Báo cáo khoa học: The two IQ-motifs and Ca2+/calmodulin regulate the rat myosin 1d ATPase activity pptx

The two IQ-motifs and Ca 2+ /calmodulin regulate the rat myosin 1d ATPase activity Danny Ko ¨ hler*, Sandra Struchholz* and Martin Ba ¨ hler Institute for General Zoology and Genetics, Westfa ¨ lische Wilhelms University, Mu ¨ nster, Germany Myosins are actin-based motors that serve a variety of cellular functions. They generally consist of a head and a tail. The head represents the motor part and contains nucleotide and actin binding sites. In addition, it encompasses at its C-terminus a light chain binding domain. The tail provides additional activities like, e.g. cargo binding. The motor activity of various myosins can be regulated by a number of different mechanisms. In several myosins the light chain binding domain and the associated light chains have been demonstrated to exhibit a regulatory function [1–3]. The light chain binding domains comprise different numbers of IQ-motifs [1] that represent binding sites for the Ca 2+ - sensor protein calmodulin (CaM) and CaM-related EF-hand proteins. Many unconventional myosins have between one and six CaM light chain(s) associated with them. CaM contains four Ca 2+ -binding sites, one pair of lower affinity sites in the N-terminal lobe and one pair of higher affinity sites in the C-terminal lobe, respectively [4,5]. Initially, IQ-motifs were identified as Ca 2+ -independent CaM-binding sites [6,7]. However, in some instances IQ-motifs have been shown to bind CaM in a Ca 2+ -dependent manner [1,8,9]. Based on myosin head sequence comparisons, myo- sins have been subdivided into 18 classes [10–12]. The mammalian class I myosins studied so far have all been shown to contain CaM light chains. Analysis of the regulation of class I myosins by Ca 2+ ⁄ CaM has provided different results with no evidence for a com- mon mechanism operating in class I myosins. Myosins 1a (brush border myosin I), 1b (myr 1) and 1c (myosin Ib, myr 2) contain 3–6 CaM binding sites [13–17]. Ele- vation of the free Ca 2+ concentration leads to a par- tial loss of CaM from these myosins in vitro. However, it is not known whether CaM dissociation occurs also in vivo. Provided that CaM-binding is saturated by the addition of exogenous CaM, an increase in free Ca 2+ concentration stimulates the basal (actin-independent) Keywords calmodulin; Ca 2+ regulation; IQ-motif; myosin; Myo1d Correspondence M. Ba ¨ hler, Institut fu ¨ r Allgemeine Zoologie und Genetik, Westfa ¨ lische Wilhelms- Universita ¨ t, Schlossplatz 5, 48149 Mu ¨ nster, Germany Fax: +49 251 8324723 Tel: +49 251 8323874 E-mail: baehler@nwz.uni-muenster.de *These authors contributed equally to this work (Received 24 January 2005, revised 25 February 2005, accepted 4 March 2005) doi:10.1111/j.1742-4658.2005.04642.x The light chain binding domain of rat myosin 1d consists of two IQ-motifs, both of which bind the light chain calmodulin (CaM). To analyze the Myo1d ATPase activity as a function of the IQ-motifs and Ca 2+ ⁄ CaM binding, we expressed and affinity purified the Myo1d constructs Myo1d- head, Myo1d-IQ1, Myo1d-IQ1.2, Myo1d-IQ2 and Myo1dDLV-IQ2. IQ1 exhibited a high affinity for CaM both in the absence and presence of free Ca 2+ . IQ2 had a lower affinity for CaM in the absence of Ca 2+ than in the presence of Ca 2+ . The actin-activated ATPase activity of Myo1d was 75% inhibited by Ca 2+ -binding to CaM. This inhibition was observed irrespective of whether IQ1, IQ2 or both IQ1 and IQ2 were fused to the head. Based on the measured Ca 2+ -dependence, we propose that Ca 2+ - binding to the C-terminal pair of high affinity sites in CaM inhibits the Myo1d actin-activated ATPase activity. This inhibition was due to a conformational change of the C-terminal lobe of CaM remaining bound to the IQ-motif(s). Interestingly, a similar but Ca 2+ -independent inhibition of Myo1d actin-activated ATPase activity was observed when IQ2, fused directly to the Myo1d-head, was rotated through 200° by the deletion of two amino acids in the lever arm a-helix N-terminal to the IQ-motif. Abbreviation CaM, calmodulin. FEBS Journal 272 (2005) 2189–2197 ª 2005 FEBS 2189 ATPase activity in Myo1a-c and also the actin-activa- ted ATPase activity of Myo1b. The translocation of actin filaments by these class I myosins in the gliding assay was either abolished, slowed or not