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To whom correspondence should be addressed: Department of Cellular Physiology and CeNS, Ludwig-Maximilians-University of Munich, Schillerstrasse 44, 80336 Munich, Germany. Tel.: 49-89-2180-75511; Fax: 49-89-2180-75512
* This work was funded by the Deutsche Forschungsgemeinschaft SFB 863, by the Friedrich-Baur Stiftung, and by the Munich Centre for Nanoscience (CeNS). This work was also supported by an EMBO long-term fellowship (to C. P. T.).
The genome of the Leishmania parasite contains two classes of myosin. Myosin-XXI, seemingly the only myosin isoform expressed in the protozoan parasite, has been detected in both the promastigote and amastigote stages of the Leishmania life cycle. It has been suggested to perform a variety of functions, including roles in membrane anchorage, but also long-range directed movements of cargo. However, nothing is known about the biochemical or mechanical properties of this motor. Here we designed and expressed various myosin-XXI constructs using a baculovirus expression system. Both full-length (amino acids 1–1051) and minimal motor domain constructs (amino acids 1–800) featured actin-activated ATPase activity. Myosin-XXI was soluble when expressed either with or without calmodulin. In the presence of calcium (pCa 4.1) the full-length motor could bind a single calmodulin at its neck domain (probably amino acids 809–823). Calmodulin binding was required for motility but not for ATPase activity. Once bound, calmodulin remained stably attached independent of calcium concentration (pCa 3–7). In gliding filament assays, myosin-XXI moved actin filaments at ∼15 nm/s, insensitive to both salt (25–1000 mm KCl) and calcium concentrations (pCa 3–7). Calmodulin binding to the neck domain might be involved in regulating the motility of the myosin-XXI motor for its various cellular functions in the different stages of the Leishmania parasite life cycle.
Leishmaniasis affects over 12 million people worldwide, with about 2 million newly infected patients each year. The disease is caused by the flagellated protozoan parasite Leishmania and can manifest itself as visceral leishmaniasis, which is potentially fatal, or cutaneous leishmaniasis, which can leave disfiguring mucocutaneous scars (
) showed that myosin-XXI is essential for survival of Leishmania promastigotes in culture and that a reduction in expression levels of myosin-XXI results in the loss of endocytosis within the flagellar pocket and impairment of other intracellular trafficking processes. In addition, myosin-XXI heterozygous cells failed to form the paraflagellar rod. The paraflagellar rod is a structure that runs along the length of the flagellum and contains a variety of proteins, including actin, but its functional role remains unclear (
). The detection of only a single myosin isoform in the parasite suggests that this myosin must carry out a variety of functions. Two distinct myosin-XXI populations have been identified. For the membrane-bound population, the tail domain localizes the motor molecules at the base of the flagellum, whereas the detergent-soluble subfraction could be involved in the transport of proteins within the flagellum (
). Myosins consist of a highly conserved motor domain followed by a neck domain of variable length, often including IQ motifs for the binding of light chains of the calmodulin family, and finally a tail domain, which can contain a large variety of motifs (
). Although the motor domain is responsible for the binding to actin and hydrolysis of ATP, it is the tail domain that determines function within the cell by controlling molecular dimerization and motor processivity, motor anchoring to the membrane, and/or selection and transport of specific cargo.
Although myosin-XXI does not contain perfect IQ motifs in the neck domain, there are several less well characterized, degenerative IQ domains present. Subsequent to the converter domain, the proximal tail contains a natural “leucine zipper” motif that is followed by a predicted short coiled-coil domain (MARCOIL) (
(Fig. 1A). UBAs are found in a variety of proteins, including members of the ubiquitination pathway, UV excision repair proteins, and certain protein kinases. However, their function is not entirely clear (
). Within the myosin family, UBAs are unique to myosin-XXI, and their role is unknown.
Controlling expression levels is one mechanism for regulating where and when a myosin is used. However, in a simplified system with only two myosins present in the genome, there clearly must be other mechanisms that allow myosin-XXI to perform its different roles within the cell. The presence of two myosin heavy chain kinases (NCBI accession numbers CBZ38915.1 and CBZ34699.1) and calmodulin in the L. donovani genome suggests other possible regulatory mechanisms.
