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• W2025251010 abstract "MI1IQ is a complex of calmodulin and an epitope-tagged 85-kDa fragment representing the amino-terminal catalytic motor domain and the first of 6 calmodulin-binding IQ domains of the mammalian myosin I gene, ratmyr-1 (130-kDa myosin I or MI130). We have determined the transient kinetic parameters that dictate the ATP hydrolysis cycle of mammalian myosin I by examining the properties of MI1IQ. Transient kinetics reveal that the affinity of MI1IQ for actin is 12 nm. The ATP-induced dissociation of actin-MI1IQ is biphasic. The fast phase is dependent upon [ATP], whereas the slow phase is not; both phases show a Ca2+ sensitivity. The fast phase is eliminated by the addition of ADP, 10 μm being required for half-saturation of the effect in the presence of Ca2+ and 3 μm ADP in the absence of Ca2+. The slow phase shares the same rate constant as ADP release (8 and 3 s−1 in the presence and absence of Ca2+, respectively), but cannot be eliminated by decreasing [ADP]. We interpret these results to suggest that actin-myosin I exists in two forms in equilibrium, one of which is unable to bind nucleotide. These results also indicate that the absence of the COOH-terminal 5 calmodulin binding domains of myr-1 do not influence the kinetic properties of MI130 and that the Ca2+ sensitivity of the kinetics are in all likelihood due to Ca2+ binding to the first IQ domain. MI1IQ is a complex of calmodulin and an epitope-tagged 85-kDa fragment representing the amino-terminal catalytic motor domain and the first of 6 calmodulin-binding IQ domains of the mammalian myosin I gene, ratmyr-1 (130-kDa myosin I or MI130). We have determined the transient kinetic parameters that dictate the ATP hydrolysis cycle of mammalian myosin I by examining the properties of MI1IQ. Transient kinetics reveal that the affinity of MI1IQ for actin is 12 nm. The ATP-induced dissociation of actin-MI1IQ is biphasic. The fast phase is dependent upon [ATP], whereas the slow phase is not; both phases show a Ca2+ sensitivity. The fast phase is eliminated by the addition of ADP, 10 μm being required for half-saturation of the effect in the presence of Ca2+ and 3 μm ADP in the absence of Ca2+. The slow phase shares the same rate constant as ADP release (8 and 3 s−1 in the presence and absence of Ca2+, respectively), but cannot be eliminated by decreasing [ADP]. We interpret these results to suggest that actin-myosin I exists in two forms in equilibrium, one of which is unable to bind nucleotide. These results also indicate that the absence of the COOH-terminal 5 calmodulin binding domains of myr-1 do not influence the kinetic properties of MI130 and that the Ca2+ sensitivity of the kinetics are in all likelihood due to Ca2+ binding to the first IQ domain. epitope-tagged motor domain and first IQ domain of 130-kDa myosin I 130-kDa myosin I actin myosin myosin I 4-morpholinepropanesulfonic acid ADP ATP subfragment 1 smooth muscle myosin Class I myosins in mammals are mechanochemical molecules with an amino-terminal motor domain containing an ATP and actin-binding region, a neck region with one or more so-called IQ domains to which calmodulin binds, and a carboxyl-terminal tail region (1.Coluccio L.M. Am. J. Physiol. 1997; 273: C347-C359Crossref PubMed Google Scholar). One member of the class I myosins, MYR-1, is ubiquitously expressed in mammalian cells. myr-1 contains up to 6 IQ domains; alternate splice forms containing 4, 5, or 6 IQ domains exist (2.Ruppert C. Kroschewski R. Bähler M. J. Cell Biol. 1993; 120: 1393-1403Crossref PubMed Scopus (98) Google Scholar). The 130-kDa myosin I isolated from rat liver is a myr-1 gene product (3.Coluccio L.M. Conaty C. Cell Motil. Cytoskel. 1993; 24: 189-199Crossref PubMed Scopus (33) Google Scholar, 4.Coluccio L.M. J. Cell Sci. 1994; 107: 2279-2284Crossref PubMed Google Scholar). Although quantitation of calmodulin has indicated that this preparation contains 6 mol of calmodulin/130-kDa myosin I heavy chain (4.Coluccio L.M. J. Cell Sci. 1994; 107: 2279-2284Crossref PubMed Google Scholar), isoforms corresponding to the 5 IQ and 4 IQ variants are also expressed in liver (2.Ruppert C. Kroschewski R. Bähler M. J. Cell Biol. 1993; 120: 1393-1403Crossref PubMed Scopus (98) Google Scholar) and may be present.The 130-kDa myosin I translocates actin filaments slowly and in a Ca2+-sensitive manner (5.Williams R. Coluccio L.M. Cell Motil Cytoskel. 