affected [18– 20]. For myosin 1c it has been demonstrated that bind- ing of Ca 2+ to the C-terminal pair of Ca 2+ -binding sites in CaM inhibits its actin-translocating activity [19]. Myosin 1e, another class I myosin, contains a single CaM-binding site and an increase in free Ca 2+ concentration did not cause a release of the CaM bound to this site, but induced a decrease in basal ATPase activity [21]. Rat myosin 1d (Myo1d, formerly called myr 4) exhibits a light chain binding domain with two IQ- motifs. In gel overlay assays, the IQ-motif 1 (IQ1) has been found to bind CaM with higher affinity in the absence of Ca 2+ whereas IQ-motif 2 (IQ2) bound CaM in a Ca 2+ -dependent manner [8]. Currently, it is not known whether the light chain binding domain and the CaM light chains serve a regulatory function in Myo1d. Single molecule mechanical measurements demonstrated that the light chain binding domain of Myo1d functions as a rigid mechanical lever rotating by 90° during the working stroke [22]. In analogy to other myosins it is assumed that the IQ-motifs adopt an a-helical conformation that is stabilized by the associated light chains [1,23]. In the present study we analyzed the regulation of Myo1d ATPase activity by Ca 2+ ⁄ CaM and the two IQ-motifs. We affinity purified recombinant Myo1d constructs from stable transfected HeLa cells that dif- fered in number and position of IQ-motifs and deter- mined the binding of CaM to these IQ-motifs. The effects on Myo1d ATPase activity upon Ca 2+ -binding to CaM associated with the IQ-motifs were investi- gated. We now report that Ca 2+ -binding to the C-ter- minal pair of Ca 2+ -binding sites in CaM bound to the IQ-motif directly following the converter domain inhibits Myo1d actin-activated ATPase activity. Dele- tion of two amino acids at the interface between con- verter and IQ-motif led to a Ca 2+ -independent inhibition of the actin-activated ATPase activity. Results Binding of Ca 2+ ⁄ CaM to the two Myo1d IQ-motifs The light chain binding domain of rat Myo1d contains two IQ-motifs that are quite distinct in sequence (Fig. 1B). To investigate their interaction with the Ca 2+ -sensor molecule CaM, we expressed different recombinant Myo1d proteins in HeLa cells (Fig. 1A). The recombinant Myo1d proteins included the head domain and either no, one or both IQ-motifs. In addi- tion, we expressed a construct that lacked the first IQ-motif and contained only the second IQ-motif (Myo1d-IQ2). This construct was further modified in that the two C-terminal amino acids (LV) of the con- verter domain not present in the Myo1d-head con- struct were deleted (Myo1dDLV-IQ2, Fig. 1). Because these two amino acids are part of a a-helix continued by the IQ-motifs, the IQ-motif is rotated counterclock- wise by 200° and moved  3A ˚ closer towards the head domain. Following expression of these Myo1d con- structs and affinity purification in the absence of free Ca 2+ , we assessed the stoichiometry of CaM bound to the two IQ-motifs. The stoichiometries of CaM asso- ciated with Myo1d-head (0 : 1; Fig. 2B), Myo1d-IQ1 (1 : 1; Figs 2A and 3) and Myo1d-IQ1.2 (1.74 : 2), respectively, have been described previously [22]. Adjusting the free Ca 2+ concentration to 0.1 mm did not cause a release of CaM bound to IQ 1 in the Myo1d-IQ1 heavy chain construct (Fig. 3). This dem- onstrates that IQ 1 has a high affinity for CaM both in the absence and presence of free Ca 2+ . In contrast, the binding of CaM to IQ 2 was clearly distinct. The deletion constructs Myo1d-IQ2 and Myo1dDLV-IQ2 could be purified under identical conditions using anti- bodies directed against the C-terminal FLAG epitope A B Fig. 1. Schematic representation of rat Myo1d constructs and IQ-motif sequences. (A) Domain organization of Myo1d: IQ motifs (white) are labeled with numbers indicative of their respective posi- tion in relation to the motor domain (gray) and tail domain (black). All recombinant constructs contain a C-terminal FLAG-epitope (cir- cle). Amino acids of rat Myo1d, linker residues (italics) and the FLAG-epitope sequence (underlined) for each of the five different constructs are indicated. (B) Aligned sequences of the two IQ-motifs in Myo1d and the generalized consensus IQ-motif are shown. Regulation of Myo1d ATPase activity D. Ko ¨ hler et al. 2190 FEBS Journal 272 (2005) 2189–2197 ª 2005 FEBS albeit with a lower protein yield (Figs 2A and 4). Den- sitometric analysis of the CaM content in affinity puri- fied Myo1d-IQ2 and Myo1dDLV-IQ2 preparations revealed a ratio of only 0.24 ± 0.03 CaM per Myo1d- IQ2 heavy chain and 0.39 ± 0.14 CaM per Myo1dDLV-IQ2 heavy chain, respectively (Figs 2A and 4). However, a higher protein yield and a stoichio- metric association of CaM with Myo1d-IQ2 were achieved when this construct was purified in the pres- ence of free Ca 2+ (data not shown), indicating that IQ2 binds CaM more tightly in the presence of Ca 2+ . To test if the IQ2 in Myo1d-IQ2 and Myo1dDLV- IQ2 could be saturated with CaM in the absence or presence of free Ca 2+ , we mixed purified Myo1d-IQ2 or Myo1dDLV-IQ2 with 10 lm exogenous CaM (Figs 4 and 5). Free CaM was separated from CaM bound to Myo1d-IQ2 or Myo1dDLV-IQ2 by cosedimentation of the myosin with F-actin. In the absence of free Ca 2+ Myo1d-IQ2 and Myo1dDLV-IQ2 had stoichiometric amounts of CaM bound (molar ratio of 0.92 ± 0.12 CaM ⁄ Myo1d-IQ2 heavy chain and of 1.1 ± 0.1 CaM ⁄ Myo1dDLV-IQ2 heavy chain) (Figs 4 and 5). The 1 : 1 ratio of CaM binding to Myo1d-IQ2 and Myo1dDLV-IQ2 did not change upon the addition of 100 lm free Ca 2+ to the assay mixture (Figs 4 and 5). Therefore, we supplemented purified Myo1d-IQ2 and Myo1dDLV-IQ2 preparations for all further experiments with 10 lm CaM to guarantee the stoichio- metric binding of CaM. Regulation of the actin-activated ATPase of recombinant Myo1d proteins by Ca 2+ ⁄ CaM In the absence of free Ca 2+ , the basal ATPase activity of the Myo1d-head without an IQ-motif was 0.01 s )1 . The V max of the actin-activated ATPase was 2.6 s )1 and the K actin 38 lm [22] with an apparent second-order rate AB Fig. 2. Different amounts of calmodulin are copurified with Myo1d- IQ1, Myo1dDLV-IQ2 and Myo1d-head. Affinity-purified Myo1d con- structs were analyzed on Coomassie blue stained 7.5–15% SDS ⁄ polyacrylamide gradient gels for their content of copurified calmodulin. (A) Myo1dDLV-IQ2 (lane 1), Myo1d-IQ1 (lane 2) and cal- modulin (lane 3); (B) Myo1d-head (lane 1) and calmodulin (lane 2). Molecular masses are indicated to the left. The arrowheads mark the position of the Myo1d heavy chains and the asterisks highlight the copurified light chain calmodulin. Fig. 3. CaM binds stoichiometrically to Myo1d-IQ1 irrespective of the free Ca 2+ -concentration. Myo1d-IQ1 purified in the presence of EGTA was either left in EGTA (EGTA, lanes 3 and 4) or buffer con- ditions were adjusted to 100 lM free Ca 2+ (Ca 2+ , lanes 1 and 2) fol- lowed by the addition of F-actin. Samples were centrifuged and separated into supernatants (S) and pellets (P). Myo1d-IQ1 and associated calmodulin was cosedimented with F-actin to monitor a potential release of calmodulin. Proteins were separated on a 7.5% - 15% SDS-polyacrylamide gradient gel and stained with Coomassie blue. Electrophoresis of CaM (lane 5) served as a marker and is indicated by an asterisk. The arrowhead indicates Myo1d-IQ1. Fig. 4. Stoichiometric binding of exogenous calmodulin to purified Myo1d-IQ2 in the absence and presence of free Ca 2+ . SDS ⁄ PAGE (7.5–15%) analysis revealed that affinity purified Myo1d-IQ2 (lane 1) does not contain stoichiometric amounts of CaM. Purified Myo1d-IQ2 (0.5 l M) was incubated with 10 lM CaM either in the presence of 100 l M free Ca 2+ (Ca 2+ , lanes 2 and 3) or in the absence of free Ca 2+ (EGTA, lanes 4 and 5). To determine the amount of calmodulin bound to Myo1d-IQ2, F-actin was added to the samples and they were centrifuged. Supernatants (S) and pellets (P) were analyzed by SDS ⁄ PAGE. As a control, calmodulin was centrifuged with F-actin alone (EGTA, lanes 6 and 7). Purified CaM served as a marker (lane 8) and is indicated by an asterisk. The position of Myo1d-IQ2 heavy chain is indicated by an arrow- head. The molar ratio of CaM to Myo1d-IQ2 determined by densi- tometry in the actin pellets was 0.92 ± 0.12 in EGTA and 0.87 ± 0.25 in 100 l M Ca 2+ , respectively. D. Ko ¨ hler et al. Regulation of Myo1d ATPase activity FEBS Journal 272 (2005) 2189–2197 ª 2005 FEBS 2191 constant K app of 0.68 · 10 5 s )1 Æm )1 (Fig. 6). These val- ues were virtually identical for the Myo1d-IQ1 and Myo1d-IQ1.2 constructs that contained in addition either IQ1 or IQ1 and IQ2. The two IQ-motifs were even exchangeable, as the Myo1d-IQ2 fusion protein also exhibited identical basal and actin-activated