In this study, we expressed Leishmania full-length myosin-XXI and a truncated minimal motor domain in an Sf21/baculoviral system for biochemical and biophysical analysis. We show that myosin-XXI is an actin-activated ATPase that binds a single calmodulin that is required for motility but not for ATPase activity. EM imaging shows a monomeric molecule that seems to bind cooperatively to actin filament ends.
We have successfully expressed full-length myosin-XXI as well as a truncated motor domain construct to investigate the structural and chemomechanical properties of this novel myosin motor found in Leishmania parasites. Sequence analysis of the protein suggested 16 potential calmodulin binding motifs on the myosin heavy chain. However, the stoichiometry between myosin-XXI heavy chain and bound light chains, combined with the calmodulin-binding studies to different tail fragments, indicated that myosin-XXI binds only a single calmodulin to a calmodulin-binding motif at aa 809–823 in the heavy chain sequence.
Comparing the sequence of calmodulin and calmodulin-like proteins in the L. donovani genome, we found a high similarity to Xenopus calmodulin (Fig. 4C), which is why we used Xenopus calmodulin for coexpression with the Leishmania myosin-XXI heavy chain. In addition, Leishmania calmodulin has only a small number of differences (12 amino acids) compared with Drosophila calmodulin, which has been shown to change conformation upon calcium-binding, to expose the hydrophobic patches required for typical target binding (
). The sequences of Drosophila and Xenopus calmodulin, on the other hand, are essentially identical. They differ only in two amino acids (aa 144 and 148). Furthermore, many of the differences in the calmodulin sequence between Leishmania on the one hand and Drosophila or Xenopus on the other are conservative (e.g. isoleucine for valine, Fig. 4C). The calcium-binding EF-hand domains in the Leishmania sequence (aa 21–68 and aa 94–141) and in the Drosophila or Xenopus sequences are almost identical (Fig. 4C, green sections). None of the very small differences in sequence (five amino acids) in these sections are expected to affect the ability of the protein to bind calcium. This is entirely consistent with our finding that Xenopus calmodulin was found to bind to Leishmania myosin heavy chain in a calcium-dependent manner but would not detach from the target sequence once bound to it, neither by changes in ionic strength nor by changes in calcium concentration (
). The target sequence (aa 809–823) on the myosin heavy chain that we identified for calmodulin binding is somewhat unusual in the sense that it contains a lot of negative charge. It is also interesting to note that this calmodulin binding motif is not the strongest motif predicted from sequence alignments (Fig. 4A). However, it does have bulky hydrophobic residues at the appropriate positions, such as positions 1–4, 10–11, and 14, as shown in Fig. 4A. It is therefore expected that this sequence will bind calmodulin and that calmodulin-binding would be calcium-dependent, as described in the literature (
In the sequence, the calmodulin-binding motif is preceded by a section identified as a natural leucine zipper motif (Fig. 4B), indicating the possibility of dimerization at this part of the structure. However, the size exclusion studies suggested that, at least in the conditions applied in this study, myosin-XXI as well as the tail fragments were monomeric. Closer inspection of the sequence between the end of the converter (aa 750) and the calmodulin-binding motif at aa 809–823, revealed that this section would also be consistent with an SAH domain (
). In the in vitro motility assays, monomeric full-length myosin-XXI with calmodulin bound was mechanically functional and able to move actin filaments. This is consistent with a mechanically stable domain (such as a SAH domain perhaps) bridging the gap between the end of the converter and the calmodulin-binding lever arm region of the motor molecule. If that bridging section were flexible, it would be hard to envisage how conformational changes in the catalytic domain could be transduced into productive movement at the end of the lever arm. On the other hand, however, the motor truncated at the end of the predicted SAH domain showed nearly unchanged ATPase activity compared with the full-length molecule but was not mechanically functional. This would suggest that the predicted SAH domain on its own (that means in the absence of the following calmodulin-binding region) is unable to form a mechanically functional lever arm and that both the predicted SAH domain and the subsequent calmodulin-binding region are necessary to form a functional lever arm structure. However, at this stage we cannot exclude the possibility that the truncated motor simply bound to the nitrocellulose surface in an unfavorable way so that lever arm movement was impaired for that reason. High-resolution structural studies are currently underway to characterize these domains of the myosin molecule in detail.
