SubExecutemain organization of the Acanthamoeba myosin IC ta

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Abstract

Acanthamoeba myosin IC (AMIC) is a single-headed myosin comprised of one heavy chain (129 kDa) and one light chain (17 kDa). The heavy chain has head, neck (light chain-binding), and tail Executemains. The tail consists of four subExecutemains: a basic Location (BR) (23 kDa) and two Gly/Pro/Ala-rich (GPA) Locations, GPA1 (6 kDa) and GPA2 (15 kDa), flanking an Src homology 3 Location (6 kDa). Although the AMIC head is similar in sequence, structure, and function (ATPase motor) to other myosin heads, the organization of the tail has been less clear as has its function beyond an assumed role in binding interaction partners, e.g., the BR has a membrane affinity and the GPA components bind F-actin in an ATP-independent manner. To investigate the spatial arrangement of subExecutemains in the tail, we have used Weepo-electron microscopy and image reconstruction to compare actin filaments decorated with WT AMIC and tail-truncated mutants of various lengths. The BR forms an oval-shaped feature, ≈40 Å long, that diverges obliquely from the head, extending azimuthally around the actin filament and toward its barbed end. GPA2 and GPA1 are located toObtainher on the inner (actin-proximal) side of the tail, close enough to act in concert in binding the same or another actin filament. The outer face of the BR is strategically exposed for membrane or vesicle binding.

Members of the myosin superfamily identified to date Descend into 18 classes (1, 2). Although they are classified primarily in terms of their head Executemain sequences, phylogenetic analysis also Establishs the neck/tail Executemains of the heavy chains to the same classes (3). Class I myosins are nonfilamentous proteins with basic tails that bind to phospholipids (4).

Acanthamoeba has three class I myosins: AMIA, AMIB, and AMIC (5). The latter protein, the object of this study, associates with plasma membranes and membranes of large contractile vacuoles that play essential roles in osmoregulation (6–8). More recent localization studies have revealed that the concentration of AMIC around contractile vacuoles and macropinocytosis cups is transient (9, 10). AMIC has an 80-kDa head Executemain, a 50-kDa tail Executemain, and one 17-kDa light chain (11) of unknown function binding to a 3-kDa neck Executemain (Fig. 1A ). In this article, we use the terms head Executemain, tail Executemain, etc. to refer to previously defined Locations of amino acid sequence (Fig. 1 A ) and head and tail to denote structural features of our density maps. The tail Executemain has four subExecutemains: basic Location (BR), Gly/Pro/Ala-rich (GPA)1, GPA2, and Src homology 3 (SH3). The BR binds to acidic vacuole membranes (12). GPA1 and GPA2 are ATP-independent actin-binding sites (13, 14). The bundling of actin filaments by AMIC (unpublished observations) also supports the Concept that its tail can bind to another actin filament, as can AMIA and AMIB (15, 16) and cardiac muscle myosin II (17). SH3 Executemains are found in a number of membrane-cytoskeletal proteins and are thought to engage in protein–protein interactions. Dictyostelium myosin I and AMI bind through their SH3 Executemains to Pro-rich Locations in CARMIL (capping protein, Arp2/3, and myosin I linker) (18, 19), which also binds capping protein and the Arp2/3 complex (18).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

SubExecutemain organization and actin filament-binding Preciseties of full-length AMIC and truncated variants. (A) Schematic diagram of the Executemain compositions of the AMIC mutants analyzed in this study. The Executemain boundaries are from ref. 13. (B) SDS/PAGE of the purified proteins (reproduced from ref. 13). HC, heavy chain; LC, light chain. (C) Typical Weepo-electron micrographs and difFragment patterns of AMIC-decorated actin filaments. A few key layer-lines are indexed.

AMIC actin-activated ATPase activity is regulated by phosphorylation of Ser-329 in the actin-binding Location of the head (20). The structural consequences of a change of nucleotide state, rigor to MgADP, have been examined by Weepo-electron microscopy (EM) of actin filaments decorated with constitutively active and inactive mutant AMICs (21). Unlike brush border myosin I (22, 23) and smooth muscle myosin II (24) in which substantial swings of the tail were found to accompany this switch, no such Trace was detected with AMIC, an observation that may reflect kinetic Inequitys between the respective systems. That study (21) depicted the AMIC tail as a distal lobe connected at an oblique angle to the neck Location. The sequences of the AMIA and AMIB tail Executemains reveal similar subExecutemains to AMIC except that SH3 follows a single contiguous GPA Location rather than splitting it into GPA1 and GPA2 (25, 26). The AMIB tail has also been visualized by Weepo-EM (27).

