Calcium can mobilize and activate myosin-VIChristopher Battersa,b, Dario Bracka,b, Heike Ellricha,b, Beate Averbecka, and Claudia Veigela,b,1

aDepartment of Cellular Physiology, Ludwig-Maximilians-Universität München, 80336 Munich, Germany; and bCenter for Nanosciences München, 80799Munich, Germany

Edited by Edward D. Korn, National Heart, Lung and Blood Institute, Bethesda, MD, and approved December 22, 2015 (received for review October 1, 2015)

The ability to coordinate the timing of motor protein activation liesat the center of a wide range of cellular motile processes includingendocytosis, cell division, and cancer cell migration. We show thatcalcium dramatically alters the conformation and activity of themyosin-VI motor implicated in pivotal steps of these processes.We resolved the change in motor conformation and in structuralflexibility using single particle analysis of electron microscopicdata and identified interacting domains using fluorescence spec-troscopy. We discovered that calcium binding to calmodulinincreases the binding affinity by a factor of 2,500 for a bipartitebinding site on myosin-VI. The ability of calcium-calmodulin toseek out and bridge between binding site components directs amajor rearrangement of the motor from a compact dormant stateinto a cargo binding primed state that is nonmotile. The lack ofmotility at high calcium is due to calmodulin switching to a higheraffinity binding site, which leaves the original IQ-motif exposed,thereby destabilizing the lever arm. The return to low calcium caneither restabilize the lever arm, required for translocating the cargo-bound motors toward the center of the cell, or refold the cargo-freemotors into an inactive state ready for the next cellular calcium flux.

unconventional myosin | electron microscopy | calmodulin

In human cells, cytoskeletal motor proteins move along mi-crotubules and actin filaments to generate complex cellular

functions that require a precise timing of motor activation andinactivation. Myosin-VI is thought to have unique propertiesbecause it is the only myosin in the human genome shown tomove toward the minus end of actin filaments (1). Apart from itsroles in the formation of stereocilia in cells of the auditory sys-tem (2, 3), membrane internalization (4–6), and delivery ofmembrane to the leading edge in migratory cells (7), myosin-VIis an early marker of cancer development, aggressiveness, andcancer–cell invasion because of its dramatically up-regulatedexpression in breast, lung, prostate, ovary, and gastresophaguscarcinoma cells (7–11). How this motor might promote cancer–cell migration, proliferation, and survival is unknown.In migrating cells, localized calcium transients (∼50 nM to

∼10 μM) (12, 13) have been reported to play a multifunctional role insteering directional movement (14), cytoskeleton redistribution,and relocation of focal adhesions (15). The effect of calciumtransients on the mobilization and cargo binding of myosin-VIand on its mechanical activation, however, are not understood.In the current model, the catalytic head domain hydrolyzes ATP,whereas the tail domain anchors the motor to specific com-partments. In vitro studies have shown that calcium affects my-osin-VI binding to phospholipids (6), as well as the kinetics andmotility rate of the motor (16, 17). The underlying molecularmechanisms, however, are unknown. It has also been discussedthat myosin-VI might be able to adopt an inactive folded state(18, 19), perhaps similar to nonmuscle myosin II and myosin-V(20–22), with folding and unfolding regulated by some unknownmechanism. When activated, the myosin-VI head domain bindsto actin, generating conformational changes that are transducedby the converter to the lever arm or neck domain and amplifiedto nanometer displacements. The neck consists of an extendedα-helix stabilized by the binding of calmodulin (23), which pointedto the intriguing possibility that the calcium sensor calmodulinbound to the myosin-VI neck domain might constitute a molecular

mechanism to control both the cellular mobilization and activationof myosin-VI in migrating cells. We therefore set out to investigatethe effect of calcium on the structural conformation, mechanicalproperties, and activation of single myosin-VI motor moleculesusing electron microscopy (EM), spectroscopic, and mechanicalexperiments.

ResultsCalcium Binding to Calmodulin Induces a Structural Rearrangement ofMyosin-VI. We characterized the effect of calcium on the struc-ture and function of full-length myosin-VI using single particleanalysis of negatively stained EM images (24, 25). We chosecalcium concentrations close to the physiological range, whichensured homogeneous populations of molecules (SI Text). Thenucleotide-free motor molecules adhered to the carbon-coatedgrids in two main orientations (Fig. 1 and Fig. S1), providing afront view with the neck along the long axis of the head domain(=straight conformation) and a side view with the head and neck atan angle of ∼53° (=bent conformation). The images in frontview did not fit to any crystal structures and were thereforenot analyzed in further detail (Fig. S1 and SI Text). The images inside view revealed detailed information on the conformation ofthe calmodulins and the tail domain. To interpret the EM data,we modeled a structure that combined the crystal structures ofthe myosin-VI head domain (23) and the neck region comprisingtwo calmodulins and the subsequent three-helix bundle of thetail domain (26). At low calcium, the modeled structure in theoptimized spatial orientation could account for the contour ofthe EM image (Fig. 1B, class average of n = 2,998, and Figs. S1and S2). Zooming into the neck region (Fig. 1C) showed anexcellent agreement between the crystal structure of both

