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Crystal structure of the motor domain of a class-I myosin

2002; Springer Nature; Volume: 21; Issue: 11 Linguagem: Inglês

10.1093/emboj/21.11.2517

1460-2075

Martin Kollmar,

Trypanosoma species research and implications

Article3 June 2002free access Crystal structure of the motor domain of a class-I myosin Martin Kollmar Martin Kollmar Department of Biophysics, Max Planck Institute for Medical Research, Jahnstraße 29, D-69120 Heidelberg, Germany Search for more papers by this author Ulrike Dürrwang Ulrike Dürrwang Department of Biophysics, Max Planck Institute for Medical Research, Jahnstraße 29, D-69120 Heidelberg, Germany Search for more papers by this author Werner Kliche Werner Kliche Department of Biophysics, Max Planck Institute for Medical Research, Jahnstraße 29, D-69120 Heidelberg, Germany Search for more papers by this author Dietmar J. Manstein Dietmar J. Manstein Department of Biophysics, Max Planck Institute for Medical Research, Jahnstraße 29, D-69120 Heidelberg, Germany Search for more papers by this author F.Jon Kull Corresponding Author F.Jon Kull Present address: Department of Chemistry, Dartmouth College, Hanover, NH, 03755 USA Search for more papers by this author Martin Kollmar Martin Kollmar Department of Biophysics, Max Planck Institute for Medical Research, Jahnstraße 29, D-69120 Heidelberg, Germany Search for more papers by this author Ulrike Dürrwang Ulrike Dürrwang Department of Biophysics, Max Planck Institute for Medical Research, Jahnstraße 29, D-69120 Heidelberg, Germany Search for more papers by this author Werner Kliche Werner Kliche Department of Biophysics, Max Planck Institute for Medical Research, Jahnstraße 29, D-69120 Heidelberg, Germany Search for more papers by this author Dietmar J. Manstein Dietmar J. Manstein Department of Biophysics, Max Planck Institute for Medical Research, Jahnstraße 29, D-69120 Heidelberg, Germany Search for more papers by this author F.Jon Kull Corresponding Author F.Jon Kull Present address: Department of Chemistry, Dartmouth College, Hanover, NH, 03755 USA Search for more papers by this author Author Information Martin Kollmar1, Ulrike Dürrwang1, Werner Kliche1, Dietmar J. Manstein1 and F.Jon Kull 2 1Department of Biophysics, Max Planck Institute for Medical Research, Jahnstraße 29, D-69120 Heidelberg, Germany 2Present address: Department of Chemistry, Dartmouth College, Hanover, NH, 03755 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:2517-2525https://doi.org/10.1093/emboj/21.11.2517 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The crystal structure of the motor domain of Dictyostelium discoideum myosin-IE, a monomeric unconventional myosin, was determined. The crystallographic asymmetric unit contains four independently resolved molecules, highlighting regions that undergo large conformational changes. Differences are particularly pronounced in the actin binding region and the converter domain. The changes in position of the converter domain reflect movements both parallel to and perpendicular to the actin axis. The orientation of the converter domain is ∼30° further up than in other myosin structures, indicating that MyoE can produce a larger power stroke by rotating its lever arm through a larger angle. The role of extended loops near the actin-binding site is discussed in the context of cellular localization. The core regions of the motor domain are similar, and the structure reveals how that core is stabilized in the absence of an N-terminal SH3-like domain. Introduction Myosins are molecular motors that use the energy of ATP hydrolysis to power movement along actin filaments. They power not only muscle contraction, but also function in cell division, cellular locomotion and vesicle transport. Since the first myosin was identified from skeletal muscle tissue, a large number of related myosins and myosin isoforms have been discovered in eukaryotic cells. To date, 17 classes have been identified (Hodge and Cope, 2000), with the myosin-II subfamily forming the largest class. Myosin-IIs have historically been referred to as 'conventional' myosins, with all other classes referred to as 'unconventional' myosins. Among these, the monomeric class-I myosins, which were the first class of unconventional myosins to be identified (Pollard and Korn, 1973), have the most members, followed by class-XI myosins, which are only found in plants, and class-V myosins. Based on the amino acid sequences of the motor domain, the class-I myosins have been grouped into four subclasses (Berg et al., 2001). The role of the myosin-Is in cellular motility has been explored using Dictyostelium as a model system (reviewed in Uyeda and Titus, 1997). This simple organism expresses at least seven different myosin-Is: MyoA–F and MyoK. The myoB, myoC and myoD genes (Jung et al., 1989, 1993; Peterson et al., 1995) have heavy chains of ∼130 kDa. In contrast, the myoA, myoE and myoF genes (Titus et al., 1989, 1995; Urrutia et al., 1993) have shorter heavy chains of ∼115 kDa. The heavy chain of each of the latter myosin-Is can be divided into three distinct domains. The N-terminal region consists of a highly conserved motor domain and is followed by an α-helical light chain binding domain and a C-terminal region, rich