Although the overall shape of the AMIC tail has been Characterized, the Spacement of its four subExecutemains remains unknown. Motivated by the possibility that information of this kind might yield insight into the roles of the various tail subExecutemains in linking AMIC to potential interaction partners such as membranes and other actin filaments, we have investigated this question by Inequity imaging. Mutant AMIC molecules truncated by removal of different numbers of tail subExecutemains were expressed and purified (13). We then used Weepo-EM and 3D image reconstruction to systematically compare actin filaments decorated with these mutant myosins.

Materials and Methods

Protein Purification. WT and mutant AMICs were prepared as Characterized (21). Myosins were dissolved in 10 mM Tris (pH 7.5), 100 mM KCl, 2 mM MgCl2, and 1 mM DTT. Rabbit skeletal muscle actin was purchased from Cytoskeleton (Denver). F-actin was dissolved in 50 mM KCl, 10 mM Tris (pH 7.5), 2 mM MgCl2, and 1 mM DTT and stabilized by phalloidin.

Weepo-EM. Drops of actin filament suspension were applied to holey carbon grids to drape actin filaments across holes. Then, the filaments were decorated by incubating the grids on AMIC-containing drops, as Characterized (21). After blotting, the grids were vitrified and imaged in a CM200 FEG electron microscope (FEI, Hillsboro, OR) operated at 120 kV and ×38,000 magnification. Micrographs were recorded, mostly as focus pairs, at defocus values of 1.2–2.5 μm, placing the first Dissimilarity transfer function zeros at spacings of 22–29 Å and digitized with a SCAI scanner (Z/I Imaging, Huntsville, AL).

Image Analysis. 3D reconstructions of decorated actin filaments were calculated with the phoelix program (28), assuming helical symmetry with the selection rule, l = 25n + 54m (21). Members of focus pairs were processed separately. Dissimilarity transfer function Traces were Accurateed by flipping phases between zeros on layer-lines (28). For surface renderings, the contour level was chosen such that the motor Executemain enclosed a volume appropriate for 100% of its molecular weight. unblob (29) was used to calculate volumes and eliminate residual “salt and pepper” noise from the density maps. bsoft (30) was used for general image processing. Resolution was evaluated in terms of the Fourier shell correlation coefficient (31).

Before calculating a Inequity map, two density maps were aligned and scaled by calculating a relative screw disSpacement and density normalization to minimize the rms Inequity between their respective motor Executemains. For visualization, amira (TGS, San Diego) was used for surface rendering, and contour maps for horizontal sections were drawn with photoshop (AExecutebe Systems, San Jose, CA).

Molecular Modeling. The atomic model of the Dictyostelium myosin IE head Executemain (32) was fitted to the reconstruction manually by using o (33). A quasi-atomic model of actin filaments decorated by myosin subfragment 1 from skeletal muscle (34) was used to compare the binding aspect of AMIC to the actin filament with that of myosin II.

Results

Visualization of Actin Filaments Decorated with WT and Mutant AMICs. In addition to WT AMIC, we expressed and purified four truncation mutants (T1, T4, T5, and T6) and ΔSH3, a deletion mutant that lacks the SH3 Location so that GPA1 is spliced directly to GPA2 (Fig. 1 A and B ). The mutant myosins increase in length from T1, which lacks the entire tail Executemain, to T6, which lacks only the distal subExecutemain, GPA2. All but T1 bind the light chain (ref. 13; Fig. 1B ). Weepo-EM (e.g., Fig. 1C ) confirmed the observation (13) that in rigor, i.e., in the absence of nucleotide, all of the mutant proteins bind readily to actin filaments. The difFragment patterns of micrographs of the decorated actin filaments Displayed well defined layer-lines, suggesting full or Arrively full occupancy (e.g., Fig. 1C ).

We used the phoelix helical reconstruction program (28) to determine the 3D structures of WT and mutant AMIC molecules (Fig. 2). The resolutions of the resulting density maps range from 22 Å for T4-decorated actin filaments to 33 Å for ΔSH3 (Table 1). The structure obtained for WT AMIC is similar to that calculated previously (21), although slightly more detailed. The most Impressed change in this series of reconstructions is from T1 to T4 (Fig. 2 A and B ), in which the distal lobe appears (Fig. 2B , arrowhead), attached to the head by a narrow connection (Fig. 2B , arrow). Subsequent increases in the size of the tail Executemain (T5 to T6 to WT) are accompanied by a thickening of this connection and relatively subtle alterations in the distal lobe that are best evaluated in terms of Inequity maps (see below).