1994; 27: 41-48Crossref PubMed Scopus (31) Google Scholar). At 10 μm free Ca2+ and above, motility is inhibited. This decrease in motility can be reversed by the addition of exogenous calmodulin, indicating that a calcium-induced dissociation of calmodulin might be responsible for the decrease in motility. Laser trap analyses have indicated that the 130-kDa myosin I translocates actin in a two-step process (6.Veigel C. Coluccio L.M. Jontes J.D. Sparrow J.C. Milligan R.A. Molloy J.E. Nature. 1999; 398: 530-533Crossref PubMed Scopus (258) Google Scholar). The authors proposed that the two mechanical steps are coupled to Pi and ADP release, respectively. Transient kinetic analyses have indicated that the mechanical step coupled to ADP release is unlikely to contribute to force generation or to motility, but could be a system for providing a strain-sensitive ADP release mechanism. This, together with the slow ATP-induced dissociation of actin-myosin I, suggests that this myosin is best suited for maintenance of tension (7.Coluccio L.M. Geeves M.A. J. Biol. Chem. 1999; 274: 21575-21580Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar).We have coexpressed in baculovirus calmodulin and a fragment representing the first 728 amino acids of MYR-1, which codes for the amino-terminal motor domain and 1 IQ domain; we refer to this complex as MI1IQ 1 (8.Perreault-Micale C. Shushan A.D. Coluccio L.M. J. Biol. Chem. 2000; 275: 21618-21623Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). MI1IQ translocates actin filaments in vitro.Unlike the parent molecule, the rate of actin translocation is not affected by the Ca2+ concentration over the range ofpCa 4–7 and the rates of movement of MI130 and MI1IQ are comparable.The availability of a homogeneous preparation from the baculovirus expression system has allowed us to explore in detail the kinetics of this construct representing the motor domain and the first IQ domain. Our results indicate that the truncated myosin I possesses kinetic properties indistinguishable from the parent molecule. Thus, the 5 deleted IQ domains and their associated calmodulins play no role in defining the unloaded properties of the myosin I head. Furthermore, our results indicate that, when bound to actin, myosin I exists in two conformations in equilibrium and that ATP can bind to only one of the two conformations. The equilibrium between the two forms is sensitive to both Ca2+ and ADP concentrations and we propose that the conformational change between the two forms may correlate with (i) the double step observed in laser trap studies with this molecule (6.Veigel C. Coluccio L.M. Jontes J.D. Sparrow J.C. Milligan R.A. Molloy J.E. Nature. 1999; 398: 530-533Crossref PubMed Scopus (258) Google Scholar) and (ii) the ADP-dependent conformational change identified by electron microscopy for a closely related myosin I, brush border myosin I (9.Jontes J.D. Wilson-Kubalek E.M. Milligan R.A. Nature. 1995; 378: 751-753Crossref PubMed Scopus (166) Google Scholar, 10.Jontes J.D. Milligan R.A. J. Cell Biol. 1997; 139: 683-693Crossref PubMed Scopus (60) Google Scholar).RESULTSThe transient kinetics of the interaction of MI1IQwith actin and ATP were examined and compared with our previous measurements on the native protein, MI130 (7.Coluccio L.M. Geeves M.A. J. Biol. Chem. 1999; 274: 21575-21580Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). In all cases the results were very similar. Fig.1 A shows the fluorescent transient observed upon addition of 100 μm ATP to a 25 nm complex of MI1IQ and pyrene-labeled actin in the presence of Ca2+. The transient was well described by a single exponential with a k obs of 2.4 s−1 and corresponds to the dissociation of actin from the complex. At low ATP concentrations (25–200 μm), the reaction was monophasic andk obs was linearly dependent upon [ATP] with an apparent second order rate constant, K 1 k +2, of 5.3 × 104m−1s−1. Above 200 μm ATP, the reaction became biphasic