Calmodulin regulates dimerization, motility, and lipidbinding of Leishmania myosin XXIChristopher Batters, Heike Ellrich, Constanze Helbig, Katy Anna Woodall, Christian Hundschell, Dario Brack,and Claudia Veigel1

Department of Cellular Physiology and Center for Nanosciences, Ludwig-Maximilians-Universität München, 80336 München, Germany

Edited by Edward D. Korn, National Heart, Lung, and Blood Institute, Bethesda, MD, and approved November 13, 2013 (received for review October 18, 2013)

Myosin XXI is the only myosin expressed in Leishmania parasites.Although it is assumed that it performs a variety of motile func-tions, the motor’s oligomerization states, cargo-binding, and mo-tility are unknown. Here we show that binding of a singlecalmodulin causes the motor to adopt a monomeric state and tomove actin filaments. In the absence of calmodulin, nonmotiledimers that cross-linked actin filaments were formed. Unexpect-edly, structural analysis revealed that the dimerization domainsinclude the calmodulin-binding neck region, essential for the gen-eration of force and movement in myosins. Furthermore, mono-meric myosin XXI bound to mixed liposomes, whereas the dimersdid not. Lipid-binding sections overlapped with the dimerizationdomains, but also included a phox-homology domain in the con-verter region. We propose a mechanism of myosin regulationwhere dimerization, motility, and lipid binding are regulated bycalmodulin. Although myosin-XXI dimers might act as nonmotileactin cross-linkers, the calmodulin-binding monomers might trans-port lipid cargo in the parasite.

unconventional myosin | motor properties

Over 12 million people worldwide are affected by leishman-iasis, which is caused by the flagellated protozoan parasite

Leishmania. The disease manifests itself in a cutaneous form,which leaves disfiguring scars, or as visceral leishmaniasis, whichis potentially fatal (1). The parasite has a life cycle in two stages.The elongated, flagellated, and motile promastigotes are foundin the gut of the sand fly host. The egg-shaped, nonmotileamastigotes possess only a rudimentary flagellum, are found inmammalian macrophages, and are responsible for the humandisease pathology (2, 3). In contrast to other eukaryotic cells thatexpress at least 11 different isoforms (4), Leishmania donovaniseems to express only a single isoform, myosin XXI (5). A laterclassification assigned myosin XXI to class XIII, a kinetoplas-tide-specific class of myosins (6). Previous attempts to identifyand localize myosin Ib in L. donovani parasites using anti-Leishmania myosin Ib antibodies were unsuccessful (3). In-triguingly, only myosin XXI was shown to be expressed in boththe motile promastigote and the nonmotile amastigote stages ofthe parasite’s life cycle (3). In the promastigote stage, this motorpreferentially localized to the proximal part of the flagellum,although it was also found along the entire length of the flagel-lum and in other cell-body compartments (7). The myosin-XXIhomozygous knockout is lethal. The heterozygous cells wereunable to form the paraflagellar rod, a structure of unknownfunction that runs along the length of the flagellum and containsa variety of proteins including actin and myosin XXI (8). It hasalso been reported that reduced expression levels cause the lossof endocytosis within the flagellar pocket and affect other in-tracellular trafficking processes (7). This makes myosin XXI anintriguing candidate to study modes of structural adaptation ofa single myosin isoform for a variety of cellular acto-myosin–based motile functions. Two distinct cellular fractions of myosinXXI have already been identified: a membrane-bound fractionthat localized to the base of the flagellum and a cytosolic fractionpossibly involved in transporting proteins within the flagellum

(3). However, it is unknown in which way the motor is targeted todifferent cargo, which oligomerization states it can adopt, andhow transitions between different functional states are regulated.The design of myosin XXI follows the general structure of

myosin motors, which comprises a conserved N-terminal motordomain, followed by a neck region including IQ motifs for thebinding of light chains of the calmodulin family, and finally a C-terminal cargo-binding tail domain (9) (Fig. 1A). Although themotor domain is responsible for the binding to actin and hy-drolysis of ATP, it is usually the tail domain that determines itsfunction within the cell by controlling dimerization or oligo-merization, motor anchoring to membrane compartments, andselection and transport of specific cargo. The neck region,mechanically stabilized by binding calmodulins, is thought toserve as a lever arm to transduce force and movement to thecargo. In a previous study we identified six potential calmodulin-binding IQ motifs outside the motor domain of myosin XXI. Thedata suggested that only the motif closest to the motor domainbound Xenopus/human calcium-calmodulin (10). Intriguingly,sequence analysis indicated a coiled-coil domain (11, 12) witha strong propensity to dimerize in between the end of the motordomain and what we initially thought to be the first calmodulin-binding IQ motif. Closer inspection of this predicted coiled-coildomain, however, suggested a further potential calmodulin-binding IQ motif within the predicted dimerization site and closeto the converter (yellow, Fig. 1A). This suggested that this my-osin might be able to dimerize, but in doing so would lose itsmechanically essential lever arm structure to the formation ofa coiled coil and, as a consequence, make a transition froma motile monomeric form to a nonmotile dimer. Consistentwith this, we found in a previous study that calcium-calmodulin

Significance

Myosin XXI is the only myosin isoform expressed in theLeishmania parasite. The myosin-XXI homozygous knockout islethal, and a reduction in expression levels leads to loss ofendocytosis and affects other intracellular trafficking pro-cesses. In this paper we show that myosin XXI can adopta monomeric or dimeric state. The states are determined bycalmodulin binding to an IQ motif that, when bound, preventsdimerization of a coiled-coil motif. In the monomeric state themotor binds phospholipids and is motile whereas the dimericstate is unable to bind lipids or to generate motility, but cancross-link actin filaments. Regulation of dimerization, motility,and lipid binding by calmodulin is a mechanism for the myosinfamily of motor proteins.

Author contributions: C.B. and C.V. designed research; C.B., H.E., C. Helbig, K.A.W.,C. Hundschell, and D.B. performed research; C.B. and C.V. analyzed data; and C.B. and C.V.wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: claudia.veigel@med.uni-muenchen.de.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1319285110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1319285110 PNAS | Published online December 30, 2013 | E227–E236

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binding was not required for the ATPase activity of the motor,but for myosin XXI in vitro motility (10).To investigate the mechanism of dimerization and the me-

chanical properties of myosin XXI, we expressed L. donovanifull-length myosin XXI, a truncated minimal motor domain, anda series of tail constructs. We also expressed L. donovani-specificcalmodulin-like (CamL) proteins to characterize calcium-de-pendent binding to target peptides on the myosin-XXI heavychain. Using size exclusion chromatography (SEC), analyticalultracentrifugation, Förster resonance energy transfer (FRET),motility assays, and electron microscopy (EM), we studied theeffect of calmodulin binding on motor dimerization and motility.We used liposome pull-down experiments and phospholipid blotassays combined with sequence analysis to identify specificphospholipid-binding motifs in the head, neck, and tail domainof the motor. The data suggest a form of myosin regulationwhere dimerization, motility, and phospholipid binding of themotor are determined by binding of calmodulin.

ResultsCalmodulin Binding Prevents Dimerization of Myosin XXI. By com-paring the sequence of the myosin-XXI motor domain withmyosins for which crystal structures are available (e.g., refs. 13–15), we localized the highly conserved elements involved inchemo-mechanical energy transduction, including the ATP-binding pocket, switches I and II, and the relay loop (Fig. 1A).The converter domain (645–747) is thought to transform smallconformational changes in the nucleotide-binding pocket tochanges in the orientation of the calmodulin-binding neck