ATPase activities (Fig. 6A,B). However, deletion of the two C-terminal amino acids of the converter domain in the construct Myo1dDLV-IQ2 lead to a pronounced inhibition of the actin-activated ATPase activity while the basal ATPase activity was unaltered (Fig. 6B). The actin-activated ATPase activity increased almost linearly in the range of the actin concentrations tested. It exhibited a K app of 0.12 · 10 5 s )1 Æm )1 that was about six-fold reduced in comparison with Myo1d-IQ2, indicating a change in coupling between the actin and nucleotide binding sites. To investigate whether the Myo1d ATPase is regula- ted by Ca 2+ ⁄ CaM, we determined the actin-activated ATPase of Myo1d constructs in the presence of 22 lm- free Ca 2+ . Interestingly, the V max of the actin-activated ATPase activity of the Myo1d-head was reduced by Fig. 5. Stoichiometric binding of exogenous CaM to purified Myo1dDLV-IQ2 in the absence and presence of free Ca 2+ . Purified Myo1dDLV-IQ2 (0.5 l M) was incubated with 10 lM CaM either in the absence of free Ca 2+ (EGTA, lanes 3 and 4) or in the presence of 100 l M free Ca 2+ (Ca 2+ , lanes 5 and 6). To determine the amount of calmodulin bound to Myo1dDLV-IQ2, F-actin was added to the samples and they were centrifuged. Supernatants (S) and pellets (P) were analyzed by SDS ⁄ PAGE (7.5–15%). As a control, calmodulin was centrifuged with F-actin alone (EGTA, lanes 1 and 2). Purified CaM served as a marker (lane 7) and is indicated by an asterisk. The position of Myo1dDLV-IQ2 heavy chain is indicated by an arrowhead. Fig. 6. The actin-activated Mg 2 ATPase activity of different Myo1d- constructs is inhibited by Ca 2+ . Actin-activated ATPase activity was determined at 37 °C in a buffer containing 30 m M KCl, 10 mM Hepes pH 7.4, 2 mM MgCl 2 ,3mM EGTA, 1 mM 2-mercaptoethanol, 2m M NaN 3 and 2 mM ATP. CaCl 2 (3 mM) was added where indica- ted to obtain a free Ca 2+ concentration of 22 lM. Samples con- tained 54–270 n M of the respective Myo1d constructs, 0–67 lM F-actin and in the case of IQ2 containing constructs exogenous cal- modulin. (A) Actin dependence of Myo1d-head ATPase activity (s) in EGTA (solid line) and 22 l M free Ca 2+ (dashed line) conditions, respectively. Also shown is the actin dependence of Myo1d-IQ1.2 ATPase activity (n) in EGTA conditions. (B) and (C) Actin depend- ence of Myo1d-IQ1 (d), Myo1d-IQ2 (m) and Myo1dDLV-IQ2 ( ) ATPase activities in EGTA conditions (B) and 22 l M free Ca 2+ conditions (C). Data points were fitted according to Eqn (1) in the Experimental procedures. Regulation of Myo1d ATPase activity D. Ko ¨ hler et al. 2192 FEBS Journal 272 (2005) 2189–2197 ª 2005 FEBS roughly 20% whereas the K actin was unaltered (Fig. 6A). This Ca 2+ -dependent inhibition was not reversible when free Ca 2+ was chelated with EGTA (data not shown). In the Myo1d-IQ1 construct, free Ca 2+ inhibited the actin-activated ATPase activity by  75% (Fig. 6C). The calculated K app was 0.12 · 10 5 s )1 Æm )1 . As the two Myo1d IQ-motifs have different CaM-binding properties, we analyzed if IQ1 and IQ2 regulate the Myo1d ATPase activity differ- ently. However, in the construct Myo1d-IQ2 that has IQ1 exchanged for IQ2, free Ca 2+ caused a compar- able inhibition of the ATPase with a V max of 0.7 s )1 (Figs 6C and 7). The actin affinity was not affected significantly with a determined K actin ¼ 44 lm. The K app derived from the initial slope of the hyperbola was 0.15 · 10 5 s )1 Æm )1 . In the construct Myo1dDLV-IQ2 that exhibited already a reduced ATPase activity in the absence of free Ca 2+ , the addition of free Ca 2+ reduced its ATP- ase activity further by about 40% and a K app of 0.07 · 10 5 s )1 Æm )1 was determined. Inhibition of the Myo1d actin-activated ATPase activity as a function of the concentration of free Ca 2+ Next, we determined the ATPase activities for the dif- ferent Myo1d constructs as a function of the free Ca 2+ concentration (0.001–158 lm) (Fig. 7). The slight reduction of the actin-activated ATPase activity of the Myo1d-head exhibited an IC 50 of pCa  6 (0.3 lm free Ca 2+ ). All of the Myo1d constructs that contained either one or two IQ motifs, specifically Myo1d-IQ1, Myo1d-IQ1.2 and Myo1d-IQ2, were inhibited with an IC 50 of pCa 7 (0.05–0.08 lm Ca 2+ ) (Fig. 7). This IC 50 value corresponds to the affinity of the pair of Ca 2+ -binding sites in the C-terminal lobe of CaM. The