and could be described by a two exponential term (Fig. 1 B). The slow phase was independent of [ATP] with a k obs of 8 s−1and an amplitude of 25–30% of the total transient. The fast phase showed a hyperbolic dependence upon [ATP], and the best fit gave a maximum observed rate constant of 79 s−1 with 1.5 mm ATP required for half saturation ofk obs (see Fig. 1 C). By analogy with other actomyosin systems, the maximal rate and the ATP concentration at half the maximal rate were assigned to k +2, and1/K 1, respectively, the rate constant for an isomerization of the actin-myosin complex, which limits the dissociation of actin and the affinity of ATP for the actin-myosin complex (Table I).Table ITransient kinetic analysis of native rat MI 130 and expressed fragment MI 1IQRate/eq constantUnitsMI130(7)MI1IQRb S1 (22)+Ca2+−Ca2++Ca2+−Ca2+Nucleotide binding to acto-MK 1 · k +2μm−1s−10.0230.0170.0530.032.11/K 1mm3.21.91.51.25.7k +2s−17432.5793612,000K ADμm≤10≤10103200k −ADs−1628.03.5>500k +ADμm−1s−10.81.1Actin binding −ADPK Anm13131230 +ADPK DAnm60–11060–1101000ATP bindingK 1 · k +2μm−1s−10.10.10.10.12.3k +3 +k −3s−1130Mant ATPK 1 · k +2μm−1s−10.10.13.2 Open table in a new tab Repeating the measurement in the absence of Ca2+ gave similar results, except that the slow component of the transient comprised 50% of the total amplitude with k obsof 3.4 s−1 and the best fit to the [ATP] dependence of the k obs of the fast component gave k +2 = 36 s−1and 1/K 1 = 1.2 mm for an apparent second order rate constant (−Ca2+) of 3 × 104m−1s−1 (Fig. 1 C).We had previously observed for MI130 that the fast phase of the transient was eliminated by incubating the proteins with 20 μm ADP before initiating the reaction by mixing with ATP. A similar result was observed here for the expressed myosin I fragment (Fig. 2 A). In this case we were able to titrate the fast phase of the transient, and a plot of the amplitude against [ADP] in the presence and absence of Ca2+ is shown in Fig. 2 B. A fit of the amplitude to a hyperbola gave a best fit of 10 and 3 μm, respectively, in the presence and absence of Ca2+, and this is assigned to the affinity of ADP for the A.MI1IQ complex ( K AD). The k obs of the slow phase remained almost constant over the range of [ADP] used (8.5 s−1 in Ca2+; 2.45 s−1, −Ca2+) and was assigned to k −AD, the rate of ADP dissociation from A.MI1IQ.ADP. Since K AD = k −AD/ k +AD, it is possible to calculate k +AD, the apparent second order rate constant of ADP binding to A·MI1IQ. This gave values of 0.8 × 106m−1 s−1(+Ca2+) and 1.1 × 106m−1 s−1(−Ca2+), i.e. the apparent second order rate constant of ADP binding is Ca2+ independent and 15–40 fold faster than the apparent second order rate constant of ATP binding to A·MI1IQ.Figure 2Influence of ADP on the ATP-induced dissociation of actin-MI130. A, addition of 2.5 mm ATP to 25 nmpyrene-actin-MI130 in the absence of ADP resulted in a rapid increase in fluorescence best described by a double exponential (k obs = 43.8 and 8.5 s−1 and amplitudes of 18.8 and 9.4%, respectively). In the presence of 20 μm ADP, the change in fluorescence can be described by a single exponential with ak obs = 10.3 s−1 and an amplitude of 19.8%; however, the data can be equally described by a double exponential with k obs = 37.7 and 8.5 s−1 and amplitudes of 3.8% and 17.3%, respectively. B, titration of the amplitude of the fast phase against [ADP] added to the protein before mixing with ATP in the presence and absence of Ca2+. The data were fitted to a hyperbola in each case, and the apparent affinity ( K AD) for ADP is 10 μm in the presence of Ca2+ and 3 μm in its absence.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To verify that the rate of ADP binding is faster than ATP binding, we examined the rates of competitive binding of ADP and ATP to A·MI1IQ. A·MI1IQ at 30 nm was mixed in the stopped flow apparatus with 1 mm ATP and 0–40 μm ADP. The amplitude of the fast phase decreased, whereas k obs increased with increasing [ADP] (data not shown). This demonstrates that ADP effectively competes with ATP even at less than one-tenth of the concentration. If the rate of ADP dissociation from the A·M·D complex is much less than the rate at which ADP and ATP bind to A·M (i.e.