforms in the cell nucleus.PLoS ONE. 2013; 8: e61406Crossref PubMed Scopus (24) Google Scholar). In rodents and primates, myosin-1C0 and myosin-1C16 isoforms are ubiquitously produced. In contrast, myosin-1C35 shows a tissue-dependent expression profile, suggesting a role in tissue-specific functions (14Kahle M. Pridalová J. Spacek M. Dzijak R. Hozák P. Nuclear myosin is ubiquitously expressed and evolutionary conserved in vertebrates.Histochem. Cell Biol. 2007; 127: 139-148Crossref PubMed Scopus (26) Google Scholar, 15Sielski N.L. Ihnatovych I. Hagen J.J. Hofmann W.A. Tissue specific expression of myosin IC isoforms.BMC Cell Biol. 2014; 15: 8Crossref PubMed Scopus (15) Google Scholar). Isoform-specific functions of myosin-1C include roles of myosin-1C16 and myosin-1C35 as nuclear cofactors in chromatin remodeling and transcription activation and a role of myosin-1C16 in plasma membrane tension adaptation (3Ihnatovych I. Migocka-Patrzalek M. Dukh M. Hofmann W.A. Identification and characterization of a novel myosin Ic isoform that localizes to the nucleus.Cytoskeleton. 2012; 69: 555-565Crossref Scopus (28) Google Scholar, 6Venit T. Kalendová A. Petr M. Dzijak R. Pastorek L. Rohožková J. Malohlava J. Hozák P. Nuclear myosin I regulates cell membrane tension.Sci. Rep. 2016; 6: 30864Crossref PubMed Scopus (12) Google Scholar, 16Philimonenko V.V. Zhao J. Iben S. Dingová H. Kyselá K. Kahle M. Zentgraf H. Hofmann W.A. Lanerolle P. de Hozák P. Grummt I. Nuclear actin and myosin I are required for RNA polymerase I transcription.Nat. Cell Biol. 2004; 6: 1165Crossref PubMed Scopus (301) Google Scholar, 17Sarshad A. Sadeghifar F. Louvet E. Mori R. Böhm S. Al-Muzzaini B. Vintermist A. Fomproix N. Östlund A.-K. Percipalle P. Nuclear myosin 1c facilitates the chromatin modifications required to activate rRNA gene transcription and cell cycle progression.Plos Genet. 2013; 9Crossref PubMed Scopus (48) Google Scholar, 18Schwab R.S. Ihnatovych I. Yunus S.Z.S.A. Domaradzki T. Hofmann W.A. Identification of signals that facilitate isoform specific nucleolar localization of myosin IC.Exp. Cell Res. 2013; 319: 1111-1123Crossref PubMed Scopus (9) Google Scholar). The underlying regulatory mechanisms that support isoform-specific functional behavior and controlled partitioning between the nucleus and cytoplasm have not been identified. It has been shown that the different myosin-1C isoforms can at least partially complement or replace each other in their function (13Venit T. Dzijak R. Kalendová A. Kahle M. Rohožková J. Schmidt V. Rülicke T. Rathkolb B. Hans W. Bohla A. Eickelberg O. Stoeger T. Wolf E. Yildirim A.Ö. Gailus-Durner V. et al.Mouse nuclear myosin I Knock-out shows Interchangeability and Redundancy of myosin isoforms in the cell nucleus.PLoS ONE. 2013; 8: e61406Crossref PubMed Scopus (24) Google Scholar). Biochemical studies show that the myosin-1C0 isoform produced in rodents is a low-duty-ratio myosin under low-load conditions (19Adamek N. Coluccio L.M. Geeves M.A. Calcium sensitivity of the cross-bridge cycle of Myo1c, the adaptation motor in the inner ear.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5710-5715Crossref PubMed Scopus (46) Google Scholar, 20Lin T. Greenberg M.J. Moore J.R. Ostap E.M. A hearing loss-associated myo1c Mutation (R156W) decreases the myosin duty ratio and force sensitivity.Biochemistry. 