domain that serves as a mechanical lever arm. Intriguingly, thesequence suggests a coiled-coil domain (11, 12) with a strongpropensity to dimerize between the end of the motor domain andwhat we previously thought to be the first calmodulin-binding IQmotif (809–823) (10). This predicted coiled-coil region (730–810)revealed a further potential calmodulin-binding site (754–769,yellow in Fig. 1A) close to the converter. Downstream of thatcoiled-coil region we identified three more sections with poten-tial to form a dimerizing coiled-coil or even to trimerize (bluesections, negative score for predicted trimers in Fig. 1A). Toinvestigate whether calmodulin binding had an effect on di-merization, we coexpressed full-length myosin XXI (FL-XXI)with human calmodulin using a baculovirus/SF21-based expres-sion system and analyzed the oligomerization state by SEC asdescribed previously (Materials and Methods) (10) (Fig. 1 B andC). The SEC study showed that FL-XXI heavy chain, coex-pressed with high levels of calmodulin virus, was monomeric.Using the approach of Coluccio (16), we had found in a previousstudy that the stoichiometry between myosin-XXI heavy chainand human calmodulin was 1:1 for these conditions of expression(10). In contrast, in the absence of added calmodulin virus, mostof the purified myosin-XXI sample eluted earlier, indicating theformation of dimers or higher oligomers (Fig. 1B). The elutedfractions were analyzed by SDS/PAGE, and silver staining con-firmed that calmodulin only eluted together with the myosin-XXI heavy chain in the monomeric fraction at intermediate orhigh levels of calmodulin expression (fractions 12–15, Fig. 1C,Lower). In the absence of added calmodulin virus, the levels ofendogenous calmodulin in the Sf21 insect cells were too low tobe detected by a Western blot (Fig. 1D, low levels of calmodulinexpression). This suggests that the expression level of endoge-nous calmodulin in Sf21 was too low for a 1:1 stoichiometry ofcalmodulin to myosin-XXI heavy chain. For these conditions,SEC analysis showed a large peak for the dimeric or oligomericfraction of myosin XXI and a very small peak for the monomericfraction, presumably with endogenous insect calmodulin bound(Fig. 1B). This small peak was the only peak observed previouslywith the Superdex 200 column (10). The major peak eluting hereat 10 mL was in the void volume on the Superdex 200 column inthe previous study. The expression levels of calmodulin could bevaried by varying the amount of added calmodulin virus and arereflected in a qualitative fashion in the signal strength of theWestern blot in Fig. 1D (high and intermediate levels of cal-modulin expression). The experiments showed that a change inthe level of coexpressed calmodulin in the Sf21 cells resulted ina different oligomerization state of myosin XXI.

Two Different Leishmania CamL Proteins Bind to the Same TargetSequence on Myosin XXI. To investigate in more detail how cal-modulin binding affects dimerization of myosin XXI, we studiedthe binding of human calmodulin and of Leishmania-specificcalmodulin-like proteins to predicted target sequences on themyosin-XXI heavy chain. Sequence analysis showed that thereare only eight CamL proteins in the L. donovani genome. Cal-modulin is highly conserved between species, and Xenopus orhuman calmodulin (CamH, NCBI Gene ID 4502549) is 90%identical to L. donovani CamL1 (NCBI Gene ID 13392113).Using change in tryptophan fluorescence of the target peptide asdescribed previously (17), we investigated CamH and CamL1binding to the target sequence 754–769 that is close to the my-osin-XXI converter domain and within the strongest predicteddimerization domain of myosin XXI (Fig. 2A). Titrations of in-creasing concentrations of CamH or CamL1 against the targetpeptide saturated at a stoichiometry of 1:1, consistent with thetarget peptide binding a single CamL1 or CamH protein. Non-linear least-squares fitting of the fluorescence signal at increasingCamH or CamL1 concentrations (18) yielded similar dissocia-tion constants with Kd values of 21 ± 5 nM for CamH and 18 ± 3

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Fig. 1. Calmodulin binding prevents the dimerization of myosin XXI. (A)Domain structure of full-length myosin XXI. By comparing the sequence withwell-studied myosins, we identified the highly conserved regions of themotor domain (gray). In addition to the calmodulin-binding IQ motifsidentified in a previous study (10), we found another IQ motif (yellow) im-mediately following the converter domain. Sequence analysis revealed fourpotential coiled-coil regions (in blue). The predicted propensity to oligo-merize was scored between 0 and 10 using Scorer 2.0 (11); positive numberspredict dimer and negative numbers predict trimer formation. (B) Size ex-clusion chromatography using a Superose-6 column and silver staining ofSDS/PAGE gels (C) of the elution fractions indicate that the motor is mo-nomeric when expressed in high levels of calmodulin and dimeric or ina higher oligomeric state when expressed at low levels of calmodulin. Ag-gregated myosin XXI would appear in the void volume. (D) The Western blotdoes not resolve endogenous calmodulin in the purified myosin-XXI prepa-rations that were expressed in the absence of added calmodulin virus (i.e., atlow levels of calmodulin). By varying the amount of calmodulin virus addedduring expression, the amount of calmodulin bound to the expressed myosinXXI could be controlled. The apparent difference in molecular weight ofcalmodulin in C and D is due to the differences in buffers.

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nM for CamL1. Binding to the target peptide was dependentupon calcium and fully reversible (Fig. 2B). In contrast, anothercalmodulin-like protein from the L. donovani genome, whichshares only 30% sequence identity with human calmodulinCamL2 (Gene ID 13386921), also bound to the target peptide inthe presence of calcium, but, once bound, remained bound ina calcium-independent fashion (Fig. 2C). This showed that atleast two different Leishmania-specific CamL proteins can bindto the same target peptide in the strongest predicted coiled-coildomain in myosin XXI and could affect myosin-XXI dimerization.To characterize oligomerization of the myosin-XXI tail in theabsence of calmodulin in more detail, we expressed a number ofmyosin-XXI tail constructs. The oligomerization state of theconstructs was analyzed by a combination of SEC, analytical ul-tracentrifugation, and FRET studies (Fig. 3 and Table 1).

Dimerization Involves Neck and Tail Domains of Myosin XXI. Tosimplify, we will call all constructs “tails” that begin downstreamof the converter region (645–747) of myosin XXI. The tailconstructs in Fig. 3A were designed to study the dimerizationpropensity of the four predicted coiled-coil segments. The pre-dicted propensity was scored between 0 and 10 using Scorer 2.0(11) (Fig. 1A; positive numbers predict dimer and negativenumbers predict trimer formation). The SEC experiment in Fig.3A was carried out in the absence of calcium-calmodulin andshows that all four constructs eluted as a single peak, consistentwith the samples adopting predominantly a single oligomericstate. As expected for proteins of near-globular shape, the 800-,830-, and 930-tail constructs ran through the SEC columns ap-proximately to their calculated molecular weight, following thecalibration curves for standard proteins of approximately glob-ular shape, as described previously (10) (Materials and Methods).The Stokes radii of these constructs were determined by a com-bination of SEC and analytical ultracentrifugation (Materials andMethods and Table 1). The results from both methods wereconsistent and suggested that the distal 800-, 830-, and 930-tailconstructs were monomeric. Following this argument we alsoinferred the oligomeric states of the largest tail construct directlyfrom the Stokes radius, which was calculated from the elutionvolume (19) (Table 1 and Materials and Methods). This 730-tailconstruct eluted too early to be monomeric and, when sized,strongly suggested a dimeric state. The 730-tail constructs com-posed the strongest predicted coiled-coil segment in full. To studythe oligomeric state of the different sections of the myosin-XXItail at lower protein concentrations, we used FRET. For thesestudies, we generated tail constructs with an N-terminal red fluo-rescent protein (RFP) or cyan fluorescent protein (CFP) tag. SECof the RFP-tagged constructs showed that the RFP-730, RFP-830,and RFP-930 tails eluted with two peaks, consistent with a mixedmonomeric and dimeric population (Fig. 3 B and C and Table 1).In the presence of the N-terminal RFP tag, all tail constructs in

fact adopted mixed monomeric and dimeric populations (Table 1).The only exception was RFP-830–930, which remained purelymonomeric. The coiled-coil segment of this construct seemedtoo weak to dimerize, even in the presence of the RFP tag. Thisshowed that the RFP tag on its own was not able to induce di-merization. The RFP-tagged sequences needed a propensity todimerize with a score >0.6 to induce the formation of dimers.

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Fig. 2. Analysis of calmodulin binding to predicted calmodulin-binding motifs. Tryptophan fluorescence (excitation at 290 nm, emission at 323 nm) was usedto study calmodulin binding to target peptide sequences. (A) The synthetic peptide of the first predicted calmodulin-binding motif C-terminal of the con-verter (aa 754–769) bound to human calmodulin (CamH) and to Leishmania CamL1 with a 1:1 stoichiometry and a Kd of 20 nM in the presence of calcium (pCa4). (B) For this target peptide, calmodulin binding was reversible and calcium-dependent. (C) Leishmania CamL2 also bound to the peptide in the presence ofcalcium, but remained bound when free calcium was subsequently lowered to less than 100 nM.