Significance

The timing of motor protein activation is central to a broadrange of cellular motile processes including endocytosis, celldivision, and cancer cell migration. The cytoskeletal motormyosin-VI is involved in these processes and is the only myosinin the human genome shown to move toward the minus end ofactin filaments. Using electron microscopy, fluorescence spec-troscopy, and motility assays, we demonstrate that calcium isthe cellular switch that directs the rearrangement of the motorfrom a dormant, inactive state at low calcium to a cargo-bindingnonmotile state at high calcium. The return to low calciumgenerates either cargo-bound active motors that translocateto the center of the cell or refolded inactive motors ready forthe next cellular calcium flux.

Author contributions: C.B., B.A., and C.V. designed research; C.B., D.B., and H.E. per-formed research; C.B., D.B., H.E., and C.V. analyzed data; and C.B., B.A., and C.V. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.

See Commentary on page 2325.1To whom correspondence should be addressed. Email: claudia.veigel@med.uni-muenchen.de.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1519435113/-/DCSupplemental.

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calmodulins with a cross-correlation coefficient (CCC) of 0.99and 0.96, respectively, plus the helix bundle (represented here bythe first helix in dark green; CCC, 0.89) and the EM outline(solid white line). At high calcium (Fig. 1 D–F), in a completereversal to the low calcium condition, most molecules adhered tothe grid in a side view, indicating a major change in surfacecharge and structure (Fig. S3). The crystal structure of the sec-ond calmodulin (CCC, 0.76) and helix bundle (CCC, 0.68; Fig. 1E and F) were no longer in agreement with the EM contour(dashed white line, n = 2,392). The data indicated that the sec-ond calmodulin had rotated clockwise by ∼30° in the projectionplane, whereas the helix bundle had rotated anticlockwise by∼30° (black arrows).

Ca2+-Calmodulin Binds to a Bipartite Binding Site. To investigate theconformational change induced by calcium, we performed ti-tration experiments (Fig. 2, Table 1, and Fig. S4) using apo- andCa2+-calmodulin and calmodulin mutants with either the N- orC-terminal calcium binding site eliminated (16), against 16 dif-ferent peptides containing potential calmodulin binding sites(Table S1) (27, 28). We used the intrinsic tryptophan fluores-cence in these target peptides to detect calmodulin binding (SIText and Fig. S4) (29). We found that apo-calmodulin bound tothe target peptide P2 (Fig. 1B, yellow) with a stoichiometry of 1:1(Kd ∼ 290 nM). For Ca2+-calmodulin, however, the stoichiometrywas 0.5:1, which indicated that the coordination between the

N- and C-terminal lobes was lost in the Ca2+ state (Fig. 2A), i.e.,that only one calmodulin lobe could bind. Peptide P3 had a veryweak affinity for Ca2+-calmodulin and did not bind apo-calmodulinat all (Table 1). Intriguingly, the binding stoichiometry for thedouble peptide P2–3 was 1:1, for both apo- and Ca2+-calmodulin(Fig. 2B), with a high affinity for the Ca2+ state (Kd ∼ 83 nM). Thisresult revealed that the double peptide must contain a bipartitebinding site available only for Ca2+-calmodulin (Fig. 2E). Usingpeptides omitting the N-terminal half of P2, the C-terminal half ofP3, and C-terminal mutations in the peptide P2-3‡, we localizedthe two nonadjacent halves of this composite holo-calmodulinbinding site (contained in the peptide P½ 2–3, Kd ∼ 38 nM; Fig. 2D and E). Interestingly, comparison with the structure of the three-helix bundle (26) revealed that the distal half of this binding sitewas located at the extended loop between the first and the secondhelix of the bundle. This structural comparison indicated that, inthe presence of calcium, the partly detached Ca2+-calmodulin canseek out and form a bridge between P2 and the loop in the helixbundle, with a change in affinity for the bipartite binding site by afactor of 2,500 (Fig. 2D and Table 1, for P½ 2–3 Kd ≥ 100 μM atpCa 8 and 38 nM at pCa 4). This binding pattern is consistent withthe second calmodulin rotating clockwise and the helix bundlebeing pulled anticlockwise in the EM images (Fig. 1 E and F). Thecalcium-dependent transitions between the different binding modesof apo- and Ca2+-calmodulin to the peptide P2–3 were fully re-versible with a rearrangement, but no detachment, of calmodulin