2011; 50: 1831-1838Crossref PubMed Scopus (24) Google Scholar). Biochemical studies on murine myosin-1C0 show that external loads increase the duty cycle by means of a force-sensitive mechanism (21Greenberg M.J. Lin T. Goldman Y.E. Shuman H. Ostap E.M. Myosin IC generates power over a range of loads via a new tension-sensing mechanism.Proc. Natl. Acad. Sci. 2012; 109: E2433-E2440Crossref PubMed Scopus (54) Google Scholar). In a previous study aimed at dissecting the impact of the 16- and 35-residue NTEs of myosin-1C16 and myosin-1C35, we described the kinetic properties of the full-length myosin-1C splice isoforms, provided a detailed model of the differential distribution among the isoforms with respect to the close and open state of the actomyosin ADP-bound state during cycling, and related these findings to a structural model where the NTEs form a compact structural domain that crosses the cleft between the converter domain and the calmodulin bound to IQ-repeat 1, thereby enabling a contact between the 35-residue NTE and the relay loop (22Zattelman L. Regev R. Ušaj M. Reinke P.Y.A. Giese S. Samson A.O. Taft M.H. Manstein D.J. Henn A. N-terminal splicing extensions of the human MYO1C gene fine-tune the kinetics of the three full-length myosin IC isoforms.J. Biol. Chem. 2017; 292: 17804Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Thus, the NTEs affect the specific nucleotide-binding properties of myosin-1C splice isoforms, adding to their kinetic diversity (22Zattelman L. Regev R. Ušaj M. Reinke P.Y.A. Giese S. Samson A.O. Taft M.H. Manstein D.J. Henn A. N-terminal splicing extensions of the human MYO1C gene fine-tune the kinetics of the three full-length myosin IC isoforms.J. Biol. Chem. 2017; 292: 17804Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Here, we describe the isoform-specific changes in the enzymatic and motor properties of the different myosin-1C isoforms by using both full-length and TH1-truncated myosin-1C constructs (Fig. 1C). Our results show distinct differences for ADP release, duty cycle, filament sliding velocity, and force-sensing behavior between the isoforms. Furthermore, we observed that in the presence of saturating concentrations of the myosin-1C35–derived peptide NTE35, the sliding velocity of the Myo1C0-ΔTH1·NTE35 complex closely resembles that of Myo1C35-ΔTH1. The Myo1C0-ΔTH1·NTE16 complex propelled actin filaments at an intermediate velocity. The changes in motor activity mediated by the different NTEs are consistent with the different roles of myosin-1C isoforms, which range from slow transporter to molecular tension holder (1Gillespie P.G. Cyr J.L. Myosin-1c, the hair Cell's adaptation motor.Annu. Rev. Physiol. 2004; 66: 521-545Crossref PubMed Scopus (129) Google Scholar, 6Venit T. Kalendová A. Petr M. Dzijak R. Pastorek L. Rohožková J. Malohlava J. Hozák P. Nuclear myosin I regulates cell membrane tension.Sci. Rep. 2016; 6: 30864Crossref PubMed Scopus (12) Google Scholar, 18Schwab R.S. Ihnatovych I. Yunus S.Z.S.A. Domaradzki T. Hofmann W.A. Identification of signals that facilitate isoform specific nucleolar localization of myosin IC.Exp. Cell Res. 2013; 319: 1111-1123Crossref PubMed Scopus (9) Google Scholar, 19Adamek N. Coluccio L.M. Geeves M.A. Calcium sensitivity of the cross-bridge cycle of Myo1c, the adaptation motor in the inner ear.