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Fig. 3. Analysis of myosin-XXI tail constructs to investigate domains in-volved in dimerization. (A) SEC data of myosin-XXI tail constructs. The UVsignals (in arbitrary units) were normalized to the highest peak for eachexperiment. Each construct was loaded at ∼30 μM. (B) SEC showed that, inthe presence of the N-terminal RFP tag, both monomer and dimer con-formations were observed for the tail constructs. The ratio of monomer todimer was dependent on protein concentration. The calculated molecularweight for RFP and CFP is 27 kDa. (C) For the RFP-830–930 construct, thepresence of the N-terminal RFP tag was insufficient to induce dimerization.(D) FRET study of a control CFP-RFP fusion protein containing a cleavableTEV site. CFP-donor excitation was performed at 445 nm. The emissionspectrum was recorded from 450 to 650 nm (in gray for the uncleaved fusionprotein and in black for the fusion protein following enzymatic cleavage).Protein concentration before cleavage was 7 μM; measurements were doneat 22 °C. (E) CFP-donor emission at 475 nm (blue) and RFP-acceptor emissionat 605 nm (red) for a mixture of 2-μM CFP-730 tails and 7-μM RFP-730 tailconstructs in the presence of 100 μM calmodulin and 1 mM CaCl2. (F) In theabsence of calcium calmodulin, the mixture of 2-μM CFP-730-tails and 7-μMRFP-730 tail constructs resulted in a FRET signal due to the formation ofheterodimers. (G) Summary of the FRET experiments with 1:1 mixtures ofRFP-730 and CFP-730 tails (2 μM each) in the presence and absence of 100 μMcalmodulin and of the control CFP-RFP fusion protein (2 μM) before andafter enzymatic cleavage. The FRET efficiency was calculated as described inMaterials and Methods. Each experiment was performed at least three timeswith three different preparations.

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For the FRET studies, we exploited the fact that mixed con-formations were obtained with the RFP-tagged tail constructs.This enabled us to study the formation of heterodimers by mixingCFP- and RFP-tagged tails in the presence and absence of cal-modulin. The formation of heterodimers was detected by FRETbetween the N-terminal CFP and RFP tags. As a control experi-ment, we generated a CFP-RFP fusion protein with a cleavableTEV site connecting the two fluorophores (20). Fluorescenceexcitation of the CFP donor was induced at 445 nm, and theemission spectrum was recorded between 450 and 650 nm (Fig.3D). Following cleavage at the TEV site, CFP-donor emission at475 nm was increased and RFP-acceptor emission at 605 nm wasreduced. The increased RFP-acceptor emission at 605 nm for theuncleaved protein indicated FRET between the linked CFP-RFPfluorophores. The energy transfer efficiency E was determined asdescribed (21) from the quenching of fluorescence of the donormolecule by the relation E = [1 − (Fda/Fdn)] × 100, with Fda andFdn the donor fluorescence intensities at 475 nm in the presenceand absence of the energy acceptor, respectively (Fig. 3G). WhenCFP- and RFP-730 tails were mixed in the presence of ∼10-foldmolar excess of CamH, a FRET efficiency of only about 1% wasobtained, similar to the FRET signal for the fully cleaved CFP-RFP fusion protein (0.4%, Fig. 3 E and G). This suggested thatthe binding of CamH to the monomeric CFP- and RFP-730 tailsprevented the formation of heterodimers. When the tails weremixed in the absence of CamH, we observed a fast increase inFRET efficiency, followed by a slower rate of signal change overabout 1 h until a steady state was reached (Fig. 3F). The fastsignal change was probably due to free monomers forming het-erodimers, whereas the slow rate was likely due to an exchangebetween homo- and heterodimers. As illustrated in the cartoonin Fig. 3F, this result further supports the hypothesis that cal-modulin binding regulates myosin-XXI dimerization. The next

question was whether the monomeric and/or dimeric confor-mations of the motor would bind to lipids and therefore couldaccount for the membrane-bound fraction of myosin XXI ob-served in the parasite.

Lipid-Binding Sections of Myosin XXI Include the Converter, Neck, andTail Domains. To test whether myosin XXI would bind to lipidsincorporated into a bilayer maintaining geometry and curvatureof a physiological membrane, we performed pull-down experi-ments with liposomes produced from bovine brain extract [Folchfraction I (22); Sigma] following standard procedures (23, 24).Several freeze–thaw cycles and extrusions through 100-nm porefilters were performed to obtain unilamellar vesicles with a sizedistribution of about 150 ± 30 nm. The lipid-binding studies withFL-XXI were carried out in the presence and those with the tailconstructs in the absence of calcium-CamH. Remarkably, thepurely monomeric constructs, such as the 830-tail and the trun-cated motor domain (Trunc XXI) (10), were pulled down com-pletely with the liposomes (Fig. 4A). In contrast, the mixtures ofmonomers and dimers, such as FL-XXI expressed at inter-mediate levels of CamH (Fig. 1D), and the RFP-tagged tails,were partially pulled down with the liposomes whereas a fractionremained in the supernatant. This suggested that dimerizationand lipid binding were mutually exclusive for myosin XXI. Thesame result was obtained with a protein lipid overlay (PLO)assay where lipids were blotted onto a nitrocellulose membraneand protein binding was detected using antibodies against theHis tag of the recombinant proteins (Fig. 4B). The dimeric 730-tail construct did not bind to the Folch lipid, whereas themonomer/dimer mixtures of the RFP-tagged constructs bound ina concentration-dependent fashion. These assays in additionconfirmed that the tails bound only to the mixed lipids of theFolch preparation, but not to the pure, main Folch constituents,

Table 1. Summary of hydrodynamic studies on myosin-XXI constructs to determine their oligomerization state

Tail constructStokes radius fromSEC/AUC* (nm)

Estimated Stokes radius frommolecular weight (nm)

Molecular weight from amino acidsequence and MS† (kDa)

Calculated molecularweight (kDa)

Monomer/dimer

730-tail 3.77 3.12 41 80 D800-tail 2.81* 2.9 32.1† 28 M830-tail 2.78* 2.81 28.6† 25 M930-tail 2.73* 2.43 18† 19.5 MRFP-730 tailPeak 1 4.18 117 DPeak 2 3.42 3.61 68 56 MRFP-830 tailPeak 1 4.38 141 DPeak 2 3.48 3.42 56 59 MRFP-930 tailPeak 1 4.06 106 DPeak 2 3.33 3.23 46 50 MRFP-730–830Peak 1 4.11 110 DPeak 2 3.2 3.19 44 44 MRFP-830–930 3.17 3.17 43 51 MFL-XXILow CamH 10.43 4.65 147 DHigh CamH 5.29 4.8 164 MTrunc XXI (amino

acids 1–800)4.17 4.20 120 M

The hydrodynamic properties of full-length and truncated myosin XXI and myosin-XXI tail constructs were determined using SEC and AUC. For the full-length and truncated myosin-XXI constructs, the native molecular weight had been determined previously (10) from their Stokes radius measured by SEC andtheir sedimentation coefficient, which was determined by sucrose gradient centrifugation as described by Post et al. (19). For the tail constructs, a near-globular shape was assumed. A native molecular weight was estimated by comparing the Stokes radius determined from the SEC elution volume, AUC, andmass spectrometry (MS) to the calculated Stokes radius determined for the theoretical molecular weight of the constructs. For RFP-730, a smaller Stokes radiuswas determined compared with RFP-830, which has a lower molecular weight. This suggests that the RFP-730 construct can adopt a more compact confor-mation. D, dimer; M, monomer.

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namely phosphatidylcholine (PC) or phosphoethanolamine (PE).Furthermore, we found that the proximal (730–830), the medial(830–930), and also the distal (930-tail) myosin-XXI tail con-structs bound to mixed lipids. To check which specific lipids thetails would bind to, we used commercial lipid strips (Fig. 4C).These experiments showed phospholipid binding along the neckand tail of the motor (Fig. 4D). Although the binding of phos-phatidyl inositol monophosphates was found along the entiremolecule outside the motor domain, binding of di- and triphos-phates seemed to be more localized at the proximal part of thetail (Trunc XXI and 730–930 construct, Fig. 4C). Comparingphospholipid binding of monomeric (fraction 7) and dimeric(fraction 2) RFP-730–830 constructs further confirmed that onlythe monomeric fraction of myosin XXI bound to the lipids.