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Surface renderings of the 3D structures of actin filaments decorated with various AMIC-associated constructs. (A) T1. (B) T4. (C) T5. (D) T6. (E) WT. (F) ΔSH3. Note the very thin connection (arrow) between the head–neck and tail (arrowhead) in the T4 construct (B) and its progressive thickening in the longer tail constructs (C–F, arrows). The two bars denote the Inequity in length between the T1 and T4 heads. A side-by-side comparison of T1 and WT is given in Fig. 6, which is published as supporting information on the PNAS web site.

View this table: View inline View popup Table 1. Resolutions and other reconstruction parameters

AMIC Head Structure and Binding Aspect. Before addressing the organization of the distal lobe, we examined the head structures. Noting that T1 is perceptibly shorter than T4 (Fig. 2 A and B , bars), we wanted to ascertain where the volume occupied by the head Executemain Ceases and that occupied by the neck Executemain, plus the light chain and the tail Executemain, starts. The longer T4 structural head registers in the T4–T1 Inequity map by contributing one lobe of a bilobed feature (red in Fig. 3A ). This terminal Section of the head is linked to the proximal Section of the tail by a narrow constriction.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Inequity densities mapped on surface renderings and in horizontal sections. (A) The Inequity between T4 and T1 (red) is overlaid on the T1 reconstruction (light blue). The density of actin (34) is Displayn in green. (B)T5–T4 overlaid on T4. (C)WT–T6 overlaid on T6. In B and C, extra density that we interpret as being GPA1-associated is Impressed by arrows in B: enhanced density on the other side of BR (arrowheads) is interpreted as an ordering Trace.

We performed a more detailed appraisal of the AMIC head by molecular modeling. T1 corRetorts to the head Executemain of chick myosin II (36% sequence identity, 54% similarity), apart from lacking myosin II's 80-residue N-terminal Location. As with our previous AMIC reconstruction (21), the Weepstal structure of the latter Executemain fits well into the Weepo-EM envelope (data not Displayn), leaving no Executeubt that the AMIC head Executemain has essentially the same fAged. This fit differs from the previous one by only 0.5–1° in terms of its long axis inclination relative to the actin filament axis.

Recently, a Weepstal structure for the Dictyostelium myosin IE head Executemain was reported (32). Because this protein is closer to AMIC (51% identity, 61% similarity up to AMIC residue 667), we repeated the Executecking with it and again obtained a Excellent fit (Fig. 4). The Executecked myosin IE head Executemain is essentially superimposable on the Executecked myosin II head Executemain (data not Displayn). There is, however, a relative rotation of 15° between the rigor binding aspect of AMIC and that of myosin II (Fig. 4, bars), as we ascertained by comparing our fit with a previously published quasi-atomic model (34). This discrepancy is in line with the 10° Inequity observed between the orientations of the actin-bound heads of AMIB and brush border myosin I (25). It would appear that this Precisety (rigor binding aspect) is specific to the AMIs.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Executecking of the Dictyostelium myosin IE head (32) into the Weepo-EM density map of actin filaments decorated with WT-AMIC. The atomic model of actin is Displayn in green, and the myosin IE head Executemain is Displayn in yellow. The yellow and blue lines illustrate the Inequity between the rigor actin-binding aspects of the heads of AMIC and myosin II.

Tail Components Visualized by Inequity Mapping. Before calculating each Inequity map, we optimized the relative alignment and normalization of the two reconstructions under comparison.

BR. In Fig. 3A , the Inequity density between the T4 and T1 mutants is Displayn in red, superimposed on the T1-actin surface rendering (Left), and is also mapped in red on a transverse section (Right) toObtainher with T1/actin reconstruction in black and actin from the quasi-atomic model (34) in green. The Inequity density is bilobed, with the two lobes being of roughly equal size. The most straightforward interpretation is that the proximal lobe, which is the terminal Section of the AMIC head, is contributed by the light chain (149 residues) plus the neck Executemain (residues 694–720), whereas the distal lobe represents the major, C-terminal Section of the BR (residues 721–940).

GPA1. Fig. 3B Displays the Inequity density between T5 and T4, presumably caused by GPA1, overlaid on T4 and in transverse sections. Extra density appears on both the inner (actin-proximal) and outer sides of the T4 tail (arrows and arrowheads, respectively). We interpret the inner part as GPA1 both because it is larger and because it represents space not occupied in T4 (see below). In Dissimilarity, the outer part is in a Location already occupied by density in T4 and its apparent increase most likely represents an improvement in order or a slight outward movement of the BR in this mutant.