Myo1dDLV-IQ2 protein that exhibited already a reduced ATPase activity in the absence of free Ca 2+ did not show any significant changes in ATPase activ- ity with increasing free Ca 2+ concentrations. Discussion To obtain a complete understanding of the physiologi- cal functions of a given myosin, it is necessary to understand the mechanisms that regulate its motor activities. Here we investigated the regulation of the Myo1d ATPase activity by its light chain binding domain and the associated light chain CaM. The light chain binding domain of Myo1d consists of two IQ-motifs that were found to bind CaM with different affinities and calcium-sensitivity. Binding of Ca 2+ to the CaM bound to the first IQ-motif inhibited the actin-activated ATPase activity by  75%. When the first IQ-motif was deleted, binding of Ca 2+ to the CaM bound to the second IQ-motif inhibited the actin-activated ATPase activity by the same extent. In both cases, the inhibition of the ATPase activity was induced by virtually identical free Ca 2+ concentra- tions. Deletion of two amino acids N-terminal to the IQ-motif did not affect CaM-binding, but inhibited the ATPase activity in a Ca 2+ -independent manner to a similar extent as observed with Ca 2+ for the other constructs containing either one or two IQ-motifs. Ca 2+ -independent and Ca 2+ -dependent binding of CaM to IQ-motifs The two IQ motifs in Myo1d are supposed to belong to different classes of IQ motifs [8,23]. Whereas IQ1 in Myo1d conforms well to the consensus sequence of IQ-motifs, IQ2 is less well conserved and possesses hydrophobic residues at positions 1, 5, 8 and 14 as is typical for Ca 2+ -dependent CaM-binding motifs [1]. Indeed, we found that IQ1 has a higher affinity for ApoCaM than IQ2. Based on structural studies of ELC binding to IQ1 of a myosin II [24,25] and Mlcp1 binding to the IQ-motifs in Myo2p [23], we presume that the C-terminal lobe of ApoCaM binds to the N-terminal parts of IQ1 (LQKVWR) and IQ2 Fig. 7. Inhibition of the actin-dependent ATPase activities of differ- ent Myo1d-constructs as a function of free Ca 2+ concentrations. Calcium dependence of ATPase activity of Myo1d-head (s), Myo1d-IQ1.2 (n), Myo1d-IQ1 (d), Myo1d-IQ2 (m) and Myo1dDLV- IQ2 ( ). ATPase activities were measured at 37 °C in a solution containing 24 l M F-actin, 2 mM ATP, 30 mM KCl, 10 mM Hepes pH 7.4, 2 m M MgCl 2 ,3mM EGTA, 1 mM 2-mercaptoethanol and 2m M NaN 3 . To adjust free Ca 2+ concentrations (0–158 lM), corres- ponding amounts of CaCl 2 were added. D. Ko ¨ hler et al. Regulation of Myo1d ATPase activity FEBS Journal 272 (2005) 2189–2197 ª 2005 FEBS 2193 (IIRYYR), respectively, in a semi-open conformation. We expect that the N-terminal lobe of ApoCaM binds in a closed conformation to the C-terminal part of IQ1 (GTLAR). The reduced affinity of IQ2 for ApoCaM as compared to IQ1 is probably due to a lack of inter- action between the N-terminal lobe of CaM and the C-terminal part of IQ2 (RYKVK). The exchange of Gly at position 7 for an Arg with a bulky side chain in IQ2 is likely to interfere sterically with the binding of the N-lobe of ApoCaM as has been demonstrated for Mlc1p binding to IQ4 of Myo2p [23]. ApoCaM has also been reported to bind weakly to the single IQ motif present in Myo VI [26]. This IQ motif deviates from the consensus sequence (IQXXXRGXXXR ⁄ K) in that the glycine consensus residue at position 7 is changed to a methionine. Charge repulsion due to a stretch of positively charged amino acids in the C-ter- minal part of IQ2 (Arg733, Lys735, Lys737) may addi- tionally repel the N-lobe farther away from IQ2. The N-lobe of CaM might thus be free to interact with sequences in the Myo1d tail or with other proteins. The affinity of IQ2 for ApoCaM was not affected when the two C-terminal amino acids (LV) of the con- verter domain a-helix were deleted. This deletion is predicted to introduce a counterclockwise rotation by 200° and a shift by  3A ˚ towards the head domain of the CaM bound to the IQ-motif. We conclude that no steric hindrance for CaM binding was introduced by this deletion. We have shown previously that 1.74 CaM molecules were associated with affinity purified Myo1d-IQ1.2. This stoichiometry of bound CaM is higher than the sum of CaM molecules bound to Myo1d-IQ1 (1.1) and Myo1d-IQ2 (0.24). This result indicates that CaM binds in a cooperative manner to the light chain