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Fig. 1. Calcium-induced conformational change of calmodulin revealed using negative stain EM of myosin-VI. (A) The nucleotide-free molecules adhered tothe carbon-coated grids in a straight conformation (class average of n = 13,583) or a bent conformation (class average of n = 2,998), pCa 8. (B) The optimizedprojection of a modeled structure (catalytic domain blue, converter gray, first calmodulin red, second calmodulin yellow, first helix of the three-helix bundledark green) was overlaid onto the inverted image of the bent conformation shown in B. (C) The EM image was color-coded with high intensities in blue andlow intensities in green and yellow. The contour lines were created using an intensity threshold. (D–F) Class averages at pCa 4 in the straight (n = 1,405) andbent conformations (n = 2,392). Note the differences (marked by arrows) between the EM image at high calcium and the overlaid modeled structure in aprojection optimized for the catalytic domain.

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observed in either condition (Fig. 2C). We did not observe thebinding of a third calmodulin to this region, as described for artificially

zippered myosin-VI dimers (30), where modified tertiary structuresmay expose extra binding sites. We found the peptide P1 binding tocalmodulin to be extremely calcium sensitive at pCa 4 (Kd = 11 nM),with no binding observed at pCa 8 (Fig. S4). Our EM data, however,support the report by Bahloul et al. (31), who showed that whenadditional parts of the converter were included, calmodulin waslocked into position regardless of the calcium concentration. Tolocate any further C-terminal calmodulin binding sites, we studied sixfurther peptides covering the entire myosin-VI tail up to the cargobinding domain (Table S1). No binding was observed for any of thepeptides with either apo- or Ca2+-calmodulin (all Kd ≥ 100 μM).

Apo-Calmodulin Binds the Tail Segment aa1060–1125 and Causes theMotor to Backfold. Closer inspection of the head domain at lowcalcium (Fig. 1 B and C) also revealed some extra mass in theaveraged EM between the converter (gray), the catalytic domain(blue), and the first calmodulin (red), which was not accountedfor by the modeled structure and indicated the presence of abackfolded myosin-VI tail. Intriguingly, at high calcium this extramass was not observed (Fig. 1 E and F), implying that in theseconditions the tail was more flexible. To test the idea of calciumregulating the backfolding of the myosin-VI tail, we performedpull-down and microscale thermophoresis (MST) experimentswith truncated myosin-VI head and tail constructs. At low calcium,the Head 913 (aa1–913) was pulled down together with F-actin(pellet P), whereas the Tail 1125 (a1125–1276) remained in thesupernatant (S), indicating that this part of the tail did not bind tothe head (Fig. 3 A and C). In contrast, the Tail 1060 (aa1060–1276, GST-tagged) bound to the Head 913 (Kd = 8.21 ± 0.51 μM;Fig. 3 B and C). These experiments identified the segmentaa1060–1125 as the part of the myosin-VI tail that bound to the

A

E

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Fig. 2. Ca2+-calmodulin binds to a bipartite site, bridging between sites P2 and P3 and rearranging the conformation of the tail domain. Tryptophanfluorescence (λex = 290 nm, λem = 323 nm) for (A) 2 μM peptide P2, (B) 2 μM P2-3, or (D) 2 μM P½ 2–3 and increasing calmodulin concentrations was measuredat pCa 4 and pCa 8. (C) Tryptophan fluorescence in response to a change in calcium concentration was measured with 2 μM P2-3 and 2 μM calmodulin.(E) Predicted calmodulin binding sites P1–P4, and Helix 1, Helix 2 of the three-helix bundle (26), and single α helical domain (SAH) (44). The apo-calmodulinbinding site identified on P2 and the bipartite Ca2+-calmodulin binding site on P2–P3 are indicated. For P2-3‡, the C-terminal serine and valine were deleted.For HB1‡, the residues on HB1 labeled by * were replaced by alanine or glycine.