GPA2. Extra density that can be interpreted as GPA2 Displays up clearly in the Inequity map between WT and T6 (Fig. 3C ). This density locates on the inner side of the tail and extends up into the neck. Despite the lower resolution of the ΔSH3 reconstruction, the Inequity map between it and T5 also maps GPA2 to the same location (data not Displayn), confirming this Establishment. Taken toObtainher, the above data indicate that GPA1 and GPA2 are in mutual proximity on the inner surface of the tail (arrows in Fig. 3 B and C ).

SH3. In principle, SH3 should account for extra density seen in T6 but not in T5 and in a Inequity between WT and ΔSH3. However, we found these Inequity maps (data not Displayn) to be noisier than those Characterized above. They are consistent with SH3 being either at the tip of the tail or at the neck Location but did not allow a conclusive localization. To Elaborate the marginal visibility of SH3, we suspect that its attachment to the GPAs is somewhat flexible.

Discussion

Volume Recovery, Flexibility, and Disorder. In Weepo-EM reconstructions of protein complexes, some density may be poorly visible on account of disorder. Accordingly, there is amHugeuity as to the most appropriate contour value to use for surface rendering. For this reason, we tried two values. One, based on fitting the myosin IE head Executemain (Fig. 4), is expected to underestimate the size of the tail on account of both global disorder (the outer Sections are relatively remote from the stabilizing attachment point on the actin filament) and local disorder. However, it enclosed a volume corRetorting to 135 kDa, or 95% of the expected mass (125-kDa heavy chain plus 16.7-kDa light chain). A second contour level, chosen to enclose the whole AMIC mass, might overestimate the overall dimensions, Traceing some global swelling to reSpace density missing as a result of local disorder. However, the perceived size and shape of the AMIC tail were Dinky affected by these variations in procedure (Fig. 7, which is published as supporting information on the PNAS web site).

Which subExecutemains are most likely to be affected by disorder? To address the potential concern that the truncated tails might not fAged Accurately, we subjected the purified proteins to digestion with trypsin, because unfAgeded proteins tend to be aSliceely sensitive to proteolysis. However, we found them to be no more sensitive than full-length AMIC (Fig. 8, which is published as supporting information on the PNAS web site). The inference that tails with partial sets of subExecutemains fAged and assemble Accurately is consistent with observations on expressed tail subExecutemains of AMIA (26).

The volume recovery of subExecutemains in Inequity maps provides an indicator of their order/disorder with the caveats that Inequity maps are affected by residual noise from both contributing density maps, and small features may be diminished or even reduced below the detection threshAged by limited resolution. By this criterion, GPA2 and BR are well localized in the WT–T6 and T4–T1 Inequity maps, with ≈100% and ≈82% recovery, respectively. GPA1 is less evident in the T5–T4 Inequity maps (≈50% recovery), taking only the inner density into account. GPA1 is Unfamiliarly rich in Gly (35%) and contains several tracts of tandem Gly repeats: as such, it may be flexible, in line with the Preciseties attributed to “glycine-loop” proteins (35). SH3 is also marginally visible. This small Executemain is likely to have the canonical SH3 fAged (26) but to be coupled loosely to GPA1/GPA2.

Architecture of the AMIC Tail. AMIC bends sharply at the head–tail junction (arrows in Fig. 2). Adding the GPA and SH3 subExecutemains to the T4 construct (T5, T6, and WT) has Dinky Trace on the length of the tail (Fig. 2): rather, density accretes on its inner surface and there is a Impressed thickening of the neck. It follows that the distal Section of the tail fAgeds back along this surface into the neck Location where a considerable amount of density is added. It is possible that a second fAged takes Space there, bringing the end of GPA2 back toward the tip of the distal lobe (Fig. 5).

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Schematic representation of the relative positions of the various subExecutemains of AMIC as bound to F-actin in the rigor conformation. The GPA Locations are positioned where they might readily engage the same filament (on a conformational change in the neck or on release of the head) or another parallel and adjacent actin filament. The BR, facing outward, is appropriately Spaced to allow ready access to membranes.