Phospholipid-Binding Motifs in the Neck and Tail of Myosin XXIOverlap with the Dimerization Domains. To identify probablephospholipid-binding motifs in the neck and tail of myosin XXI,we compared the sequence to known motifs that have been de-scribed for other myosins. We found six nonspecific phospho-lipid-binding domains (LBDs) (Fig. 5A) with basic residuesflanking a central hydrophobic patch (Fig. 5C), as described forclass I myosins (25, 26). Two of those domains overlapped withpotentially unstructured lipid-binding domains (ULBDs in Fig.5A) rich in basic and hydrophobic residues, which were identifiedby calculating a basic-hydrophobic score over a running windowof 20-amino-acid residues along the sequence, as described byBrzeska et al. (27) (Fig. 5D). Consistent with the experimentalresults, the predicted lipid-binding sites were found along theentire sequence C-terminal of the myosin-XXI motor domain.Furthermore, they nearly fully overlapped with the dimerizationdomains (predicted coiled-coil, Fig. 5B).

Myosin-XXI Converter Contains a Phospholipid-Binding Phox-HomologyDomain. We also identified a phox-homology (PX) consensussequence [R (Y/F) x23–30 K x13–23 R] for phosphatidylinositol3-phosphate [PI(3)P] binding (28) that overlapped with the endof the converter domain of the motor head (Fig. 5A). The aminoacid residues of the PX consensus sequence are thought to forman electro-positive basic patch to bind the negatively chargedphosphate groups of phosphoinositides (29). To investigatewhether the critical residues of the PX consensus sequence werelocated at the surface of the converter domain and potentiallyaccessible for interaction with lipid membranes, we used thecrystal structure of scallop muscle myosin II (30) and replacedthe sequence in the converter domain by the sequence of L.donovani (Fig. 6). The four critical residues R (Y/F) x23–30 K x13–23 Rwere in nonconserved regions and were all located at the surfaceof the converter domain and should therefore be able to forma patch for lipid binding without interfering with the structure andfunction of the motor. We analyzed 130 sequences of the con-verter domain of 21 other myosin classes (Dataset S1) but onlymyosin XXI contained the PX-domain consensus sequence forphospholipid binding in this region (Fig. 6). We expressed a con-struct comprising the myosin-XXI converter domain (600–758),including the WT PX domain, as well as a PX-mutant constructwith the first two amino acids of the consensus sequence, RY,replaced by LS. We found that the WT converter construct boundnot only PI(3)P monophosphate, which is specific for PX domains(28), but also the two other monophosphates, PI(4)P and PI(5)P,as well as the diphosphate PI(3,5)P2. A weak interaction was alsofound for other anionic lipids, such as phosphatidylserine (PS, Fig.4C). For other PX domains, this has been related to the presenceof adjacent hydrophobic residues that might form an additionalpositively charged surface patch (28, 29). For the PX-mutantconstruct, however, the binding of the monophosphates was

A

S PS P

Trunc-XXI FL-XXIRFP-

930-Tail

S P

BLipid-binding

domains of

monomeric

myosin-XXI tails

TriglycerideDAG

PAPSPEPCPG

Cardiolipin

PIPtdIns(4)PPtdIns(4,5)P2PtdIns(3,4,5)P3CholesterolSphingomyelinSulfatideBlank

LPALPC

PtdInsPtdIns(3)PPtdIns(4)PPtdIns(5)P

PEPC

S1PPtdIns(3,4)P2PtdIns(3,5)P2PtdIns(4,5)P2PtdIns(3,4,5)P3PAPSBlank

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D

RFP-

730-830RFP-

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Fraction 2 Fraction 7 Elution volume (ml)1311 15

100

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3 71 5 957423125

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3 71 54 82 6 9Fractions

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u)

{

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RFP-

830-930RFPPEPC

FolchProtein

0.5 0.35

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nmoles RFP-

730-830

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C 730-930 830-Tail

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S P S P

RFP-

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PiP-binding

FL-XXI

pull down in the presence

of Folch liposomes

S Ppull down

without lipids

110 240 17561 49 2714 9 1017 6 3.4

125180< 0.1 <0.1Protein

(ng) {{

control

Fig. 4. Lipid binding of myosin XXI. (A) Pull-down experiments of Folch mixed liposomes and myosin-XXI constructs showed that monomeric constructsbound to liposomes. (B) PLO experiments indicated lipid binding along the entire tail of monomeric myosin XXI, whereas dimers (730-Tail) did not bind tolipids. The PLO data also showed that myosin XXI binds to Folch mixed lipid preparations but not to pure PC or PE lipids. (C) Binding to lipid membrane stripsshowed that all constructs bound to PIP monophosphates. Binding to di- and triphosphates was found N-terminally of amino acid 830. Binding studies of RFP-730–830 confirmed that the monomeric form (fraction 7) bound to phospholipids, whereas the dimeric form (fraction 2) did not. The fractions collected froma SEC experiment on the RFP-730–830 construct were analyzed using silver-stained SDS/PAGE (Inset) and confirm that both peaks originate from a singleprotein. (D) The cartoon indicates capacity for phospholipid binding along the entire myosin-XXI tail, including the converter region (645–747).

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retained whereas the binding of the diphosphate PI(3,5)P2 andof PS was completely abolished. This strongly supports thehypothesis that the PX domain in the converter is involved inphospholipid binding.

Mechanical Properties of Monomeric and Dimeric Myosin XXI. Fi-nally, we studied the mechanical properties of monomeric anddimeric myosin XXI in the presence of actin using in vitro mo-tility assays and electron microscopy. The model in Fig. 7Asummarizes our current results. At least two different calmod-ulin-like proteins (CamL1 and CamL2) in L. donovani can bindto the same calmodulin-binding motif (754–769) following theconverter and interfere with dimerization. Their binding differsin Kd and calcium sensitivity. Monomeric, CamH-binding myosinXXI (Fig. 1C, Lower, fraction 14) is motile and binds to phos-pholipids. Immobilized unspecifically on a nitrocellulose surface(31) in the presence of a fivefold molar excess of calcium-CamHand at 2 mM ATP, monomeric myosin XXI moved rabbit skel-etal actin filaments (n= 52) at 18 ± 3 nm·s−1 (Fig. 7B and MovieS1). Intriguingly, the velocity was about four times higher when

the monomeric myosin XXI was bound to Folch membranes.The concentrations of myosin XXI required to produce smoothmotility were reduced 30-fold, suggesting that, when bound tolipids via the tail, myosin XXI is in an improved configuration tosupport motility (Fig. 7B and Movie S2). In contrast, dimericmyosin-XXI motors (Fig. 1C, Upper, fraction 10) immobilized onnitrocellulose bound actin filaments, but were unable to movethem. In the presence of 2 mM ATP, the actin filaments werebroken into smaller fragments by the interaction with the dimericmyosin-XXI motors (Movie S3). To obtain structural infor-mation on monomeric myosin XXI, we performed negative-stainelectron microscopy on monomeric full-length myosin-XXImolecules, adsorbed to carbon-coated EM grids in rigor (ATP <1 μM). The molecules were classified as described previously(32). The two representative class averages (52 and 56 images,respectively) in Fig. 7C are shown with overlaid crystal structuresof a truncated myosin-V motor domain (red) in rigor witha single bound calmodulin (green) [Protein Data Bank (PDB)1OE9]. Although the class averages of the myosin-XXI motordomain are consistent with different surface projections of the

PX-Domain760

818

878

934

992

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. .

converter (aa 645-747) UBA-domains

A

B DLipid binding domains (LBDs)

LBD1

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LBD4LBD5

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Lipid binding domainscalmodulin

binding site LBD6

Unstructured lipid

binding domains

0

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BH-Score

ULBD1 ULBD2

FKQGRYSDASQDFLQRHQRLYSWAEPNYAVGKTKVFLRAEVWSALERLVLRRRAQLLH

RCKPYLRRWIDELRERRRIEEQKRLEAARKLREAREAKAADAANGVPAEKLQWVEEAS

NMFPDFDTDTLLDVAVEADTREEALSAILAIQADRLDKQTASGFMEVMAAANVRRGVI

NNFISADIKTVSALSRLQPEDMKSLGASEMEVVAITKKLAEQQGQRVKYQRLAEAIGT

DSEYAAAGAVQRAEVARHQEDFDAKVQTLASMGFDEPTGRLVLAHYNGDVQRTAARLLUBA

YGVDSRKMRNNARKHKNFNTTDPNVQQLISLGATKQDAKMALRRNNGDANAAVKMLFKVS

LBD2 LBD3

LBD5 LBD6

LBD1

LBD4

R R W I D E L R E R R RK R L E A A R K L R E A RK A A D A A N G V P A E KK S L G A S E M E V V A I T K KR K M R N N A R K H KR R N N G D A N A A V K

Fig. 5. Sequence analysis of myosin XXI to localize lipid binding and dimerization. (A) We identified a number of potential lipid-binding sites, including a PXdomain that overlapped with the converter region of the motor. (B) The potential lipid-binding sites (green) overlapped with the potential dimerizationdomains (blue). (C) Six lipid-binding domains (LBD1–6) with basic residues flanking a hydrophobic patch of amino acids (25, 26) were found. (D) Using thebasic-hydrophic scale (25), we identified a potential lipid-binding site following the PX domain and confirmed LBD5 as a site with high potential for phos-pholipid binding.