[A·M·D] k −AD ≪ [ATP] K 1 k +2 + [ADP] k +AD), thenk obs = [ATP] K 1 k +2 + [ADP] k +AD. At a fixed ATP concentration,k obs increased from 23 to 42 s−1 at 20 μm ADP and further increased to 73 s−1 at 40 μm. The data over this limited range are therefore compatible with a value of k +AD of 0.95–1.25 × 106m−1s−1 in agreement with the estimate above.The slow phase of the ATP-induced dissociation of A·MI1IQhas a k obs that is very similar to k −AD, the rate constant of ADP dissociation from the A·MI1IQ·D complex even in the absence of added ADP. It is possible, therefore, that the slow phase represents a fraction of A·MI1IQ that is isolated with ADP bound. Extensive treatment of the protein with apyrase did not eliminate the slow phase of the reaction. In contrast, if the protein was treated with 20 μm ADP such that the fast phase was eliminated, then treatment with apyrase restored the fast phase but only to the same extent as in the original measurement. Thus, apyrase treatment does eliminate the protein bound ADP effectively. We therefore conclude that the slow phase is not caused by the presence of ADP bound to the protein.Another possibility is that ATP could be the source of contaminating ADP. ATP normally contains about 1% ADP. If ADP binds to the protein faster than ATP (as shown above), then the contaminant ADP could bind a fraction of the protein to produce the slow phase. The true substrate for myosin is MgATP, and the product is MgADP. Since ATP binds Mg2+ more tightly than ADP, reducing the free Mg2+ concentration to a minimum should reduce the contaminant MgADP concentration; however, under limiting Mg2+ concentrations, the slow phase remained constant (data not shown). Furthermore, addition of up to 5% ADP into the ATP had no effect on the amplitude of the slow phase (see above). These results indicate that the slow phase is not due to ADP contamination in the ATP.The effect of Ca2+ on the rate of ADP release from MI130 was measured by determining in buffers containing fixed amounts of Ca2+, the rate of the ATP-induced dissociation of pyrene-labeled actin-MI in the presence of saturating amounts of ADP (Fig. 3). Thek obs was plotted as a function of [Ca2+], and the line represents the best fit to the Hill equation with midpoint of 6.8 ± 0.6 μmCa2+ and k obs in the presence and absence of Ca2+ of 8.0 and 1.5 s−1, respectively. The slope of the graph that defines the Hill coefficient was poorly defined as 3 ± 1.5. These data indicate that binding of Ca2+ to the myosin I complex is positively cooperative.Figure 3Calcium dependence of the observed rate of ADP displacement from A· MI130 (squares) and A· MI1IQ (circles). The observed rate constant for ADP release was estimated from the observed rate of ATP-induced dissociation of 25 nm pyrene-labeled actin-MI in the presence of 50 μm [ADP]. Thek obs was plotted as a function of [Ca2+], and the line represents the best fit of the MI130 data to the Hill equation with a Ca2+ affinity of 6.9 ± 0.8 μm withk obs in the presence and absence of Ca2+ of 8.3 ± 0.4 and 1.5 ± 0.2 s−1, respectively. The Hill coefficient was not well defined by the data, and an acceptable fit could be achieved with any value from 2 to 5.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The binding of ATP to MI1IQ was followed using intrinsic protein fluorescence and gave results that were indistinguishable from those of MI130 (data not shown), indicating a second order rate constant for ATP binding of 0.1 × 106m−1 s−1that was independent of Ca2+. There was no evidence of a slow component of the reaction; however, the signal to noise ratio was very poor in these measurements, and the possibility of a slower component cannot be eliminated.The affinity of MI1IQ for actin was measured by monitoring the amplitude of the ATP-induced dissociation of pyrene-labeled A·MI1IQ as a function of [MI1IQ] (15.Kurzawa S.E. Geeves M.A. J. Muscle Res. Cell Motil. 