Table 1. Calmodulin binding to myosin-VI target peptides

Peptide

pCa 4.0 pCa 8.0

Kd (nM)StoichiometryCaM:peptide Kd (nM)

StoichiometryCaM:peptide

CaM CaM CaM CaM

WT WT Mutants WT WT

P1 11 ± 0.7 1:1 2:1 NA NAP2 170 ± 6 0.5:1 1:1 290 ± 5 1:1P2-3 83 ± 4 1:1 1:1 142 ± 11 1:1P2-3‡ 278 ± 13 0.5:1 NA 143 ± 10 1:1P ½ 2–3 38 ± 17 1:1 2:1 ≥100,000 NAP 2–½ 3 167 ± 19 0.5:1 2:1 165 ± 9 1:1P3 ≥6,000 NA NA NA NAP4 NA NA NA NA NAHB1 NA NA NA ≥100,000 NAHB1‡ NA NA NA ≥500,000 NA

The titrations of target peptide sequences (Fig. 2E) with calmodulin wereperformed at 20 °C. The dissociation constants Kd in Table 1 for the Trp-containing peptides were determined by direct titration, and the data wereanalyzed as previously described (29). NA, Kd was bigger than 500 μM. Thebinding stoichiometry for calmodulin binding to the peptide at pCa 8 was1:1 for all peptides. At pCa4, the binding stoichiometry is specified for eachpeptide. For each experiment, four independent titrations were performed,and the average Kd value is reported with its SD.

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catalytic domain and/or the neck region, enabling the molecule tobackfold. The Western blot-based binding studies with peptidesand calmodulin confirmed that it was in fact calmodulin, and notthe calmodulin target sites P1 or P2, that bound the tail segmentTail 1060 (Kd = 17.79 ± 10.5 nM; Fig. 3 C and D). To investigatewhether calmodulin with the peptide bound, as found in thephysiological complex, would affect the binding of the tail weprobed increasing ratios of the complex formed by calmodulin andpeptide P2 in the presence and absence of calcium. Intriguingly,the experiments showed that the tail segment only bound to thecalmodulin-peptide complex in the apo-calmodulin state and notthe Ca2+ state, supporting the idea that backfolding is regulated bycalcium (Fig. 3E).

Calcium Induces the Release of the Myosin-VI Tail and Increases LipidCargo Binding. We then investigated whether the release of thebackfolded tail, induced by calcium binding to calmodulin, alsoincreased the binding affinity of the myosin-VI tail for lipidcargo. We performed pull-down experiments with myosin-VIand liposomes prepared from mixed bovine brain lipids (Folchfraction 1) in the presence and absence of calcium (Fig. 4A). Atlow calcium, ∼30% of myosin-VI remained in the supernatant.At high calcium, essentially the entire myosin-VI sample waspulled down together with the liposomes, consistent with an in-creased availability of the lipid-binding domains (6) on the myosin-VI tail. The fat blots (Fig. 4B) confirmed that the lipid binding ofthe recombinant full-length myosin-VI used in this study was

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Fig. 3. Backfolding of the tail segment aa1060–1125 onto apo-calmodulin. (A) Cosedimentation of the myosin-VI construct Head 913 (aa1–913) with F-actin inthe presence of the Tail 1125 (aa1125–1276), analyzed using SDS/PAGE. The Head 913 was pulled down with actin into the pellet P, whereas the Tail 1125remained in the supernatant (S). Controls of the proteins alone are labeled in red. (B) In contrast, the Head 913 was pulled down together with the GST-tagged Tail 1060 (aa1060–1276), indicating that the tail segment aa1060–1125 was binding to the Head 913. Controls of the proteins alone labeled in red.(C) MST. Fluorescently labeled proteins indicated in red, and the binding partner in black. Values are mean values ± SD for three separate experiments.Squares: Kd 17.79 ± 10.5 nM; triangles: 8.21 ± 0.51 μM; circles: 554.1 ± 165.6 μM. (D) In the dot far Western blot, the Tail 1125 did not interact with calmodulin,whereas the Tail 1060 did. The Tail 1060 did not interact directly with the calmodulin-binding peptides P1 or P2. The Tail 1125 and Tail 1060 were detectedwhen spotted directly onto the membrane (positive controls), whereas no reaction was detected when the tails were not applied (negative control). Allexperiments in A–D were performed at pCa 8. (E) For the dot far Western blot, the calmodulin-P2 complex was spotted onto the membrane at high and lowcalcium. Binding of the GST-tagged Tail 1060 was probed using an anti-GST antibody. The cartoon illustrates that the tail section aa1060–1125 binds tocalmodulin at low, but not high, calcium.

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calcium dependent and specific. Myosin-VI only bound to themixed brain lipids containing phospholipids as described pre-viously (6) but not to the main constituents of Folch fraction 1, i.e.,phosphatidylcholine (PC) or -ethanolamine (PE). These experi-ments showed that the calcium-induced release of the myosin-VItail can mobilize the motor and target it to bind to cargo, which ledto the question of how calcium binding would affect the mechanicalproperties of the motor.