Converter

PX Domain

Loop with

Insert

LPALPC

PtdInsPtdIns(3)PPtdIns(4)PPtdIns(5)P

PEPC

S1PPtdIns(3,4)P2PtdIns(3,5)P2PtdIns(4,5)P2PtdIns(3,4,5)P3PAPSBlank

WT

600-758

PX Mutant

600-758

Converter-Domain

PX-Domain

Consensus sequence

GF R F RY L ------ Y GKTK FFK L LEEH + H +H H Insert H + + HH+ H H--GYPVRRPLEQFC-RYFYLVMSRTTASLFKQGRYSDASQDFLQRHQRLYSWAEPNYAVGKTKVFLR-AEVWSALERLVLRRR

WT RY/F X23-30 K X13-23 RPX mutant L S K R

Myosin-XXI

Leishmania

donovani

A B

Fig. 6. Structural analysis of the PX domain in the myosin-XXI converter. (A) We used the crystal structure of scallop muscle myosin II (30) and replaced thesequence in the converter domain with the sequence of L. donovani. The amino acids specific to the PX domain (in green) form a patch on the surface of theconverter (in red), consistent with these residues interacting with membranes without interfering with the general structure and function of the converter ofthis myosin motor. Comparison of 130 sequences from 21 myosin classes (Dataset S1) suggest that myosin XXI is exceptional within the myosin family in termsof featuring a PX domain in the converter region. (B) PIP strips showing that site-directed mutagenesis of arginine tyrosine (RY) in the expressed PX mutantconverter construct completely abolished the characteristic Pi(3,5)P2 binding of the PX domain.

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myosin-V crystal structure, the calmodulin-binding region ismore difficult to localize. And, although in image (i) in Fig. 7Cthis domain seems clearly visible, a large part of it seems to behidden in image (ii). The same holds true for the class average of400 images of myosin XXI bound to actin in image (iii) (PDB1OE9 and PDB 4A7F). This suggests that at least under theseconditions the calmodulin-binding domain either is found invariable orientations with respect to the motor domain or isfolded back onto the motor domain. Negatively stained EMimages of dimeric myosin XXI in rigor and in the presence ofactin showed that this myosin had a tendency to cross-link actinfilaments as shown in Fig. 7D.

DiscussionIn summary, our experiments have provided insights into thestructural and functional properties of the unusual myosin motorin the protozoan parasite Leishmania, myosin XXI. In less an-cient eukaryotic cells, such as mammalian cells, more than 10different isoforms are expressed and responsible for various formsof motility (33). Loss of any particular myosin may or may notreveal a clear phenotype owing to the functional compensation byother myosins. With only two myosins in the Leishmania genomeand with only a single isoform expressed, which, however, is vitalfor parasite survival (3), myosin XXI provides an intriguing op-portunity to study modes of regulation and structural-mechanicaladaptation for diverse motile functions of a single myosin. In thisstudy we have focused on myosin-XXI oligomerization and lipidcargo binding.

Myosin-XXI Dimerization. An unexpected finding was revealed bysequence analysis. The motor has eight potential calmodulin-binding sites, only one of which bound human calmodulin in ourprevious study (10). We have now identified a binding site closeto the converter domain that is located within an extended di-merization site covering the entire C terminus of the moleculefrom the converter region to the C-terminal end. This site bound

the L. donovani-specific calmodulin-like proteins CamL1 andCamL2 in a calcium-dependent fashion. This was unexpectedbecause in other myosins that dimerize or oligomerize, for ex-ample, myosin class II, V, or VI (34, 35), the predicted coiled-coil domain starts only downstream of the calmodulin-bindingneck region, which serves as a mechanical lever arm to transduceforce and movement to the cargo-binding C terminus. The leverarm structure is thus unaffected by motor dimerization. Formyosin XXI, however, we found that calcium-calmodulin bindingnot only prevented dimerization by precluding the formation ofa dimerizing coiled-coil but also seemed to enable motility in thefirst place by generating a functional lever arm. Consistent withthis hypothesis, we found in a previous study that, in contrast tomonomeric full-length myosin XXI with calmodulin bound, thetruncated construct (1–800) was also monomeric, but did notbind calmodulin and was not motile, although it had an actin-activated ATPase activity similar to that of the full-length motor(10). This construct comprised the catalytic head domain in-cluding 53 amino acids downstream of the converter. However, itdid not bind CamH and therefore probably could not forma functional lever arm. Its inability to bind calmodulin might berelated to some sort of backfolding of the C-terminal sequenceonto the catalytic domain, such as the EM images suggested forthe full-length motor (Fig. 7C). In the absence of calmodulin, theentire myosin-XXI C terminus downstream of the converter re-gion seemed to dimerize. Here, loss of the lever arm due to theformation of a coiled-coil could explain why the dimer was ableto bind to and cross-link actin filaments, but unable to generateany movement. Still binding of actin filaments in the presence ofATP caused the actin filaments to break, and the biochemicalactin-activated ATPase cycle of the dimer seemed to be at leastpartly uncoupled from the mechanical output of the motor.

Myosin-XXI Dimerization and Lipid Binding Are Mutually Exclusive.The second finding was that the predicted coiled-coil sections ofthe molecule nearly fully overlapped with lipid-binding domains.

B

[Myosin XXI] (nM)1 10 100 1000

0

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40

60

80

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ty (

nm

/s)

PI(3,4,5)P

PI(3,5)P

CamL1

CamL2 Non-Motile

Non lipid binding

Actin binding and ATPase

Motile

Lipid binding

insensitive

Ca2+

sensitive

A

C

Ca2+

4005652

PIP

(i) (ii) (iii) D

Fig. 7. Model of myosin-XXI regulation of dimerization, lipid binding, and motility. (A) The model summarizes our findings. At least two different cal-modulin-like proteins (CamL1 and CamL2) in L. donovani can bind to the same calmodulin-binding motif (754–769) following the converter and preventdimerization. Their binding differs in Kd and calcium sensitivity. Monomeric, calmodulin-binding myosin XXI is motile and binds to phospholipids, whereas thedimeric motor does not and is nonmotile. (B) The velocity of actin filaments driven by monomeric myosin XXI unspecifically adsorbed to nitrocellulose is fourtimes lower compared with motors bound to Folch lipid bilayers. (C) Negatively stained electron micrographs of single, monomeric myosin XXI in rigor (ATP <1 μM) adsorbed to carbon-coated EM grid on their own and when bound to actin. Representative class averages are shown. Crystal structures of truncatedmyosin V in rigor with a single light chain bound (green) have been overlaid. The numbers state the number of images contributing to the class average. (D)Representative negatively stained electron micrograph of two actin filaments cross-linked by dimeric myosin XXI in rigor (ATP < 1 μM).