1996; 17: 669-676Crossref PubMed Scopus (69) Google Scholar). Using 30 nm pA and 400 μm ATP, the amplitude of the reaction was measured for MI1IQ from 10 to 150 nm (Fig. 4). Analysis of the data gave a value of K A of 12 nm, in good agreement with the value obtained for the native protein by a less direct method (7.Coluccio L.M. Geeves M.A. J. Biol. Chem. 1999; 274: 21575-21580Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar).Figure 4Titration of actin with MI.A, 30 nm phalloidin-stabilized pyrene-labeled actin was incubated with 10, 20, and 150 nmMI1IQ before mixing with 200 μm ATP. In each case the reaction was well described by a single exponential withk obs of 3.8 s−1. The amplitude increased with increasing MI concentration, and a plot of the amplitudes against [MI] is in B. The best fit to the quadratic equation describing the binding isotherm (see “Experimental Procedures”) gave a K d of 12 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONOur results demonstrate that MI1IQ is essentially indistinguishable from MI130 in terms of its interaction with actin and nucleotide, indicating that the presence of the 5 additional IQ domains and associated calmodulins in the parent molecule has no effect on these properties (Table I). The truncated myosin I shows the same actin-activated Mg2+-ATPase activity as the parent molecule (8.Perreault-Micale C. Shushan A.D. Coluccio L.M. J. Biol. Chem. 2000; 275: 21618-21623Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) and the ATP, ADP, and actin binding to A·MI1IQ are identical to the parent. Since the actin and nucleotide binding to MI1IQ are unchanged, the coupling between actin and nucleotide binding is also expected to remain unaffected by the missing IQ domains and their associated calmodulins. In addition, the Ca2+ sensitivity of all of the above properties is identical to that of the parent molecule, MI130. These results demonstrate that the Ca2+sensitivity of these properties is not a function of the missing calmodulins and must result from Ca2+ binding to the first calmodulin or from a novel independent Ca2+ binding site in the motor domain.All of the events in the acto-MI ATPase cycle that we have measured are significantly faster than the turnover rate and are therefore not rate-limiting in the ATPase reaction. It is therefore likely that Pi release is rate-limiting, as has been observed for other myosins, and in this case the Pi release must be regulated by Ca2+, as previously observed for scallop muscle myosin II (19.Wells C. Bagshaw C.R. Nature. 1985; 313: 696-697Crossref PubMed Scopus (43) Google Scholar). A biphasic fluorescent transient was observed upon introduction of ATP to complexes of pyrene-labeled actin with both MI130 and MI1IQ. The amplitudes and rates of the two phases were similar for the two proteins, and in both cases the rate constant of the slow phase was very similar to the rate constant limiting ADP release from A·MI. To address the possibility that the slow component was due to ADP present either in association with the expressed myosin I or as a trace contaminant in the ATP, several experiments designed to reduce ADP levels were performed. In all cases, the slow phase remained unaltered.For the native protein we considered the alternative possibility that the slow phase in the ATP dissociation reaction was due to the presence of a fraction of damaged myosin I, e.g. missing one or more of the calmodulins, or that a contaminant myosin I could be present and therefore responsible for the biphasic nature of the ADP-induced dissociation. It seems unlikely, however, that the same contaminant would be present in both myosin I preparations (i.e. the native protein isolated from liver and the expressed fragment from insect cells) since they derive from different cell types and involve different purification schemes. We are therefore forced to conclude that this represents an intrinsic property of both the expressed protein and the parent MI130.The simplest explanation of the biphasic nature of the ATP-induced dissociation is that the protein exists in two forms: one to which ATP can bind readily (at an apparent second order rate constant of 5.3 × 104m−1s−1 in the presence of Ca2+) and the other, which cannot bind ATP without first isomerizing. One possible model is shown in Fig. 5, where the nucleotide pocket must open before nucleotide can bind. (In this model, we have assumed a direct link between the opening of the nucleotide pocket and an ADP-induced structural change, i.e.