Ca2+-Calmodulin Destabilizes the Myosin-VI Lever Arm. To investigatethe effect of calcium on the mechanical stiffness of the myosin-VIneck, we reexamined the two calmodulins bound to the extendedα-helix in the EM averages at high and low calcium (Fig. 1). Incontrast to their close proximity at low calcium (Fig. 1 A and C),the two calmodulins in the EM images at high calcium seemedto have lost contact as a consequence of the rotation of the sec-ond calmodulin (Fig. 1 D and F). This loss of contact indicated apossible loss of mechanical stability of the neck domain, whichserves as a mechanical lever arm. To further explore this possibility,we realigned and classified the EM images using a mask covering

only the catalytic domain (Fig. 1B, blue). The outlines of the ex-treme orientations of the neck region (Fig. 4 D and E and H and I)at low and high calcium, respectively, were overlaid in Fig. 4 F andJ. At low calcium, the overlay revealed some flexibility arising froma single pivot within the converter domain (Movie S1), whereas thecalmodulin binding neck domain itself appeared inflexible. At highcalcium, a second pivot point emerged between the two calmodu-lins. This second pivot indicated that the rearrangement due tocalmodulin binding to the bipartite site caused a break in thestructure of the lever arm (Movie S2). To test whether such a leverarm was still mechanically stable and functional, we performedgliding filament assays (32, 33). In these experiments, fluorescentlylabeled actin filaments were imaged while gliding over a lawn ofmyosin-VI motors attached to the surface of the experimentalchamber in the presence of ATP. The maximum intensity projec-tions in Fig. 4 G and K illustrate the motility. The tracks of actinfilaments gliding over myosin-VI in the absence of calcium (Fig.4G) are shown in red, whereas the actin filaments in the firstframe of the movie are shown in yellow. In the absence of cal-cium, the maximum gliding velocity was ∼60–80 nm/s and largely

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Fig. 4. Calcium regulates the binding to lipid cargo and mechanical activity of myosin-VI. (A) Pull-down of full-length myosin-VI (FL-MyoVI) with Folchliposomes at low and high calcium, analyzed using SDS/PAGE. At low calcium, ∼70% of myosin-VI was pulled down, whereas at high calcium, the pull-downwas virtually complete. (B) The protein-lipid-overlay (PLO) showed calmodulin binding to Folch mixed lipids in the presence and absence of calcium, but not tothe pure main Folch constituents, phosphatidylcholine (PC) or -ethanolamine (PE). (C) Actin filament gliding observed with myosin-VI immobilized via aglobular tail antibody (green), via a biotin-streptavidin linker (blue), and when attached to a surface coated with Folch liposomes (black). (D) To study thestructural flexibility of myosin-VI, EM images at high and low calcium were aligned and classified using a mask covering the catalytic domain and groupedaccording to the orientations of the neck and tail domains. Extreme orientations of the neck are shown in D and E and H and I for low and high calcium,respectively. The outlines of the classes are overlaid in F and J, catalytic domain blue, converter gray, first calmodulin red, second calmodulin yellow. Themechanical pivot points at low and high calcium are indicated by a black dot. Note, that the rotations also include movement in and out of the projectionplane. (G) Maximum intensity projection showing the tracks of actin filaments gliding over myosin-VI in red, with the first frame shown in yellow, pCa 8.Myosin-VI was immobilized on the surface via a biotin-streptavidin linker. (K) As in G, but at pCa 4; the filaments remained attached to the myosin-coatedsurface but did not move.

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independent of the mode of motor attachment to the surface of theexperimental chamber. We tested three conditions of myosin at-tachment (Fig. 4C), namely an antibody against the myosin-VIglobular tail domain (green), a C-terminal biotin-streptavidin-linker(blue), and Folch fraction 1 liposomes deposited onto the surface(black). When calcium was added, the motility stopped completely,consistent with a mechanically unstable lever arm. This calcium-regulated motility is illustrated in the experiment in Fig. 4K, wheremyosin-VI attached to streptavidin via a C-terminal biotin at highcalcium was able to bind actin filaments. As long as calcium washigh, the motor remained target bound but mechanically inactive.The position of the actin filaments in the first (yellow) and last (red)frame of the 600-s movie overlapped. Only when calcium waslowered did the lever arm gain the mechanical rigidity required toallow the generation of force and movement as seen in Fig. 4G.The structural flexibility of the calmodulin-binding lever arm athigh calcium in the EM images (Fig. 4J) indicated that it was notthe detachment, but in fact a conformational change of the secondcalmodulin, including a loss of contact between the calmodulins,that caused the loss in rigidity of the lever arm structure and thusthe loss of mechanical functionality at high calcium.