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Furthermore, lipid binding and dimerization were mutually ex-clusive. This suggested a mechanism of myosin regulation andtargeting to cargo: in the absence of calmodulin, the myosin-XXImotor seemed to be dimeric and nonmotile, but able to bind toand cross-link actin filaments. The dimer is expected to be cy-tosolic because it was unable to bind to lipids. In contrast, cal-modulin-binding brought the motor into a monomeric state thatcan generate force and movement and bind to lipid cargo. Lipidbinding at various sites along the neck and tail of the moleculemight target the monomer to specific lipid compartments. In-terestingly, the lipid-binding studies with the tail constructssuggested that calcium-CamL binding to different target sitesalong the tail might have a regulatory effect on tail binding tospecifc compartments.The third finding was that membrane binding of myosin XXI

was not necessarily mediated in a stereospecific fashion, as de-scribed, for example, for myosin-I pleckstrin homology domainsthat interact specifically with PI(4,5)P2 (36–38). For other my-osin-I isoforms, however, membrane targeting does not seem torequire stereospecific phosphoinositide recognition (25, 26, 39).They seem to bind via more unspecific electrostatic interactionswith a variety of charged phospholipids. This is consistent withour observation for myosin XXI, where all three monophos-phates bound to the converter (645–747) and all of the tailconstructs. The diphosphates PI(3,5)P2 and PI(4,5)P2 seemed tobind N-terminally of amino acid 830 and the triphosphate PI(3,4,5)P3 between 758 and 830. Furthermore, our sequenceanalysis predicted six lipid-binding domains along the myosin-XXI C terminus following the converter, with basic residuesflanking a central hydrophobic patch, as described for non-specific charged interactions with phospholipids for Acantha-moeba, Dictyostelium, and human myosin class I (25–27). Two ofthose regions scored significantly on the basic-hydrophobic scale,as described by Brzeska et al. (27), to identify putative un-structured lipid-binding sites in myosin tail sequences. Theconsensus sequence for a phospholipid-binding PX domain thatoverlaps with the converter region of myosin XXI seemed to beunique among myosins. PX domains in Saccharomyces cerevisiaereportedly have high specificity, and mammalian ones showa preference for PI(3)P monophosphate (40). However, for somePX domains, binding of mono, di-, and triphosphoinositides havebeen reported (28, 40). This is consistent with our finding formyosin XXI where the respective sequence (600–758 construct)bound to all three monophosphates, PI(3,5)P2, and also tophosphatidyl serine (PS) whereas for the PX-mutant binding tothe PI(3,5)P2 diphosphate and to PS was completely abolished.For the kinesin family of motors, a PX domain has been reportedfor KIF16B (41). This PX domain was located at the C terminusof the molecule and targeted the motor to early endosomes viabinding to PI(3)P monophosphate. As in our case, however, thePX domain in KIF16B did not show high selectivity for PI(3)P. Italso bound other phosphoinositides with nanomolar affinity, inthis case PI(3,4)P2 and PI(3,4,5)P3, which are typically found atthe plasma membrane.

Potential Cellular Functions of Myosin-XXI Monomers and Dimers.With lipid-binding sections along the converter, neck, and taildomain of the molecule, monomeric myosin XXI seems to bewell suited to be targeted to lipid compartments with diverselipid compositions. This might include the plasma membrane,but also organelles or vesicular cargo involved in intraflagellartransport, where myosin XXI might anchor these structures toand transport along the actin cytoskeleton (7). As shown in thecartoon in Fig. 6A, we speculate that the monomeric, motile, andlipid-binding conformation corresponds to a myosin-XXI frac-tion that has been localized at the base of the Leishmania fla-gellum (3, 7). In contrast, the free, cytosolic dimeric fraction ofmyosin XXI might correspond to the detergent-labile compo-

nent that has been localized within the flagellum where it couldcontribute to the structural organization of the actin network (3,7). Apart from demonstrating the presence of actin filaments inthe main cell body and the flagellum using immuno-labeling offixed Leishmania parasites, to date little is known about thestructure and dynamics of the actin cytoskeleton in this system(3). Future studies investigating colocalization and distributionof actin and myosin-XXI monomers and dimers in the parasitewill be revealing. As illustrated in the cartoon, the monomericform might bind to single monophosphates or form ensemblesat membrane clusters of PIP2 with its different lipid-bindingdomains. Our lipid-binding studies with full-length myosin XXIand tail constructs in the presence or absence of calcium-cal-modulin indicated that calmodulin binding was not required forthe tail domains to bind to lipids. However, calcium-calmodulinbinding might be involved in sorting the motor to different targetmembranes. Consistent with this, it was recently reported thatcalmodulin is required for paraflagellar rod assembly and fla-gellum–cell-body attachment in trypanosomes (42).In conclusion, we found that Leishmania myosin XXI is a

system, where calcium-calmodulin regulates dimerization, mo-tility, and lipid binding of the motor. The following observationssuggest that expression levels of different Leishmania-specificCamLs and cytosolic calcium might regulate myosin function inthis system: (i) two very different Leishmania CamL proteinsbound to a single-peptide sequence in the strongest dimerizationdomain on myosin XXI, and these proteins differ in Kd andcalcium sensitivity; (ii) in vitro (human) calmodulin binding af-fected myosin-XXI dimerization, lipid binding, and motility; (iii)cytosolic calcium levels in the parasite are reported to be keptvery low and tightly regulated (43); (iv) the expression levels ofCamLs in the parasite are still largely unknown. However, down-regulation of cytosolic calmodulin affects the parasite’s parafla-gellar rod assembly (42), which is where myosin XXI has beenlocalized (3, 7). Intriguingly, we have now identified eight othercalmodulin-like proteins in the fully sequenced genome of theLeishmania parasite. They might interact with the seven addi-tional calmodulin-binding sequences outside the converter. Futurestudies will show which other conformations and oligomerizationstates the myosin-XXI motor can adopt, what the mechanicalproperties of these complexes are, and how they might be used forspecific cellular functions in the parasite. Apart from the interestin the basic mechanism of energy transduction and regulation ofmyosin motors, myosin XXI might also be an interesting potentialdrug target as it has been shown to be vital for Leishmaniaparasite survival.

Materials and MethodsPlasmids and Generation of Recombinant Baculovirus. FL-XXI was chemicallysynthesized, cloned into pUC57 (Genscript USA Inc.), and subcloned into the6 × His-tagged vector pFastBacHb (Invitrogen) using standard PCR methodsas described previously (10). An amino acid 1–800 truncation of myosin XXI(Trunc XXI) was created using PCR, and the N terminus of FL-XXI and TruncXXI modified by addition of EGFP (for details, see ref. 10). Recombinantbaculovirus DNA was generated using the Bac-to-Bac method beforetransfer into Spodoptera frugiperda (SF21) cells. Human calmodulin (Gene ID4502549; identical sequence in Xenopus laevis) was amplified from a P4stock before being transferred into SF21 cells for coexpression. Calmodulinsequences are highly conserved between different species. Compared withLeishmania calmodulin-like protein CamL1 (Gene ID 13392113), the se-quence of human calmodulin differs only in 15 amino acids, with eightconservative differences. Therefore, we expected similar binding propertiesfor human calmodulin compared with the endogenous Leishmania CamL1.We also expressed Leishmania CamL2 (Gene ID 13386921) that shares only30% sequence identity with human calmodulin. However, like CamL1,CamL2 colocalizes with the myosin-XXI heavy chain when coexpressed inHeLa cells. Nonfluorescent myosin-XXI tail fragments were cloned intoa pET28a vector (Invitrogen) via HindIII/XhoI restriction sites using standardcloning techniques.

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The following oligonucleotides were used for PCR (boldface indicates re-striction enzyme sites): XXI-730FP-nonF (GGGAAGCTTTAGGCAAGACGAAGGT-GTTCCTCC); XXI-800FP-nonF (GGGAAGCTTTA GACGCCGCCAATGGTGTGTGC);XXI-830FP-nonF (GGGAAGCTTTAGCCGTCGAGGCGG ACACGCGCG); XXI-930FP-nonF (GGGAAGCTTTAGGCACGGACAGCGAATATA TGCC); XXI-830RP (ACTCG-AGCTAGCGCGTGTCCGCCTCGACGGC); XXI-930RP (ACTCGAGCT AGGCATA-TTCGCTGTCCGTGCC); and XXI-ENDRP (ACTCGAGCTAGCTCACCTTGAACAGC).monomeric RFP-tagged myosin-XXI tail constructs were cloned into pET28avector containing N-terminal monomeric RFP via the following NotI/XhoI re-striction sites: XXI-730FP (AGAGGCGGCCGCCGGCAAGACGAG GTGTTCCTCC);XXI-830FP (AGAGGCGGCCGCCGCCGTCGAGGCGGACACGCGCG); and XXI-930FP (AGAGGCGGCCGCCGGCACGCGGACAGCGAATATGCC). The tail frag-ments were expressed in Escherichia coli and purified as described pre-viously (10).

Protein Expression and Purification. Protein expression and purification arebased on previously published protocols (9).