“wagging” of the myosin neck; see below.) The rate constant for the opening of the pocket is very similar to the net rate constant of ADP dissociation suggesting a similar process limits ADP release ( k +α = k +αD = 8 s−1, Fig. 5). From the data presented in Figs. 1 and 2, we can define the rate and equilibrium constants of each of the transitions shown in Fig.5. The assignment of the constants is described in the legend to Fig.5, and the values are listed in TableII.Figure 5Proposed model depicting the isomerization of the nucleotide-binding pocket that must occur before ATP or ADP can bind or ADP can be released. The conformational change is represented as a swing of the converter domain of the myosin head with respect to the actin binding domain, which is coupled to the accessibility of the nucleotide binding pocket. The data in Figs. 1 and2 allow assignment of all of the rate and equilibrium constants. Analysis of the amplitudes of the ATP-induced dissociation reaction in the presence of Ca2+ shows a 60:30 ratio of the two forms. This ratio defines the equilibrium constant between the two forms of A·M, K α, with a value of ∼2.5. Since k +α = 8 s−1(the slow phase of ATP binding) and K α = k +α/ k −α, then k −α = 3.2 s−1. In the absence of Ca2+, the amplitudes of the two phases are similar, consistent with the equilibrium lying closer to the closed form of A·M with K α = 1–2 and since k +α = 3.4 s−1, k −α is unchanged by Ca2+ at 3–4 s−1. Since (i) the rate constant of ADP dissociation from A·M·D ( k +αD) and the rate constant of the isomerization of A·M ( k +α) are similar and (ii) they are both reduced 2–3-fold on removal of Ca2+, it suggests that the two events are closely related and may represent the same isomerization of the A·M complex as shown. The displacement of ADP from A·M occurs in a single phase and suggests little occupancy of A·M′·D; therefore, in both the presence and absence of Ca2+, K αD ≤ 0.1. The affinity of ADP for the complex, K AD, is defined by K ADP· K αDand has a value of 10 μm in the presence of Ca2+ and 3 μm in the absence of Ca2+. Although K ADP and K αD are not defined individually, K αD is k +αD/ k −αDand therefore K AD= K ADP· k +αD/ k −αDor, after rearranging, k −αD/ K ADP= k +αD/ K AD. Since k +αD has a value of 8 s−1 (+Ca2+) and 3.4 s−1 (−Ca2+), the apparent ADP on rate, k −αD/ K ADP, has a value of 0.8 × 10−6m−1 s−1in the presence of Ca2+ or 1.1 × 10−6m−1in the absence of Ca2+. This is in good agreement with the directly measured values and is independent of calcium. In this interpretation the rate of the conformational change giving access to the site is Ca2+-dependent but independent of ADP bound to the pocket, whereas reversal of the conformational change is ADP-dependent and Ca2+-independent.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IIDerived rate and equilibrium constants for the reaction scheme of Fig. 5K αk +αk −αK αDk +αDk −αD/ K ADPK ADPs −1s −1s −1μm −1 s −1μm+Ca2+2–383–4≤0.180.8≥100−Ca2+1–23.43–4≤0.13.41.1≥30 Open table in a new tab The evidence points toward a significant proportion (∼30%) of the conformation with a closed nucleotide pocket being present in the absence of nucleotide. By increasing the rate constant of pocket closing, ADP causes almost all of the myosin I to be in the closed pocket form. In contrast, the presence of Ca2+ increases the proportion of the open pocket form by increasing the rate of the opening of the pocket in both the presence and absence of ADP. In terms of nucleotide binding, Ca2+ lowers the affinity of A·MI for ADP by increasing the net rate constant of ADP release ( k +αD). Ca2+ also increases the rate of ATP-induced dissociation of actin ( k +2, Scheme I and Table I) from A·MI, but does not affect the affinity of ATP for A·MI ( K 1). Thus, Ca2+ can stimulate the detachment of actin from A·MI·D by increasing the rate of ADP release and the rate of the subsequent dissociation by ATP. Note, however, that the effects of Ca2+ are small (in all cases, values do not differ by more than a factor of 3) and therefore probably do not represent an on/off switch but rather a modulator of activity.At physiological nucleotide