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5710-5715Crossref PubMed Scopus (46) Google Scholar). Constructs for the recombinant production of Myo1C0-ΔTH1 and Myo1C35-ΔTH1 were coproduced with calmodulin in the baculovirus Sf9 system and purified to near homogeneity (>95 % purity). Typical yields were 1.6 mg of Myo1C0-ΔTH1 and 0.3 mg of Myo1C35-ΔTH1 from 2 × 109 Sf9 cells. The three full-length isoforms of human myosin-1C (Myo1C0-FL, Myo1C16-FL, and Myo1C35-FL) were produced with yields of approximately 0.1 mg of homogeneous protein from 2 × 109 HEK293SF-3F6 cells. Basal and actin-activated ATP turnover were initially measured at 37 °C. The rate of ATP turnover in the absence of actin (kbasal) differs approximately 2-fold for Myo1C0-ΔTH1 and Myo1C35-ΔTH1, with values of 0.009 ± 0.003 s-1 and 0.004 ± 0.003 s-1, respectively. The actin-activated steady-state ATPase activities of Myo1C0-ΔTH1 and Myo1C35-ΔTH1 were determined at actin concentrations ranging from 0 to 50 μM and fitting of the data to the Michaelis–Menten equation (Fig. 2A). Kapp.actin is the actin concentration at half maximum activation of ATP turnover, and kcat corresponds to the maximum value of ATP turnover in the presence of saturating actin concentrations. For both Myo1C0-ΔTH1 and Myo1C35-ΔTH1, kcat corresponds to 0.37 ± 0.01 s-1. Compared to the Kapp.actin of 12.7 ± 0.7 μM measured with Myo1C0-ΔTH1, the Kapp.actin of Myo1C35-ΔTH1 is increased 2-fold to 25.6 ± 1.8 μM (Fig. 2A and Table 1).Table 1Kinetic and mechanical parameters of human Myo1C-ΔTH1ParameterSignal and measured parameterMyo1C0-ΔTH1Myo1C35-ΔTH1Steady-state ATPase (37 °C) kbasal (s-1)NADH assay; k00.009 ± 0.0030.004 ± 0.003 Kapp.actin (μM)NADH assay; K0.512.7 ± 0.725.6 ± 1.8 kcat (s-1)NADH assay; kmax0.37 ±0.010.37 ± 0.01 kcat/Kapp.actin (μM-1 s-1)NADH assay; initial slope0.024 ± 0.0010.012 ± 0.001 Kapp.actin (μM) (20 °C)NADH assay; K0.59.8 ± 0.117.9 ± 0.1 kcat (s-1)NADH assay; kmax0.09 ± 0.010.09 ± 0.01 kcat/Kapp.actin (μM-1 s-1)NADH assay; initial slope0.008 ± 0.0010.005 ± 0.001Active site isomerization (20 °C) KαPyrene-labeled actin; Afast/Aslow0.90 ± 0.033.70 ± 0.20 k+α (s-1)Pyrene-labeled actin, kmax,slow4.1 ± 0.23.9 ± 0.2 k-α (s-1)k+α/Kα (calc.)4.56 ± 0.131.05 ± 0.11ATP binding (20 °C) 1/K1 (μM)Pyrene-labeled actin, K0.5,fast154 ± 31405 ± 79 k+2 (s-1)Pyrene-labeled actin, kmax,fast37.1 ± 1.637.0 ± 2.0 K1k+2 (μM-1 s-1)aderived from the initial slope of the plot kobs,fast versus [ATP].Pyrene-labeled actin, initial slope0.16 ± 0.010.07 ± 0.01ATP hydrolysis (20 °C) k+3 + k-3 (s-1)Tryptophan, kmax74.6 ± 1.675.7 ± 0.8Actin binding and release (20 °C) (in the absence of nucleotides) k+A (μM-1 s-1)bderived from the slope of the plot kobs versus [actin].Pyrene-labeled actin, slope1.46 ± 0.072.22 ± 0.08 k-A (s-1)Pyrene-labeled actin, kobs0.019 ± 0.0010.037 ± 0.001 KA (nM)k-A/k+A (calc.)13.7 ± 0.116.9 ± 0.2Phosphate release (20 °C) kobs (s-1)cin the presence of 5 μM F-actin at 20 °C.MDCC-PBP0.021 ± 0.0010.010 ± 0.001 k+4 (s-1)NADH assay, global fit0.10 ± 0.010.10 ± 0.01ADP binding and release K5 (μM)dderived from the fit Aslow/Atotal = [ADP]/(K5 + [ADP]). (20 °C)Pyrene-labeled actin, Aslow/Atotal0.46 ± 0.080.23 ± 0.03 k+5 (s-1) (20°/37 °C)Pyrene-labeled actin, kmin,slow1.59 ± 0.07/7.8 ± 0.10.87 ± 0.03/3.8 ± 0.1 k-5 (μM-1 s-1) (20 °C)k+5/K5 (calc.)3.45 ± 0.753.78 ± 0.62Duty ratio (20 °C)k+4/(k+4 + k+5) (calc.)0.044 ± 0.0020.075 ± 0.001Motor properties (37 °C) Sliding velocity (nm s-1)In vitro motility assay52.1 ± 4.914.4 ± 4.2kf0 (s-1)ederived from Equation 1.Frictional load assay70.3 ± 3.668.2 ± 3.9ki (s-1)ederived from Equation 1.Frictional load assay8.0 ± 0.33.7 ± 0.2w (nm)ederived from Equation 1.Frictional load assay7.8 ± 0.43.7 ± 0.1Pmax (aW)fBased on the evaluation of Figure 8B and on the reported stall force for a single myosin-1C0 motor of ∼5 pN (21), we estimate that in our assay approximately 120 motors interact productively per actin filament; single-motor parameters were derived from Equation 2, which