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Calcium regulation of calmodulin binding to and dissociation from the myo1c regulatory domain - PubMed

  • ️Mon Jan 01 2007

. 2007 Oct 23;46(42):11718-26.

doi: 10.1021/bi700894h. Epub 2007 Oct 2.

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Calcium regulation of calmodulin binding to and dissociation from the myo1c regulatory domain

Slobodanka Manceva et al. Biochemistry. 2007.

Abstract

Myo1c is an unconventional myosin involved in cell signaling and membrane dynamics. Calcium binding to the regulatory-domain-associated calmodulin affects myo1c motor properties, but the kinetic details of this regulation are not fully understood. We performed actin gliding assays, ATPase measurements, fluorescence spectroscopy, and stopped-flow kinetics to determine the biochemical parameters that define the calmodulin-regulatory-domain interaction. We found calcium moderately increases the actin-activated ATPase activity and completely inhibits actin gliding. Addition of exogenous calmodulin in the presence of calcium fully restores the actin gliding rate. A fluorescently labeled calmodulin mutant (N111C) binds to recombinant peptides containing the myo1c IQ motifs at a diffusion-limited rate in the presence and absence of calcium. Measurements of calmodulin dissociation from the IQ motifs in the absence of calcium show that the calmodulin bound to the IQ motif adjacent to the motor domain (IQ1) has the slowest dissociation rate (0.0007 s-1), and the IQ motif adjacent to the tail domain (IQ3) has the fastest dissociation rate (0.5 s-1). When the complex is equilibrated with calcium, calmodulin dissociates most rapidly from IQ1 (60 s-1). However, this increased rate of dissociation is limited by a slow calcium-induced conformational change (3 s-1). Fluorescence anisotropy decay of fluorescently labeled N111C bound to myo1c did not depend appreciably on Ca2+. Our data suggest that the calmodulin bound to the IQ motif adjacent to the motor domain is rapidly exchangeable in the presence of calcium and is responsible for regulation of myo1c ATPase and motile activity.

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Figures

Figure 1
Figure 1. Domain structure of myo1c and IQ-motif expression constructs used in this study

Light gray and dark gray boxes represent 6x-His and FLAG tags, respectively, used for purification. The black box represents an Avi-tag used for site-specific biotinylation. The protein sequences of the individual IQ motifs are shown.

Figure 2
Figure 2. Effect of calcium and calmodulin on myo1cIQ1-3

(Top) Actin-activated ATPase activity of 100 nM myo1cIQ1-3 in the presence of 50 μM actin as a function of the concentration of free calcium in the (●) absence and (■) presence of 10 μM calmodulin. The first point represents the ATPase activity in the absence of added calcium. (Center) Velocity of actin filament gliding, measured by the in vitro motility assay, as a function of calmodulin concentration in the (■) absence and presence of (●) 10 μM and (▲) 100 μM free calcium. No motility was detected in the absence of calmodulin and presence of 10 μM or 100 μM calcium. Each point and error bar represents the average and standard deviation of 50 filaments (Bottom) Number of calmodulin molecules bound to myo1cIQ1-3 as a function of calmodulin concentration in the (●) absence and (■) presence of 100 μM free calcium. The calmodulins bound at the zero point co-purifed with the myosin. Error bars represent the range of duplicate experiments.

Figure 3
Figure 3. Binding of calmodulin to IQ12 and IQ23 as determined by changes in intrinsic tryptophan fluorescence

(Top) Fluorescence intensity (λex = 295 nm, λem = 350 nm) of (left) 0.5 μM IQ12 and (right) 0.5 μM IQ23 titrated with 0 – 6 μM calmodulin in the (●) absence and (■) presence of 100 μM free calcium. Data were normalized to the average maximum intensity of the fluorescence emission in the absence of calcium. Lines are drawn by eye to show stoichiometric binding of calmodulin to the IQ motifs. (Bottom) Fluorescence emission spectra of (left) IQ12 and (right) IQ23 in the absence and presence of calmodulin.

Figure 4
Figure 4. Binding of L-CaM to IQ12 and IQ23 as determined by FRET

FRET emission intensity (λex = 295 nm, λem = 490 nm) of 0 – 3 μM L-CaM and (top) 0.5 μM IQ12 and (bottom) 0.5 μM IQ23 in the (●) absence and (■) presence of 100 μM free calcium. Lines are drawn by eye and show stoichiometric binding of calmodulin to the IQ motifs.

Figure 5
Figure 5. Kinetics of L-CaM binding to IQ12 and IQ23

Time courses of FRET emission intensity obtained after mixing 0.05 μM IQ12 or IQ23 with 0.1 μM L-CaM in the (Top) absence or (Bottom) presence of 100 μM free calcium. The solid lines are the best fits to the data as described in the text. Transients are the averages of 14 – 26 individual traces.

Figure 6
Figure 6. Kinetics of L-CaM dissociation from IQ12 and IQ23

Time courses of FRET emission intensity obtained after mixing equilibrated mixtures of 1.25 μM L-CaM plus (top) 0.5 μM IQ12 or (bottom) 0.5 μM IQ23 in the absence or presence of 100 μM free calcium with a 50-fold excess of unlabeled calmodulin. Solid lines are the best fits to two exponential rate functions. Transients are the averages of five traces.

Figure 7
Figure 7. Kinetics of calcium-induced dissociation of calmodulin from IQ12

Time course of FRET emission intensity obtained after mixing an equilibrated mixture of 0.75 μM L-CaM and 0.3 μM IQ12 with a 50-fold excess of unlabeled calmodulin and 100 μM free calcium. The solid line is the best fit to a two exponential rate function.

Figure 8
Figure 8. Time-resolved fluorescence anisotropy

(Top) Time course of fluorescence decay after brief excitation at 337 nm. The samples were L-CaM in the absence of myo1cIQ1-3 (left), L-CaM in the presence of myo1cIQ1-3 and 1 mM EGTA (center), and L-CaM in the presence of myo1cIQ1-3, 1.1 mM calcium, and 1 mM EGTA (right). The instrument response function (IRF) is the ~2 ns intensity spike near zero time. The upper red decay curve in each panel is IVV and the lower blue decay curve is IVH. Symbols are the recorded intensities and the lines are the convolutions of the IRF with a restricted rotational diffusion model. (Center) Plots of normalized residuals [(photon counts - fit)/fit]. (Bottom) Time-resolved anisotropies calculated with a restricted rotational diffusion model with r(0) and r(∞) values given in the text (solid black lines). A model assuming a single rotational decay for L-CaM in the absence of myo1cIQ1-3 (blue line) gives identical results.

Figure 9
Figure 9

Scheme of calmodulin dissociation from myo1c.

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