Liposome Cosedimentation. Liposomes were prepared from bovine brainextract, type I, Folch fraction I (Sigma) following the protocol by Spudich et al.(2007) (23). Five freeze–thaw cycles were applied to obtain unilamellarvesicles, and the liposomes extruded 11 times using a 100-nm pore filter toobtain a size distribution of 150 ± 30 nm. The liposomes (1 mg·ml−1) weremixed with myosin XXI (250 nM) in liposome buffer (in mM: 20 Hepes, pH7.4, 150 NaCl, 1 DTT) and incubated at room temperature for 10 min beforecentrifugation at 160,000 × g for 15 min at 4 °C. The pellet (resuspended inliposome buffer) and supernatant were run on SDS/PAGE and stainedwith Coomassie.

PLO. The assay was performed essentially as described by Dowler et al. (44).We prepared 1-mM stocks of the lipids (Avanti) in chloroform. A total of500, 350, or 200 pmol of the lipids and 0.5 μg of the target protein wereblotted onto the nitrocellulose membrane (Hybond-C Exra, Healthcare).The membrane was allowed to dry for 1 h at room temperature beforebeing incubated for 1 h in blocking buffer [in mM: 50 Tris·HCl, pH 7.5, 150NaCl, 0.1% Tween 20 plus 2 mg·ml−1 fatty acid-free BSA (Sigma)]. This wasfollowed by an overnight incubation with the target protein (5–10 nM) inblocking buffer plus 2 mg·ml−1 BSA at 4 °C. The membrane was then washed10 times in blocking buffer before incubation for 1 h at room temperaturewith anti–His-HRP antibody (Abcam ab1187) at a 1:2,000 dilution. Themembrane was washed again 12 times with blocking buffer. Antibodybinding was detected using an ECL kit (Invitrogen) and imaged using a Bio-rad Geldoc system. Target protein binding to lipid membrane strips andphosphatidyl-inositol phosphate (PIP) strips (Echelon Inc.) were carried out asdescribed above for reconstituted liposomes. Here the incubation with thetarget protein was reduced to 1 h at room temperature. For the PIP strips,analysis of the RFP-tail constructs was limited because of unspecific inter-actions of RFP with the PIP strip. Therefore, we focused the experiments onthe untagged constructs.

SEC. Purified proteins (∼30–50 μM) were loaded onto a Superdex-200 (10/300GL) analytical column (GE Healthcare) or a Superose-6 column (10/300 GL).The native molecular weight of the proteins was calculated from theirStokes radius measured by SEC, and their sedimentation coefficient wasdetermined by sucrose gradient centrifugation as described previously (10,19) and by analytical ultracentrifugation (AUC).

AUC. Sedimentation velocity experiments were performed on an OptimaXL-I analytical ultracentrifuge (Beckman Inc.) using an An 60 Ti rotor anddouble-sector epon centerpieces. The proteins were studied in 50 mMTris·HCl buffer, pH 7.5, with 150 mM NaCl at 0.3 mg/mL. Buffer densityand viscosity was measured using a DMA 5000 densitometer and anAMVn viscosimeter, respectively (both from Anton Paar). Protein con-centration distribution was monitored at 280 nm at 54,000 × g and 20 °C.The time-derivative analysis was computed using the SEDFIT softwarepackage, version 12.1b (45), resulting in a continuous size distributionand an estimate for the molecular weight. This was estimated from thesedimentation coefficient and the diffusion coefficient, as inferred fromthe broadening of the sedimentation boundary, assuming that all ob-served species share the same frictional coefficient f/f0) (45).

FRET Studies. We mixed CFP-730 tails and RFP-730 tails at a 1:1 molar ratio(2 μM each) in the presence or absence of 100 μM calmodulin and measuredthe FRET signal due to heterodimer formation under equilibrium conditions.To obtain a larger proportion of heterodimers, these experiments were alsocarried out in excess of RFP-730 tails (i.e., 2 μM CFP-730 tail mixed with 7 μMRFP-730 tail). The proteins were diluted in a buffer containing (in mM) 50Tris, pH 7.4, 150 NaCl, 1 DTT, and 1 CaCl2. A fluorescence spectrophotometer(Varian Cary Eclipse) was used with a wavelength of 445 nm for donor ex-citation and of 475 nm for donor emission and 605 nm for acceptor emission,respectively; for acceptor excitation, 585 nm was used. The excitation andemission slits were 10 and 5 nm, respectively; the detector gain was setto 630 V. Data were collected every 30 s over 60 min at 22 °C using a 2-sintegration time. All experiments were carried out in duplicate and repeatedusing at least four different protein preps. The CFP-TEV-RFP control exper-iment was performed in TEV-cleavage buffer that contained (in mM) 50Tris·HCl, pH 8.0, 0.5 EDTA, and 1 DTT. Ten units of TEV protease (Invitrogen)were added and spectra were taken every 30 min until the CFP emissionpeak at 475 nm reached a plateau. The energy transfer efficiency E wasdetermined as described (21) from the quenching of fluorescence of thedonor molecule by the relation E = [1 − (Fda/Fdn)] × 100, with Fda and Fdn thedonor fluorescence intensities in the presence and absence of the energyacceptor, respectively. This experiment was repeated three times using a 1:1ratio of RFP:CFP-730 tail.

Tryptophan Fluorescence. Titrations of target peptide sequences with cal-modulin-like proteins were performed at 20 °C in buffer (in mM) of 25 Tris(pH 8), 100 KCl, and 1 DTT with 1 mM CaCl2 or 0.2 mM EDTA, using a VarianCary Eclipse fluorescence spectrophotometer; λex = 290 nm and λem = 323 nmas described (17, 18). The dissociation constants Kd for the Trp-containingpeptides were determined by direct titration, and the data were analyzed asdescribed (17).

Electron Microscopy. Images were recorded on a JEOL JEM-1011 transmissionelectron microscope (Joachim Rädler, Ludwig-Maximilians-Universität,Munich). Myosin XXI was diluted to 100 nM in a buffer containing (inmM) 25 KCl, 50 Tris, pH 7.5, 0.1 MgCl2, and 0.1 DTT. The protein wasapplied to UV-treated, continuous carbon-coated copper grids (ScienceServices) and negatively stained with 1% uranyl acetate or 1% uranylformate as described (9).

Motility Assays. Procedures were adapted from those described by Kron et al.(1991) (46) and Lister et al. (2004) (31). In brief, myosin preps (∼150 μg·ml−1)were immobilized on a nitrocellulose-coated surface (0.1% vol/vol nitrocel-lulose in amylacetate) of the experimental chamber. To prevent unspecificbinding of actin filaments, the surface was then blocked with 0.5 mg·ml−1

BSA in assay buffer [AB buffer (mM): 25 KCl, 4 MgCl2, 1 EGTA, 25 imidazole,pH 7.4]. Subsequently, the rhodamine-phalloidin–labeled and stabilizedrabbit skeletal actin filaments were introduced into the chamber in ABbuffer supplemented with a scavenger system (10 mM DTT, 0.01 mg·ml−1

catalase, 0.05 mg·ml−1 glucose oxidase, 1.5 mg·ml−1 glucose) and 2 mM ATP.For fluorescence imaging, the rhodamine label on the actin filaments wasexcited with a 532-nm laser (50 mW). Images were recorded every 0.4 s fora total period of 300 s. Only filaments moving continuously for at least 20frames were included in the data analysis to determine the sliding velocity.For lipid-based motility assays, before adding the motor, Folch vesicleswere flowed into the chamber and allowed to bind for 10 min. The ve-locities were calculated using the imaging motion analysis softwareGMimPro (Gregory Mashanov; www.nimr.mrc.ac.uk/gmimpro). All assayswere carried out at 22 °C.

ACKNOWLEDGMENTS. We thank Dr. Stephan Uebel (Max Planck Institute ofBiochemistry) for his expert support with the analytical ultracentrifugationexperiments; Stephen Martin (Medical Research Council-National Institute ofMedical Research) for his expert advice and help with the tryptophan fluo-rescence experiments; and our laboratory technicians Irene Schneider,Sascha Blumentritt, Susanne Schickle, and Roswitha Maul and our mechan-ical workshop staff members Robert Waberer and Günther Zitzelsberger forexcellent support. We thank the Munich Center for Nanosciences for stimu-lating discussions and Professor Joachim Rädler (Ludwig-Maximilians-Univer-sität) for giving us access to his transmission electron microscopy microscope.The Deutsche Forschungsgemeinschaft SFB-863, the Friedrich-Baur-Stiftung,and Münchner Medizinische Wochenschrift provided financial support.

1. Desjeux P (2004) Leishmaniasis: Current situation and new perspectives. Comp Im-

munol Microbiol Infect Dis 27(5):305–318.