The SEC experiments and EM studies suggested that, at least in the experimental conditions here, myosin-XXI is monomeric and has a compact structure with a Stokes radius comparable with muscle myosin-II S1, which has a similar molecular weight. The Stokes radius of myosin-XXI was slightly larger when calmodulin was bound, consistent with calmodulin stabilizing the extended α-helical target sequence that forms a functional lever arm in other myosins (
). The EM studies also suggested that myosin-XXI does bind along the length of actin filaments but tends to accumulate at the filament ends (Fig. 8B). Further experiments are underway to investigate this observation in more detail. In the absence of actin, myosin-XXI frequently seemed to form compact, ring-like structures possibly composed of more than one myosin-XXI molecule. Again, further studies are currently underway to characterize the conditions for ring formation in more detail.
The full-length and truncated Leishmania myosin constructs showed similar actin-activated ATPase activity. We therefore conclude that calmodulin binding and the absence of the tail in the truncation has little effect on the ATPase activity. The rates were similar to smooth muscle myosin with phosphorylated regulatory light chain (
). Interestingly, the sliding velocity was 50-fold lower compared with smooth muscle myosin. This could be due to unfavorable binding of myosin-XXI, which was adsorbed non-specifically to the nitrocellulose surface. However, under the same binding conditions, 20- to 200-fold higher sliding velocities were observed for smooth muscle myosin S1 (
), respectively. If not because of unfavorable binding, the low mechanical speed could also point toward a function as an anchor or tether for this monomeric form of Leishmania myosin-XXI that does not require high speed of movement. Further studies are in progress to immobilize full-length and truncated myosin-XXI motor molecules in a more specific fashion on the surface to address this issue.
For both the solution kinetics studies and in vitro motility assays, we used actin tissue-purified from rabbit skeletal muscle. This can be obtained more readily than the Leishmania-specific actin, which would have to be recombinantly expressed. Intriguingly, Leishmania actin and rabbit skeletal actin share only about 70% sequence identity and 87% similarity as determined by multiple sequence alignment (Macvector). However, residues on the surface of actin monomers assumed to be involved in myosin binding (
) are highly conserved (Fig. 7D). This suggests that rabbit skeletal actin might interact with myosin-XXI in a similar fashion compared with endogenous Leishmania actin. In addition this approach enables us to compare the functional assays on myosin-XXI to other unconventional myosins from different species, which have also been carried out with actin purified from rabbit skeletal muscle.
To summarize, we found that myosin-XXI, to date the only myosin motor protein detected in Leishmania parasites, can be expressed recombinantly using a baculovirus expression system. Under our conditions, this myosin is a monomeric, mechanically functional molecular motor that binds a single calmodulin and moves actin filaments at a very low speed. This would be consistent with the motor acting, at least in its monomeric form, as a tether or anchor in the parasite, possibly involved in endocytic processes within the flagellar pocket, possibly also in other intracellular trafficking processes and in the formation of the paraflagellar rod structure (
). Intriguingly, sequence analysis indicates several coiled-coil sections in the tail domain of this myosin, not to mention the ubiquitin binding domains the regulatory functions of which remain completely unclear. It is therefore possible that in different conditions myosin-XXI might well dimerize or even oligomerize to perform the variety of functions it is expected to be involved in, as it seems to be the only myosin motor expressed in the Leishmania parasite.
We thank Joachim Rädler at the Physics Department of Ludwig-Maximilians-Universität, Munich, Germany, for letting us use TEM for this study and Steve Martin at The National Institute for Medical Research London, UK, for scientific discussions.
Leishmaniasis. Current situation and new perspectives.