DiscussionA central feature of cellular motile processes lies in the abilityto coordinate the timing of motor protein activation and in-activation. The molecular mechanisms of chemo-mechanicalenergy transduction of several cytoskeletal motors have beeninvestigated at the single molecule level (34). However, themechanisms and intracellular signals coordinating motor proteinmobilization, targeting, and activation in the cell remain unclear.Here we found that the second messenger calcium fundamen-tally changes the conformation and structural flexibility of themyosin-VI motor. We discovered that this is due to calmodulinchanging affinity for binding sites on calcium binding. The rotationof calmodulin from the IQ-motif P2 to the bipartite binding sitedirects a major rearrangement of the motor from a compactdormant state into a primed, cargo binding state.To interpret the negatively stained electron micrographs, we

modeled a structure that combined the crystal structures of themyosin-VI head domain (23) and the neck region comprising twocalmodulins and the subsequent three-helix bundle of the taildomain (26). At low calcium, we found an excellent agreementbetween the crystal structure of both calmodulins plus helixbundle and the EM image. The extra mass observed in the EM atlow calcium between the catalytic domain, the converter, and thefirst calmodulin indicated the presence of a backfolded myosin-VI tail, as speculated previously based on cell biological andsmall angle X-ray scattering studies (19, 35). Intriguingly, insteadof interactions between the C-terminal cargo-binding domainand the catalytic head domain, as observed in EM studies onmyosin-V (21, 22), our spectroscopic studies revealed that in thecase of myosin-VI a C-terminal tail segment (aa1060–1125) in-teracts with apo-calmodulin at the neck region of the motor. Thisdirect interaction of the cargo-binding tail with the calciumsensor on the motor provides a molecular mechanism to couple thefolding/unfolding and targeting of the motor to the intracellularcalcium signaling pathways.The majority of our EM data at high calcium showed that the

second calmodulin had rotated clockwise by ∼30° in the pro-jection plane, whereas the helix bundle had rotated anticlockwiseby ∼30°. This rotation indicated a major structural rearrangement ofthe motor without the detachment of calmodulin, in contrast to aprevious report (36). Consistent with the previous study, we alsoobtained classes of short molecules that seemed to have lost thesecond calmodulin at high calcium (Fig. S3C). However, wefound that the loss of calmodulin was strongly dependent on freecalmodulin supplemented to the buffer solution. In the presenceof physiological levels of ∼10 μM calmodulin (37), ∼65% of the

myosin-VI molecules retained the second calmodulin at high cal-cium and underwent the conformational changes described above.In contrast, at low calcium, supplementation with free calmodulinhad little effect, with the second calmodulin remaining bound inthe majority of molecules. These results indicated that the secondcalmodulin detaches only partly at the higher, more physiologicalcalmodulin concentrations and rebinds to the bipartite bindingsite, thus rearranging the fold of the neck and tail region. Thisrearrangement leads to a release of the tail and primes the motorfor mechanical activity. In this way, calcium activates the motorfrom a dormant, backfolded state into an unfolded, primed statewith a tail free to attach to binding partners and cargo.Using tryptophan fluorescence, we identified the sites and

calcium dependence of calmodulin binding to the myosin-VIneck and tail segments. Calmodulin N- and C-terminal lobesinteract with amino acids in IQ motifs both independently and inconjunction with each other, giving rise to complex binding be-havior. Using calmodulin and calmodulin mutants, as well as acomprehensive library of peptides several of which overlap, werevealed a previously undiscovered bipartite Ca2+-calmodulinbinding site with two, nonadjacent halves contained in the pep-tide P½ 2–3. This composite binding site, comprising parts of P2and the extended loop between the first two helices in the sub-sequent helix bundle (26), enables Ca2+-calmodulin to bridgebetween the neck and the proximal tail of the motor, probablykeeping the structure of the helix bundle intact. This mechanismmakes this calmodulin binding site a very likely candidate for thestructural rearrangement triggering the release of the backfoldedtail at high calcium as observed in the EM.The lipid-binding studies showed that calcium increased my-

osin-VI binding to the mixed brain Folch liposomes, consistentwith a previous study (6). At high calcium, no actin gliding couldbe obtained with myosin-VI attached via a C-terminal biotin to astreptavidin surface. In contrast, at low calcium, the actin glidingvelocities of 60–80 nm/s at saturating ATP concentrations were

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Fig. 5. Model. The model illustrates the effect of calcium on the back-foldingand release of the tail, conformation and target binding of the secondcalmodulin, and mechanical stability of the lever arm.