2. Bates PA (2008) Leishmania sand fly interaction: Progress and challenges. Curr Opin

Microbiol 11(4):340–344.

Batters et al. PNAS | Published online December 30, 2013 | E235

BIOCH

EMISTR

YPN

ASPL

US

Dow

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ded

by g

uest

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tem

ber

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021

3. Katta SS, Sahasrabuddhe AA, Gupta CM (2009) Flagellar localization of a novel iso-form of myosin, myosin XXI, in Leishmania. Mol Biochem Parasitol 164(2):105–110.

4. Bement WM, Hasson T, Wirth JA, Cheney RE, Mooseker MS (1994) Identification andoverlapping expression of multiple unconventional myosin genes in vertebrate celltypes. Proc Natl Acad Sci USA 91(14):6549–6553.

5. Foth BJ, Goedecke MC, Soldati D (2006) New insights into myosin evolution andclassification. Proc Natl Acad Sci USA 103(10):3681–3686.

6. Odronitz F, Kollmar M (2007) Drawing the tree of eukaryotic life based on the analysisof 2,269 manually annotated myosins from 328 species. Genome Biol 8(9):R196.

7. Katta SS, Tammana TVS, Sahasrabuddhe AA, Bajpai VK, Gupta CM (2010) Traffickingactivity of myosin XXI is required in assembly of Leishmania flagellum. J Cell Sci 123(Pt 12):2035–2044.

8. Santrich C, et al. (1997) A motility function for the paraflagellar rod of Leishmaniaparasites revealed by PFR-2 gene knockouts. Mol Biochem Parasitol 90(1):95–109.

9. Krendel M, Mooseker MS (2005) Myosins: Tails (and heads) of functional diversity.Physiology (Bethesda) 20:239–251.

10. Batters C, Woodall KA, Toseland CP, Hundschell C, Veigel C (2012) Cloning, expres-sion, and characterization of a novel molecular motor, Leishmania myosin-XXI. J BiolChem 287(33):27556–27566.

11. Armstrong CT, Vincent TL, Green PJ, Woolfson DN (2011) SCORER 2.0: An algorithmfor distinguishing parallel dimeric and trimeric coiled-coil sequences. Bioinformatics27(14):1908–1914.

12. Delorenzi M, Speed T (2002) An HMM model for coiled-coil domains and a compari-son with PSSM-based predictions. Bioinformatics 18(4):617–625.

13. Rayment I, et al. (1993) Three-dimensional structure of myosin subfragment-1: Amolecular motor. Science 261(5117):50–58.

14. Coureux PD, et al. (2003) A structural state of the myosin V motor without boundnucleotide. Nature 425(6956):419–423.

15. Ménétrey J, Llinas P, Mukherjea M, Sweeney HL, Houdusse A (2007) The structuralbasis for the large powerstroke of myosin VI. Cell 131(2):300–308.

16. Coluccio LM (1994) Differential calmodulin binding to three myosin-1 isoforms fromliver. J Cell Sci 107(Pt 8):2279–2284.

17. Martin SR, Bayley PM (2004) Calmodulin bridging of IQ motifs in myosin-V. FEBS Lett567(2–3):166–170.

18. Martin SR, Bayley PM (2002) Regulatory implications of a novel mode of interaction ofcalmodulin with a double IQ-motif target sequence from murine dilute myosin V.Protein Sci 11(12):2909–2923.

19. Post PL, et al. (2002) Myosin-IXb is a single-headed and processive motor. J Biol Chem277(14):11679–11683.

20. Batters C, Zhu H, Sale JE (2010) Visualisation of PCNA monoubiquitination in vivo bysingle pass spectral imaging FRET microscopy. PLoS ONE 5(2):e9008.

21. Ubarretxena-Belandia I, et al. (1999) Outer membrane phospholipase A is dimeric inphospholipid bilayers: A cross-linking and fluorescence resonance energy transferstudy. Biochemistry 38(22):7398–7405.

22. Folch J (1949) Complete fractionation of brain cephalin: Isolation from it of phos-phatidyl serine, phosphatidyl ethanolamine, and diphosphoinositide. J Biol Chem177(2):497–504.

23. Spudich G, et al. (2007) Myosin VI targeting to clathrin-coated structures and di-merization is mediated by binding to Disabled-2 and PtdIns(4,5)P2. Nat Cell Biol 9(2):176–183.

24. Schaap IAT, Eghiaian F, des Georges A, Veigel C (2012) Effect of envelope proteins onthe mechanical properties of influenza virus. J Biol Chem 287(49):41078–41088.

25. Brzeska H, Hwang KJ, Korn ED (2008) Acanthamoeba myosin IC colocalizes withphosphatidylinositol 4,5-bisphosphate at the plasma membrane due to the highconcentration of negative charge. J Biol Chem 283(46):32014–32023.

26. Mazerik JN, Tyska MJ (2012) Myosin-1A targets to microvilli using multiple mem-brane binding motifs in the tail homology 1 (TH1) domain. J Biol Chem 287(16):13104–13115.

27. Brzeska H, Guag J, Remmert K, Chacko S, Korn ED (2010) An experimentally basedcomputer search identifies unstructured membrane-binding sites in proteins: Appli-cation to class I myosins, PAKS, and CARMIL. J Biol Chem 285(8):5738–5747.

28. Teasdale RD, Collins BM (2012) Insights into the PX (phox-homology) domain and SNX(sorting nexin) protein families: Structures, functions and roles in disease. Biochem J441(1):39–59.

29. Kurten RC, et al. (2001) Self-assembly and binding of a sorting nexin to sorting en-dosomes. J Cell Sci 114(Pt 9):1743–1756.

30. Himmel DM, et al. (2002) Crystallographic findings on the internally uncoupled andnear-rigor states of myosin: Further insights into the mechanics of the motor. ProcNatl Acad Sci USA 99(20):12645–12650.

31. Lister I, et al. (2004) A monomeric myosin VI with a large working stroke. EMBO J23(8):1729–1738.

32. Burgess S, et al. (2002) The prepower stroke conformation of myosin V. J Cell Biol159(6):983–991.

33. Veigel C, Schmidt CF (2011) Moving into the cell: Single-molecule studies of molecularmotors in complex environments. Nat Rev Mol Cell Biol 12(3):163–176.

34. Cheney RE, et al. (1993) Brain myosin-V is a two-headed unconventional myosin withmotor activity. Cell 75(1):13–23.

35. Spink BJ, Sivaramakrishnan S, Lipfert J, Doniach S, Spudich JA (2008) Long single al-pha-helical tail domains bridge the gap between structure and function of myosin VI.Nat Struct Mol Biol 15(6):591–597.

36. Hokanson DE, Laakso JM, Lin T, Sept D, Ostap EM (2006) Myo1c binds phosphoino-sitides through a putative pleckstrin homology domain. Mol Biol Cell 17(11):4856–4865.

37. Hokanson DE, Ostap EM (2006) Myo1c binds tightly and specifically to phosphatidy-linositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate. Proc Natl Acad Sci USA103(9):3118–3123.

38. Komaba S, Coluccio LM (2010) Localization of myosin 1b to actin protrusions requiresphosphoinositide binding. J Biol Chem 285(36):27686–27693.

39. Feeser EA, Ignacio CMG, Krendel M, Ostap EM (2010) Myo1e binds anionic phos-pholipids with high affinity. Biochemistry 49(43):9353–9360.

40. Lemmon MA (2003) Phosphoinositide recognition domains. Traffic 4(4):201–213.41. Hoepfner S, et al. (2005) Modulation of receptor recycling and degradation by the

endosomal kinesin KIF16B. Cell 121(3):437–450.42. Ginger ML, et al. (2013) Calmodulin is required for paraflagellar rod assembly and

flagellum-cell body attachment in trypanosomes. Protist 164(4):528–540.43. Philosoph H, Zilberstein D (1989) Regulation of intracellular calcium in promastigotes

of the human protozoan parasite Leishmania donovani. J Biol Chem 264(18):10420–10424.

44. Dowler S, Kular G, Alessi DR (2002) Protein lipid overlay assay. Sci STKE 2002(129):pl6.45. Schuck P (2000) Size-distribution analysis of macromolecules by sedimentation ve-

locity ultracentrifugation and lamm equation modeling. Biophys J 78(3):1606–1619.46. Kron SJ, Toyoshima YY, Uyeda TQP, Spudich JA (1991) Assays for actin sliding

movement over myosin-coated surfaces. Methods Enzymol 196:399–416.

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