was extended by a term representing frictional force.Frictional load assay∼0.05∼0.003FPmax (pN)fBased on the evaluation of Figure 8B and on the reported stall force for a single myosin-1C0 motor of ∼5 pN (21), we estimate that in our assay approximately 120 motors interact productively per actin filament; single-motor parameters were derived from Equation 2, which was extended by a term representing frictional force.Frictional load assay∼2.0∼0.45Stopped-flow buffer and steady-state assay buffer: 25 mM Hepes pH 7.5, 50 mM KCl, 5 mM MgCl2, 0.5 mM DTT; Motility assay buffer: 20 mM imidazole pH 7.5, 50 mM KCl, 5 mM MgCl2, 2.0 mM EGTA.a derived from the initial slope of the plot kobs,fast versus [ATP].b derived from the slope of the plot kobs versus [actin].c in the presence of 5 μM F-actin at 20 °C.d derived from the fit Aslow/Atotal = [ADP]/(K5 + [ADP]).e derived from Equation 1.f Based on the evaluation of Figure 8B and on the reported stall force for a single myosin-1C0 motor of ∼5 pN (21Greenberg M.J. Lin T. Goldman Y.E. Shuman H. Ostap E.M. Myosin IC generates power over a range of loads via a new tension-sensing mechanism.Proc. Natl. Acad. Sci. 2012; 109: E2433-E2440Crossref PubMed Scopus (54) Google Scholar), we estimate that in our assay approximately 120 motors interact productively per actin filament; single-motor parameters were derived from Equation 2, which was extended by a term representing frictional force. Open table in a new tab Stopped-flow buffer and steady-state assay buffer: 25 mM Hepes pH 7.5, 50 mM KCl, 5 mM MgCl2, 0.5 mM DTT; Motility assay buffer: 20 mM imidazole pH 7.5, 50 mM KCl, 5 mM MgCl2, 2.0 mM EGTA. To obtain explicit solutions for the mechanism shown in Figure 3, we performed numerical integration by global fitting using rate constants determined in transient kinetic experiments (Table 2). As transient kinetic experiments were performed at 20 °C, we performed additional measurements of actin-activated steady-state ATPase activities at this temperature (Table 1). In addition to the rate constants determined in transient kinetic experiments, we used the experimentally determined values for the apparent second-order rate constant for actin binding (kcat/Kapp.actin) as additional constraints during simulations, as they are well defined by the initial slope of the data fit to the Michaelis–Menten equation at [actin] << Kapp.actin (23Furch M. Geeves M.A. Manstein D.J. Modulation of actin affinity and actomyosin Adenosine Triphosphatase by Charge changes in the myosin motor domain.Biochemistry. 1998; 37: 6317-6326Crossref PubMed Scopus (141) Google Scholar). The resulting simulated data set describes the actin dependence of ATP turnover for actin concentrations up to 300 μM (Fig. 2B). The global fitting results support a model where Kapp.actin is dominated by the equilibrium constant for actin dissociation from the A·M'·D·Pi complex. Simulated modifications probing the role of changes in the rate of ADP release show only negligible effects on Kapp.actin.Table 2Kinetic parameters of human Myo1C-ΔTH1 isoforms obtained by global fit simulationIndividual reaction stepNomenclature KinTek ExplorerNomenclature used in this studyUnitsMyo1C0-ΔTH1Myo1C35-ΔTH1AM + T ⇌ AMTk+1k+1μM-1 s-14.04.1k–1k-1s-162116501/K11/K1μM156.0405.0AMT ⇌ AM′Tk+2k+2s-137.0037.00k–2k-2s-11.06.9K2K237.05.36AM′T ⇌ M′T + Ak+3k+8s-110.110.1k–3k-8μM-1 s-10.010.01M′T ⇌ M′DPik+4k+3+k-3s-175.0075.00M′DPi + A ⇌ AM′DPik+5k+9μM-1 s-10.791.2k–5k-9s-18.5326.6K5K9μM10.822.2AM′DPi ⇌ AM′D + Pik+6k+4s-10.100.10k–6k-4μM-1 s-10.080.08AM′D ⇌ AM + Dk+7k+5s-11.660.86k–7k-5μM-1 s-13.943.94K7K5μM0.420.22M + A ⇌ AMk+8k+AμM-1 s-11.472.27k–8k-As-10.0190.037K8KAnM13.0116.30AM′ ⇌ AMk+9k+αs-14.23.9k–9k-αs-14.721.05K9Kα0.903.70Shown in red are the experimentally determined parameters measured by transient kinetic experiments that were used to constrain the simulation. Conditions used were 25 mM Hepes pH 7.5, 50 mM KCl, 5 mM MgCl2, 0.5 mM DTT at 20 °C. Open table in a new tab Shown in red are the experimentally determined parameters measured by transient kinetic experiments that were used to constrain the simulation. Conditions used were 25 mM Hepes pH 7.5, 50 mM KCl, 5 mM MgCl2, 0.5 mM DTT at 20 °C. The dissociation of Myo1C0-ΔTH1 and Myo1C35-ΔTH1 from pyrene-labelled F-actin by ATP is accompanied by a biphasic increase in the fluorescence signal (Fig. 4A). The reaction is best fitted by two exponentials and was analyzed according to the model shown in Figure 3 (19Adamek N. Coluccio L.M. Geeves M.A. Calcium sensitivity of the cross-bridge cycle of Myo1c, the adaptation motor in the inner ear.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5710-5715Crossref PubMed Scopus (46) Google Scholar, 24Geeves M.A. Perreault-Micale C. Coluccio L.M. Kinetic Analyses of a truncated Mammalian myosin I suggest a novel isomerization event Preceding nucleotide binding.J. Biol. Chem. 2000; 275: 21624-21630Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The equilibrium constant Kα for the transition from the A·M to A·M' state is given by the ratio of fast to slow phase amplitude at saturating ATP concentrations (24Geeves M.A. Perreault-Micale C. Coluccio L.M. Kinetic Analyses of a truncated Mammalian myosin I suggest a novel isomerization event Preceding nucleotide binding.J. Biol. Chem. 2000; 275: 21624-21630Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Kα was determined with values of 0.90 ± 0.03 for Myo1C0-ΔTH1 and 3.70 ± 0.20 for Myo1C35-ΔTH1 (Fig. 4B). The observed rate constants for the slow phase have a hyperbolic dependence on ATP concentration (Fig. 4C). The fit curves converge toward plateau values that define the isomerization rate k+α for the nucleotide-binding pockets of Myo1C0-ΔTH1 (4.1 ± 0.2 s-1) and Myo1C35-ΔTH1 (3.9 ± 0.2 s-1). The observed rate constants for the fast phase were linearly dependent upon ATP concentrations in the range of 5 to 50 μM. The apparent second-order rate constants for ATP binding K1k+2 are defined by the respective slopes. K1k+2 is 2.4-fold reduced for Myo1C35-ΔTH1 compared with Myo1C0-ΔTH1 with values of 0.068 ± 0.002 μM-1 s-1 and 0.162 ± 0.008 μM-1 s-1, respectively. At high ATP concentrations (>2 mM), the observed rate constants saturate, and the [ATP] dependence of kobs is described by a hyperbola as predicted by Figure 3, where kmax = k+2 and K0.5 = 1/K1 (Fig. 4D). In the case of Myo1C0-ΔTH1, the affinity of ATP for the actin–myosin complex 1/K1 was determined as 154 ± 31 μM for Myo1C35-ΔTH1 and as 405 ± 79 μM for Myo1C35-ΔTH1. The rate constant k+2 for the isomerization that limits the conformational change from high to low actin affinity equals 37.1 ± 1.6 s-1 for Myo1C0-ΔTH1 and 37.0 ± 2.0 s−1 Myo1C35-ΔTH1. To measure ADP release kinetics from the acto· Myo1C-ΔTH1 constructs, we preincubated the protein with ADP and determined the rate of displacement of ADP by monitoring the biphasic exponential increase of the pyrene fluorescence signal that follows the addition of excess ATP (Fig. 5A). Since ADP is in rapid equilibrium with A·M and A·M' on the time scale of the slow phase of the reaction, the kobs of the slow phase decreases with a hyperbolic dependence on the concentration of ADP (19Adamek N. Coluccio L.M. Geeves M.A. Calcium sensitivity of the cross-bridge cycle of Myo1c, the adaptation motor in the inner ear.