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similar to those obtained under control conditions with the motormolecules attached at the C terminus via an antibody against theglobular tail (18), via Folch liposomes or a biotin-strepavidinlinker, and similar to those previously reported for myosin-VIconstructs (16, 17). An explanation for the calcium effect is pro-vided by the EM image analysis showing that, at low calcium, thecalmodulin binding lever arm pivoted around a single fulcrumnear the converter and the lever arm itself appeared largely in-flexible, consistent with a mechanically functional lever arm.Intringuingly the emergence of a second fulcrum in between thetwo calmodulins at high calcium indicated that the lever arm hadlost stiffness and had become largely flexible and therefore unableto transmit force. The inhibition of motility at high calcium isconsistent with either the binding of the second calmodulin to theneck domain at the bipartite site or the detachment of calmodulin(Fig. S3). Both scenarios are expected to destabilize the me-chanical lever arm and would provide a simple explanation for thelack of motility at high calcium, seen here and reported previously(17), and also the uncoupling of the heads in enforced myosin-VIdimers (16).We propose the following model how calcium can mobilize

myosin-VI to attach to cargo and empower the motor mechan-ically to generate motility (Fig. 5): (i) at low calcium, myosin-VIadopts a backfolded, dormant state, with the tail domainaa1060–1125 binding to apo-calmodulin; (ii) at high calcium, thesecond calmodulin detaches partly and seeks out an alternate,high-affinity bipartite binding site, coupled with the strong re-duction in affinity of the tail for Ca2+-calmodulin, myosin-VIunfolds and is now primed to bind cargo; (iii) the conformationalchange of the second calmodulin leads to a destabilization of thelever arm that prevents myosin-VI from translocating beforebinding to (lipid) cargo; and (iv) once calcium is lowered againthe second calmodulin rebinds to P2 alone, restabilizing the leverarm; the motor, attached to cargo, is now mechanically active; onthe release of cargo, or in the event that no cargo is bound, thetail folds back onto calmodulin, switching the motor off. It isconceivable in this condition that cargo could directly competewith the tail–calmodulin interaction if the Kd for the cargo isstrong enough. However, increasing calcium concentrationsmakes this much more likely, by reducing the tail-calmodulinaffinity in the backfolded conformation. Our simple model opensup new perspectives of how transients in the ubiquitous secondmessenger calcium can orchestrate the timing of localized motoractivation in this superfamily of molecular motors. The proposedmechanism uncouples the process of target/cargo binding fromthe mechanical activation. Such a two-step mechanism mightrepresent a paradigm for the problem of recruitment and timingof activation and inactivation of cytoskeletal motors in general.

Materials and MethodsMolecular Biology. Myosin-VI from chicken brush border cells containing thelarge insert (residues 1–1276) (18) was cloned into pFastbacHtB (Invitrogen).The myosin-VI heavy chain was coexpressed with human calmodulin andpurified as previously described (18, 32). The expression of excess calmodulincompared with the myosin-VI heavy chain was confirmed by the presence ofunbound calmodulin in the flow-through on loading the cell extract ontothe HisTrap-myosin-VI-purification column (GE Healthcare). To generatecalmodulin with either the N- or C-terminal calcium binding sites eliminated,the cDNA of calmodulin was mutated to make E104A, E140A for theC-terminal mutant, and E31A, E67A for the N-terminal mutant. The mutantcDNA constructs were then cloned into pET28a bacterial vector andexpressed and purified as described previously for WT calmodulin (32).

Tryptophan Fluorescence. All tryptophan fluorescence studies were performedwith target peptide sequences from human myosin-VI (National Center ofBiotechnology Gene ID 92859701; amino acids 788–1036, synthesized byGenScript) and human calmodulin. The titrations of predicted target peptides(27, 28) with calmodulin were performed and analyzed at 20 °C in the followingbuffer (in mM): 25 Tris (pH 8.0), 100 KCl, and 1 DTT, supplemented with either

1 CaCl2 or 0.2 EDTA, using a Varian Cary Eclipse fluorescence spectrophotometer:λex = 290 nm and λem = 323 nm, as previously described (29, 32). The dissoci-ation constants Kd for the tryptophan-containing peptides were determined bydirect titration, whereas peptides without tryptophans were titrated againstpreformed calmodulin–peptide complexes with a known Kd as previously de-scribed (29). The equations used to fit the data are described in SI Text.

Motility Assay. Procedures were adapted from assays as previously described(18, 32, 33) and as detailed in SI Text. Images of the fluorescently labeledactin filaments (90× magnification) were recorded every 10 s for a totalperiod of 600 s. Only filaments moving continuously for at least 20 frameswere included in the data analysis. For the assays with biotinylated myosin-VI, myosin was bound to a streptavidin-coated nitrocellulose surface. For thelipid-based assays, before adding myosin, Folch liposomes were introducedinto the chamber, as previously described (38). The gliding velocity of filamentswas calculated using the analysis software GMimPro (www.mashanov.uk). Allassays were carried out at 37 °C. A composite of the first frame (Fig. 4 G andK in yellow) overlaid on a maximum intensity projection of the following40 frames shows the tracks of the actin filaments.