bind- ing domain of Myo1d. The binding of CaM to IQ1 may induce a stabilization of the IQ2 a-helical struc- ture and thereby facilitate binding of the C-terminal lobe of ApoCaM to the N-terminal half of IQ2. We found that purified Myo1d-IQ2 and Myo1dDLV- IQ2 could be fully saturated by the addition of exogen- ous ApoCaM or Ca 2+ ⁄ CaM. This finding allowed us to investigate the effects of Ca 2+ -binding to ApoCaM associated with the IQ2. Myo1d-IQ2 affinity purified under Ca 2+ conditions contained stoichiomet- ric amounts of copurified CaM demonstrating that IQ2 actually has a higher affinity for Ca 2+ ⁄ CaM than for ApoCaM. This result is in accordance with previous observations in a gel overlay assay [8] and the above mentioned sequence similarity of IQ2 with Ca 2+ - dependent CaM-binding motifs. The two lobes of Ca 2+ ⁄ CaM may both bind in an open conformation to IQ2, explaining the increased affinity. Mechanism of inhibition of the Myo1d ATPase activity by Ca 2+ ⁄ CaM As reported previously, in the absence of free Ca 2+ Myo1d-head, Myo1d-IQ1 and Myo1d-IQ1.2 exhibited very similar basal and actin-activated ATPase activities. In the presence of actin, the ATPase activities reached V max values of 2.6–3.1 s )1 [22]. The addition of one or two IQ motifs to the head domain did not affect ATPase rates and actin affinities. In the present study, we show that IQ1 can even be replaced by IQ2 without that the ATPase rates and actin affinities get significantly altered. However, binding of Ca 2+ to CaM associated with the IQ motif directly following the head (converter) domain induced a significant inhibition of the Myo1d actin-activated ATPase activity. This inhibition was independent of whether one or two IQ motifs were pre- sent or IQ1 or IQ2 were directly fused to the head region. The free Ca 2+ concentrations that were neces- sary to induce the inhibition of the actin-activated ATPase activity correlated well with the reported affin- ity of the C-terminal lobe of CaM for Ca 2+ . The N-ter- minal lobe of CaM exhibits a 10-fold lower affinity for Ca 2+ [19]. These results support the notion that the C-terminal lobe of CaM is bound to both IQ1 and IQ2 in a semi-open conformation and switches upon Ca 2+ - binding to an open conformation. The open conforma- tion inhibits the actin-activated ATPase activity of Myo1d. The C-terminal lobe of CaM bound in an open conformation to the IQ motif directly following the head region might inhibit the actin-activated ATPase activity by specific interactions with the head region. The ELC bound to IQ1 of smooth muscle myosin II has been observed to contact in the prepower stroke state a loop in the head domain that modulates nucleotide affinity [27]. Therefore, a change in nucleotide affinity might be the reason for the reduced ATPase activity. Changes in nucleotide affinity have also been reported for Dictyostelium discoideum myosin II head constructs with varying lengths of the C-terminal a-helix of the converter domain that is continuous with the IQ-motifs [28]. Therefore, Ca 2+ ⁄ CaM may modulate the actin- activated ATPase activity by affecting the stability or flexibility of the a-helix N-terminal to the IQ-motif(s). Fusion of the IQ2 directly to the Myo1d head con- struct led to the deletion of the two amino acids immedi- ately N-terminal to the IQ-motif. This deletion caused a similar but Ca 2+ -independent inhibition of the Myo1d actin-activated ATPase activity. This deletion is predic- ted to rotate the CaM associated with the IQ-motif by 200° on the a-helix emanating from the converter domain and to shorten this a-helix by roughly 3 A ˚ . The fact that this construct demonstrates a similar reduction Regulation of Myo1d ATPase activity D. Ko ¨ hler et al. 2194 FEBS Journal 272 (2005) 2189–2197 ª 2005 FEBS in ATPase activity might either be explained by coinci- dence or by the assumption that it mimics the Ca 2+ - dependent changes induced in the other IQ-motif constructs. However, the latter possibility seems only feasible when the inhibitory mechanism does not involve a stereospecific interaction of CaM with the head region. A common mode of regulation may include effects on conformation and ⁄ or flexibility of the light chain bind- ing domain and N-terminal a-helix that are transduced to the head region. Such effects may become more pro- nounced when the head is bearing strain. As