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5710-5715Crossref PubMed Scopus (46) Google Scholar). The fit of kobs,slow converges toward a minimal plateau value that defines the rate constant for ADP release (k+5) with values of 1.59 ± 0.07 s−1 and 0.87 ± 0.03 s−1 for acto·Myo1C0-ΔTH1 and acto·Myo1C35-ΔTH1, respectively (Fig. 5B). The ADP concentrations at which half-saturation is reached define the apparent ADP affinity constant Kapp with values of 0.21 ± 0.06 μM for acto·Myo1C0 and 0.10 ± 0.03 μM for acto·Myo1C35. The relationship between K5, the dissociation equilibrium constant for ADP to acto·Myo1C, and the apparent equilibrium constant for ADP is defined by Kapp = (K5−/−(1 + 1/Kα). The resulting calculated K5 values correspond to 0.44 ± 0.12 μM for acto·Myo1C0 and 0.13 ± 0.04 μM for acto·Myo1C35. Plots of the fraction of Aslow as a function of [ADP] show a hyperbolic dependence, which at half-saturation directly defines K5 giving values of 0.46 ± 0.08 μM for Myo1C0-ΔTH1 and 0.23 ± 0.03 μM for Myo1C35-ΔTH1 with a smaller margin of error than the calculated values (Fig. 5C). The second-order rate constants for ADP binding (k−5) were calculated from k+5/K5, yielding values of 3.45 ± 0.75 μM-1 s-1 and 3.78 ± 0.62 μM-1 s-1 for acto·Myo1C0-ΔTH1 and acto·Myo1C35-ΔTH1, respectively (Table 1). We measured the Pi release kinetics for the myosin-1C isoforms in the presence of 5 μM actin (Fig. 5D). The observed rates of Pi release were 0.021 ± 0.001 s−1 from acto·Myo1C0-ΔTH1 and 0.010 ± 0.001 s−1 from acto·Myo1C35-ΔTH1. Considering that ATP-turnover measurements in the presence of 5 μM actin, performed at 20 °C and under identical buffer conditions, showed only 15 and 30% of the maximum activation level for Myo1C35-ΔTH1 and Myo1C0-ΔTH1, respectively, we estimate that both constructs share a maximum rate of Pi release of about 0.09 s−1, which limits the rate of ATP turnover. These estimates are in good agreement with values of 0.10 ± 0.01 s−1 for k+4, the rate constants for Pi release in the presence of saturating concentrations of actin, obtained for both constructs by global fitting simulation (Table 1). The rate of myosin-1C binding to actin filaments k+A was measured by recording the exponential decrease of the pyrene fluorescence signal that follows rapid mixing of the proteins. Secondary plots of the observed rate constants against the actin concentration (0.25–3.0 μM) show linear dependencies (Fig. 6A). The second-order association rate constants k+A are defined by the slope of the fit lines. In comparison with Myo1C0-ΔTH1, k+A is 1.5-fold increased for Myo1C35-ΔTH1. The dissociation rate constant k-A was determined by chasing pyrene-labeled actin with a large excess of unlabeled actin. Figure 6B shows the time course for displacement of pyrene-labeled actin from 0.35 μM pyrene-acto·Myo1C-ΔTH1 by the addition of 10 μM unlabeled actin. The time dependence of the ensuing rise in fluorescence amplitude is best described by a single-exponential function, where kobs corresponds directly to the dissociation rate constant k-A. Our results show a 2-fold slower rate of Myo1C0-ΔTH1 dissociation from F-actin than that of Myo1C35-ΔTH1. The equilibrium dissociation constant KA for the interaction of the myosin-1C isoforms with F-actin in the absence of ATP was calcula

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