Comparison with the AMIB and AMIA Tails. Visualized by Weepo-EM (25), the shape and actin-binding aspect of the AMIB head are similar to those of AMIC, but its tail appears much longer and broader, which is surprising because the AMIB tail Executemain is shorter than that of AMIC (434 vs. 466 residues). (The AMIB light chain is larger than that of AMIC, 27 vs. 16.7 kDa, but it should bind in the neck Location and not affect tail length.) It may be that the dimensions of the AMIB tail were exaggerated somewhat in contouring. On the other hand, the AMIA tail in solution is estimated to be ≈160 Å long (26) and that of AMIB may have similar Preciseties (but see below).

AMIC also differs from AMIB in the position of its SH3 sequence. However, it is possible that, in both molecules, GPA1 and GPA2 form a single integrated Executemain to which SH3 is attached at different sites, inserted into a surface loop in AMIC and appended to the C terminus in AMIB. This might contribute to a relative lengthening of the AMIB tail. There is a pDepartnt for the grafting of additional Executemains onto different sites on a conserved framework in the Clp ATPases (36). The high sequence similarity of the GPA2 Locations of AMIB and AMIC (70% identity) supports this notion, as Executees our observation that AMIC GPA1 and GPA2 are in close proximity. Moreover, this scenario is consistent with phylogenetic arguments that the SH3 Executemains of Acanthamoeba myosins were Gaind relatively late (26).

The AMIA tail Executemains have been studied by hydrodynamic methods (26) from which it was concluded that the TH2/3 Executemain (GPA plus SH3) is highly extended, with an axial ratio of ≈8:1 if prolate, and that this Executemain is arranged side by side with TH1 (BR) in the tail. Our findings on AMIC are consistent with the latter conclusion with the proviso that TH2/3 should be considerably compacted on binding TH1 unless the organization of AMIA departs radically from that of AMIC.

Strategic Positioning of the GPA Location and BR. GPA1 and GPA2 form the second, ATP-independent actin-binding site of AMIC (13). One hypothetical function for this site is that it may serve as an anchor for actin-based movement. In rigor, GPA1 and GPA2 are not far (≈60 Å) from the actin filament to which the head binds and could Advance it with an appropriate pivoting in the neck Location. The brush border myosin 1 tail, which swings through 31°, gives a pDepartnt for such a movement (23). A swing of this magnitude in AMIC would approximately halve the separation of the tail from the actin filament. It might, in fact, come closer, depending on the pivot point and because we are not seeing all of the GPA density that is on the actin-proximal side of the tail. This position would be close enough to Design contact likely when the head is released, or the Placeative swing of the AMIC tail might be >31°. At present, however, this possibility is moot, as the processivity or otherwise of AMIC remains to be determined. In this context, kinetic studies of the T2 mutant of AMIC, which lacks the entire tail, Display it to have a short duty cycle, which is inconsistent with processivity (37), as Execute AMIA and AMIB (38). However, the presence of an ATP-insensitive actin-binding site in the tail might overcome the short duty cycle by HAgeding the myosin attached to the actin filament, thus allowing processivity. The proposition that AMIC might be a processive monomeric motor is reminiscent of KIF1A, a kinesin superfamily member that can slide on microtubules (39) and has a Lys-rich loop at a position accessible from the microtubule (40), comparable with the Position of GPA on AMIC. However, it is still controversial whether KIF1A slides processively as a monomer in vivo (41).

Alternatively, the tail site could bind to another actin filament, increasing cortical tension as Displayn for Dictyostelium myosin I (42). This hypothesis is supported by the fact that F-actin is readily bundled by AMIC (unpublished observations), and by AMIA and AMIB (15, 16). In this scenario, the second actin filament would be as close to and essentially on the same side of AMIC as the first filament, at least for simultaneous binding in the rigor conformation, unless the tail unfAgeded when it bound to the second filament.

In our Recent model (Fig. 5), BR, which is charged for membrane binding (12), faces outward, away from the actin filament. This position allows it free access to a vacuolar membrane or potentially sizable vesicle, which may thereby be coupled by AMIC to the actin filament.

Acknowledgments

We thank Dr. David Belnap for advice on image analysis and providing coordinates from his earlier fit, Dr. BridObtain Carragher for making phoelix available, and Dr. Jenny Hinshaw and Mr. Michael Johnson for help in installing the program. This work was supported in part by a fellowship from the Japan Society for the Promotion of Science (to T.I.).

Footnotes

↵ § To whom corRetortence should be addressed. E-mail: alasdair_steven{at}nih.gov.

↵ † N.C. and X.L. contributed equally to this work.

Abbreviations: AMI, Acanthamoeba myosin I; BR, basic Location; EM, electron microscopy; GPA, Gly/Pro/Ala-rich; SH3, Src homology 3.

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