Cosedimentation of Myosin-VI Head Constructs with F-Actin. Interactions be-tween the myosin-VI head and tail constructs were probed with pull-downexperiments in the presence of F-actin. The Head 913 (aa1–913; 1 μM) and Tail1060 (aa1060–1276; 3 μM) were mixed with F-actin (5 μM) in a pull-downbuffer (in mM: 20 Hepes, pH 7.4, 150 NaCl, and 1 DTT) and incubated at 23 °Cfor 10 min before centrifugation at 160,000 × g for 15 min at 4 °C. The pellet(resuspended in pull-down buffer) and supernatant were run on SDS/PAGEand stained with Coomassie.

Cosedimentation of GST-Tagged Constructs, Using MagneGST Beads. Interac-tions between GST-tagged and His-tagged or nontagged protein werestudied using a MagneGST beads system (Promega). The beads with highaffinity for GST were incubatedwith GST-fusion protein in PBS plus 1mMDTTfor 10 min, including 1% BSA to reduce nonspecific binding to the beads.Washing using PBS and incubationwith the His-tagged or nontagged bindingpartner plus 10% BSA for 60 min was followed by further washing steps inPBS. Finally SDS sample buffer was added, the sample was boiled for 5 min,the beads were removed, and the sample was analyzed using SDS/PAGE. Theexperiments were performed at 23 °C.

Dot Far Western Blot. To detect protein–protein interactions, unlabeled prey-peptides were titrated against calmodulin, and samples were drawn atpredefined ratios. One microliter of a 2 μM solution was spotted onto anitrocellulose membrane and left to dry for 30 min before the membranewas incubated for 60 min in blocking buffer (in mM: 50 Tris HCl, pH 7.5,150 NaCl, 0.1% Tween 20 plus 20 mg/mL fatty acid-free BSA; Sigma). Fol-lowing incubation with the bait protein (15 nM) in blocking buffer for 60 minand five washing steps in TBST, the membrane was incubated for 60 min withan antibait antibody, which was detected using an ECL kit (BioRad) followingthe manufacturer’s instructions and imaged using a BioRad Geldoc system.

MST. The method as described previously (39) was adapted for probing theinteraction of myosin-VI tail fragments with myosin-VI motor domains andcalmodulin (SI Text).

Liposome Cosedimentation. Liposomes were prepared from bovine brain ex-tract, type I, Folch fraction I (Sigma), and the cosedimentations with myosin-VIwere performed as described previously (6, 32).

Negative Stain EM. Nucleotide-free myosin-VI was diluted to 200 nM in abuffer containing (in mM) 25 NaCl, 20 Tris·HCl, pH 7.5, 20 imidazole, pH 7.5,5 MgCl2, 1 EGTA, and 10 DTT. For studies at high calcium, instead of EGTA,CaCl2 (pH 7.5) was added to obtain 100 μM free calcium to ensure saturationof the myosin-VI–calmodulin complex, whereas for the experiments at highcalmodulin, the buffer was supplied with 10 μM calmodulin, which yieldedfour different conditions in terms of calcium and calmodulin concentrations.For each condition, the protein samples were applied to hydrophilized (glowdischarge) carbon-coated copper grids (Science Services) and negativelystained with 2% uranyl formate as previously described (32, 40). The gridswere examined using a Philips CM 100 electron microscope (Hendrick Dietz,Laboratory for Biomolecular Nanotechnology, Technische Universität Munich)operating at 100 kV, and the micrographs were recorded using a CCD cameraat a resolution of 0.33 nm/pixel.

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Single Particle Analysis. The micrographs were processed using EMAN2 (41)and SPIDER (42) software for particle picking, alignment, and classification. Ifnot stated otherwise, alignments were done reference free, and classifica-tions were obtained using K-means clustering. To reduce the influence ofbackground noise and to focus the classification onto specific parts of themolecules, alignments and classifications were preceded by the applicationof a mask, as previously described (43). Stacks of 40,000–100,000 images,processed in a first round of reference-free alignment and K-means classi-fication, led to stacks of 12,000–45,000 usable images of myosin-VI molecules

for each of the four conditions. As described further in SI Text, each stackwas further classified into four classes representing major structural differences,called bent and straight, each in two different orientations, related by mirrorsymmetry with respect to the long axis of the catalytic domain of myosin.

ACKNOWLEDGMENTS. We thank Hendrick Dietz (Technische Universität Munich)for making his Philips CM 100 electron microscope available. We thank the MunichCentre for Nanosciences and SteveMartin (Crick Institute) for stimulating discussions.We acknowledge Deutsche Forschungsgemeinschaft Grant SFB-863-B6, Friedrich-Baur-Stiftung, and Münchner Medizinische Wochenschrift for financial support.

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