reported here for Myo1d, the first of several IQ- motifs was demonstrated to control the Ca 2+ -sensitiv- ity of the kinetics of Myo1b (MI 130 ; myr 1) [29]. Myo1e (myr 3; Myosin IC) has only a single IQ-motif and Ca 2+ -binding to the CaM associated with this IQ- motif negatively regulates the basal ATPase activity [21]. Therefore, the first IQ-motif in class I myosins might serve generally as a Ca 2+ -regulatory element. On the other hand, conversion of the CaM C-lobe upon Ca 2+ -binding from a semi-open to open IQ- motif binding configuration is unlikely to provide a general inhibitory mechanism for class I myosin ATPase activities. Although binding of Ca 2+ to the C-lobe of CaM has been reported to inhibit actin gli- ding powered by Myo1c (myosin Ib, myr 2), Myo1c ATPase activity was not affected at this Ca 2+ concen- tration and was enhanced at 10-fold higher Ca 2+ con- centrations simultaneously with the dissociation of one CaM [19]. The actin-activated ATPase activities of Myo1a (brush border myosin I) and Myo1b (MI 130 ; myr 1) were actually increased in the presence of Ca 2+ ⁄ CaM [20,30]. In Myo1e the basal ATPase activ- ity was reduced with a Ca 2+ -sensitivity suggestive of a contribution by both the C- and N-terminal lobes of CaM [21]. In conclusion, the regulatory functions of Ca 2+ ⁄ CaM in different class I myosin molecules appears to be quite diverse and no common mecha- nisms have emerged yet. The molecular basis for these differences remains to be elucidated. The detailed char- acterization of Myo1d regulation by Ca 2+ ⁄ CaM provi- ded here represents a necessary step towards this goal. Experimental procedures Plasmid construction Construction of the expression plasmids Myo1d-head- FLAG, Myo1d-IQ1-FLAG and Myo1d-IQ1.2-FLAG which encode amino acids 1–697, 1–721 and 1–743 of rat Myo1d, respectively, has been described previously [22]. Plasmids Myo1d-IQ2-FLAG and Myo1dDLV-IQ2-FLAG have the first of the two IQ-motifs deleted, so that the second IQ-motif is fused to the head domain directly. Myo1dDLV-IQ2-FLAG is further missing the last two codons for amino acids 698–699 (LV) of the head. For the generation of these two deletion constructs, a two step PCR strategy was employed. At first, two overlapping frag- ments were amplified by PCR. The sequences flanking the deleted region were fused in the reverse primer used for amplification of the 5¢-fragment. To construct Myo1d-IQ2- FLAG, the two overlapping fragments were amplified with the two primer pairs 5¢-GGCAAACTTGATGATGAGCG CTGC-3¢ (forward 1) ⁄ 5¢-CAGAGCTGCCTTGACGA- GCATCTG-3¢ (reverse 1) and 5¢-CAGATGCTCGTCAA GGCAGCTCTG-3¢ (forward 2) ⁄ 5¢-ATTCCAGCACACT GGTCACTT-3¢ (reverse 2). To obtain Myo1dDLV-IQ2- FLAG, the two primer pairs forward 1 ⁄ 5¢-CAGAGC TGCCTTCATCTGGGCGCG-3¢ (reverse 1¢) and 5 ¢-ATT CCAGCACACTGGTCACTT-3¢ (forward 2¢) ⁄ reverse 2 were used, respectively. After annealing and extension of the two overlapping fragments, the resultant fragment served as the template in a final PCR using the primer pair forward 1 ⁄ reverse 2. The resulting products were cloned into pIRES Myo1d-head-FLAG using the unique BstXI and Eco47III restriction sites. PCR derived fragments were verified by sequencing. Cell culture HtTA-1 HeLa cells [31] were cultured at 37 °C and 5% (v ⁄ v) CO 2 in Dulbecco’s modified Eagle’s medium supple- mented with 10% (v ⁄ v) fetal bovine serum, 100 UÆmL )1 penicillin and 100 lgÆmL )1 streptomycin. Cells were trans- fected by the addition of plasmid DNA precipitated with calcium phosphate. Single colonies were isolated after selection in 200 lgÆmL )1 hygromycin for 2 weeks. Cells were expanded and analyzed for expression of Myo1d constructs by immunoblotting with the rat Myo1d anti- body SA 522. Selection by hygromycin was maintained continuously. Protein expression and purification Recombinant Myo1d proteins were purified as described in detail previously [22]. Briefly, cells were grown in 20–30 cul- ture dishes (diameter 150 mm) to 80% confluence, washed with NaCl ⁄ P i , collected by scraping and permeabilized with lysis buffer [150 mm NaCl, 20 mm Hepes pH 7.4, 2 mm MgCl 2 ,1mm EGTA, 0.5% (v ⁄ v) Triton X-100, 2 mm ATP, 0.1 mgÆmL )1 Pefabloc, 0.01 mgÆmL )1 leupeptin, 0.02 UÆmL )1 aprotinin] for 1 h on ice. After clearing the lysate by two subsequent centrifugation steps, the super- natant was mixed with 1 mL FLAG-antibody agarose (Sigma-Aldrich) and incubated for 2 h in the cold. The beads were washed twice with buffer WP (50 mm KCl, 10 mm Hepes pH 7.4, 2 mm MgCl 2 ,1mm EGTA, 1 mm D. Ko ¨ hler et al. Regulation of Myo1d ATPase activity FEBS Journal 272 (2005) 2189–2197 ª 2005 FEBS 2195 2-mercaptoethanol and 2 mm NaN 3 ) and finally, bound pro- tein was eluted by buffer WP supplemented with 125 lgÆmL )1 soluble FLAG peptide (Sigma-Aldrich). In some cases, Myo1d-IQ2 was purified in the presence of 50–100 lm free Ca 2+ concentrations. Occasionally, 5 lm calmodulin was added during the elution of Myo1d-IQ2 to enhance protein solubility. Eluted proteins were dialyzed against buffer WP and concentrated using microcon filters (cut-off 10 kDa) if necessary. All recombinant Myo1d pro- teins were cleared by ultracentrifugation (150 000 g for 20 min) immediately before use. To saturate all light chain binding sites, cleared Myo1d-IQ2 and Myo1dDLV-IQ2 preparations were preincubated with 10 lm calmodulin for 20 min on ice. Densitometric analysis of Coomassie-stained protein bands on SDS gels was performed with an ultrascan laser densitometer. Values represent the mean of at least three different preparations. Actin was purified from rabbit skeletal muscle as described by Pardee and Spudich [32]. Purified calmodulin was purchased from Sigma-Aldrich. ATPase assays Steady-state ATPase activities were determined at 37 °Cas described in detail previously [21]. All assay mixtures con- tained 30 mm KCl, 10 mm Hepes pH 7.4, 2 mm MgCl 2 , 2mm ATP, 3 mm EGTA, 1 mm 2-mercaptoethanol, 2 mm NaN 3 and various actin concentrations. To measure Ca 2+ - dependent activities, a constant actin concentration of 24 lm was used. Free Ca 2+ concentrations between 0.001 and 158 lm were adjusted by adding the appropriate amounts of CaCl 2 to obtain the desired value in the pres- ence of 3 mm EGTA. Actin-dependent ATPase activities were measured in the absence or presence of 22 lm free Ca 2+ in a concentration range between 0 and 75 lm actin. Purified Myo1d constructs were used in a range between 54 and 270 nm. V max and K m values were determined by fitting the measured ATPase rates (v) to the Michaelis–Menten equation v ¼ V max ½actin=ðK actin þ½actinÞ ð1Þ with the program kaleidograph. Sedimentation assays To separate myosin-associated CaM from soluble CaM, myosin was incubated with 2 lm F-actin in a buffer con- taining 30 mm KCl, 10 mm Hepes pH 7.4, 2 mm MgCl 2 , 3mm EGTA, 1 mm 2-mercaptoethanol and 2 mm NaN 3 for 15 min on ice. Where indicated, the free Ca 2+ concen- trations were adjusted accordingly. Assay mixtures with Myo1d-IQ2 purified under calcium conditions contained 50–100 lm free Ca 2+ . Free Ca 2+ was chelated in these preparations by the addition of appropriate amounts of EGTA. After high speed centrifugation at 150 000 g for 20 min, supernatants with soluble CaM were separated from pellets containing acto-myosin complexes with tightly bound CaM and analyzed by SDS ⁄ PAGE and densito- metry. Acknowledgements We thank Margrit Mu ¨ ller and Edith Bru ¨ ne for techni- cal assistance. We acknowledge the financial support of the DFG (Ba 1354 ⁄ 6–1). References 1Ba ¨ hler M & Rhoads A (2002) Calmodulin signaling via the IQ motif. 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J Biol Chem 275, 21624–21630. 30 Collins K, Sellers JR & Matsudaira P (1990) Calmodu- lin dissociation regulates brush border myosin I (110- kD-calmodulin) mechanochemical activity in vitro . J Cell Biol 110, 1137–1147. 31 Gossen M & Bujard H (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89, 5547–5551. 32 Pardee JD & Spudich JA (1982) Purification of muscle actin. Methods Cell Biol 24, 271–289. D. Ko ¨ hler et al. Regulation of Myo1d ATPase activity FEBS Journal 272 (2005) 2189–2197 ª 2005 FEBS 2197 . The two IQ-motifs and Ca 2+ /calmodulin regulate the rat myosin 1d ATPase activity Danny Ko ¨ hler*, Sandra Struchholz* and Martin Ba ¨ hler Institute for General Zoology and Genetics,. binding domain of rat myosin 1d consists of two IQ-motifs, both of which bind the light chain calmodulin (CaM). To analyze the Myo1d ATPase activity as a function of the IQ-motifs and Ca 2+ ⁄ CaM binding,. Myo1d-IQ2-FLAG and Myo1dDLV-IQ2-FLAG have the first of the two IQ-motifs deleted, so that the second IQ-motif is fused to the head domain directly. Myo1dDLV-IQ2-FLAG is further missing the last two codons