concentrations of 1 mm ATP and 10–50 μm ADP, the net rates of nucleotide binding to A·MI will be 18 s−1 for ATP and 10–50 s−1 for ADP in the absence of Ca2+. Thus, the A·MI that has released ADP is as likely to rebind ADP as to bind ATP. The low efficiency for binding ATP and detaching from actin supports our proposal that myosin I is designed for tension maintenance not motility (7.Coluccio L.M. Geeves M.A. J. Biol. Chem. 1999; 274: 21575-21580Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The presence of Ca2+ increases the net rate of ATP binding ( K 1 k +2) but does not alter the rate of ADP rebinding k −αD/ K ADP.The possibility that myosin I exists in two conformational states, as shown by the kinetic evidence presented here, is of great interest since an isomerization of A·MI·D can be responsible for the second displacement seen in laser trap assays for MI130, brush border myosin I (6.Veigel C. Coluccio L.M. Jontes J.D. Sparrow J.C. Milligan R.A. Molloy J.E. Nature. 1999; 398: 530-533Crossref PubMed Scopus (258) Google Scholar), a close relative to MI130, and smooth muscle myosin S1 (smS1) (20.Veigel C. Kendrick-Jones J. Sellers J.R. Sparrow J.C. Molloy J.E. Biophys. J. 1999; 76: A145Google Scholar). It is also required for the structural changes or “tail wagging” seen in three-dimensional reconstructions from electron micrographs for some actomyosins including brush border myosin I and smS1 (9.Jontes J.D. Wilson-Kubalek E.M. Milligan R.A. Nature. 1995; 378: 751-753Crossref PubMed Scopus (166) Google Scholar, 21.Whittaker M. Wilson-Kubalek E.M. Smith J.E. Faust L. Milligan R.A. Sweeney H.L. Nature. 1995; 378: 748-751Crossref PubMed Scopus (339) Google Scholar). If our assignment is correct, then the identified isomerization provides a direct link between the accessibility of nucleotide to its binding pocket and the mechanical transient and lends strong support for the role of nucleotide release in providing a strain-sensing mechanism (22.Cremo C.R. Geeves M.A. Biochemistry. 1998; 37: 1969-1978Crossref PubMed Scopus (148) Google Scholar). We previously argued that a MI cross-bridge bearing the typical isometric tension could have ADP release reduced up to 100-fold. In the current model, the strain would act directly on the isomerization shown in Fig. 5. Any load or strain on the cross-bridge would inhibit the swing of the tail against the load. The effect would be to reduce k +αDand hence K α.The Ca2+ sensitivity of the A·MI isomerization also establishes a link between the binding of Ca2+ (presumably to the remaining calmodulin), and the biochemical, structural, and mechanical events discussed above. If the protein isomerization is a mechanical sensor that slows down the rates of both ADP release and ATP binding in the presence of strain, then the effect of Ca2+may be far more dramatic for a head bearing strain. Ca2+could act on the strained head by binding to calmodulin to alter the elasticity of the calmodulin-IQ complex and so reduce the strain leading to acceleration of the isomerization, ADP release, and cross-bridge detachment. The other 5 calmodulin-IQ domains could also contribute to the Ca2+-assisted release of strain if they are elastically distorted in a strained cross-bridge. All that is required is for the Ca2+-induced change in calmodulin conformation to alter the rest length of each calmodulin-IQ domain such that the strain on a A·MI·D head is modulated. It has previously been noted that Ca2+ binding reduces the affinity of calmodulin for the IQ domain but probably not enough to cause calmodulin dissociation at cellular concentrations of calmodulin.A remaining question about the A·MI conformation is its relation to conformational changes in MI alone and t" @default.
• W2025251010 created "2016-06-24" @default.
• W2025251010 creator A5002133650 @default.
• W2025251010 creator A5039484661 @default.
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• W2025251010 date "2000-07-01" @default.
• W2025251010 modified "2023-09-29" @default.
• W2025251010 title "Kinetic Analyses of a Truncated Mammalian Myosin I Suggest a Novel Isomerization Event Preceding Nucleotide Binding" @default.
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