in basic residues, that binds directly via electrostatic interactions to membranes in vitro (Adams and Pollard, 1989; Neuhaus and Soldati, 2000). The cellular function of MyoA–D and MyoK has been deduced primarily from double or triple knockout mutants, because the deletion of a single myosin-I gene results in subtle phenotypes. Mutant strains show defects in pseudopod extensions, fluid phase endocytosis, streaming and terminal development. Furthermore, they oversecrete lysosomal enzymes and cannot maintain cortical tension (Novak et al., 1995; Jung et al., 1996; Temesvari et al., 1996; Schwarz et al., 2000). The cellular role of MyoE is still under investigation. In contrast to class-II myosins, which are regulated by phosphorylation of the myosin light chain or by binding of Ca2+, some of the myosin-I ATPases (the amoeboid, yeast and Aspergillus class-I myosins) as well as the ATPases of the class-VI myosins are activated by phosphorylation of the heavy chain at a single site in the cardiomyopathy (CM) loop. The site of phosphorylation, which is in the upper 50 kDa domain and thought to be part of the actomyosin interface, is called the TEDS site (Bement and Mooseker, 1995). Most myosins contain either a glutamate or an aspartate at this position, resulting in a constitutively activated form. In contrast, some of the myosin-Is contain a serine or a threonine at this position, which can be phosphorylated by specific myosin heavy chain kinases in order to regulate activity (Wang et al., 1998). High-resolution crystal structures exist for a number of class-II myosins, which have been crystallized in a variety of nucleotide states (Rayment et al., 1993; Dominguez et al., 1998; Houdusse et al., 1999). All of the crystal structures assume one of three, presumably weak actin binding, conformations: (i) near rigor, in which the nucleotide binding pocket is open and the lever arm is down; (ii) transition, in which the nucleotide pocket is closed and the lever arm is up; or (iii) detached, which is thought to be non-actin binding and in which the lever arm is very far down. In order to understand the structural basis for the differences observed among myosin classes, we have solved the crystal structure of a recombinant motor domain of Dictyostelium myosin-IE. MyoE was crystallized with MgADP·VO4 in the active site, resulting in a closed structure. The overall fold is quite similar to the smooth muscle myosin and Dictyostelium myosin-II motors, except that it lacks the N-terminal SH3-like domain. A docking to the F-actin model (Holmes et al., 1990; Lorenz et al., 1995) shows that the lever arm of MyoE moves ∼30° further up than that of myosin-II, which could result in a larger power stroke. The four crystallographically independent molecules show different movements of the converter domain and surrounding structures about the actin long axis, demonstrating that this region of myosin has additional degrees of freedom beyond those described previously for myosin-IIs. The actin-binding site of MyoE is considerably different from that of myosin-II, with all loops involved in actin binding being different in length and conformation. Results Kinetic characterization The head fragment of MyoE was cloned from genomic DNA into a Dictyostelium expression vector containing a C-terminal His8 tag. The protein was expressed in Dictyostelium cells and purified using ATP-dependent actin binding, Ni affinity and ion-exchange chromatography. In order to analyze the catalytic competence of our construct, we performed transient kinetic and steady-state experiments on the MyoE motor domain. The results summarized in Table I show that binding to nucleotide and actin is similar for MyoE, chicken smooth muscle myosin-II (GgSmS1) and rabbit skeletal muscle myosin-II (OcSkS1). In the presence of actin, the ADP affinity of MyoE is ∼100-fold greater than that of DdMyoII and OcSkS1. As indicated by the ratio of the dissociation equilibrium constants KAD/KD (1.5), coupling between nucleotide binding and actin binding is weak. In this respect, MyoE resembles GgSmS1, for which a KAD/KD ratio of 4.2 has been determined (Cremo and Geeves, 1998). In the presence of saturating amounts of actin, the steady-state ATPase activity of MyoE is activated from a basal level of 0.04/s to 1.3/s (kcat). Additionally, we demonstrated the motile competence of MyoE by generating a construct in which the motor domain was fused to an artificial lever arm consisting of two α-actinin repeats (Anson et al., 1996; Kliche et al., 2001) and showed that it was able to move actin filaments in vitro. The specific kinetic and mechanical properties of this construct, in its phosphorylated and unphosphorylated form, will be described in detail elsewhere (U.Dürrwang, S.Fujita-Becker and D.J.Manstein, manuscript in preparation). Table 1. Transient kinetic analysis Rate constant MyoE DdMyoII GgSmS1a OcSkS1a Nucleotide binding to myosin ATP K1k2 (per μM/s) 1.0 0.6 2.1 2.3 ADP KD (μM) 7.1 14 1.2 2.0 Nucleotide binding to actomyosin ATP K1k+2 (per μM/s) 0.40 0.16 0.47 2.1 ADP KAD (μM) 11 253 5 200 KAD/KD 1.5 18 4.2 100 Actin binding k+A (per μM/s) 2.4 0.8 1.24 6.7 k−A (per s) 0.006 0.002 0.004 0.187 KA (nM) 2.5 2.5 3.5 28.2 KA, KAD and KD are defined as dissociation equilibrium constants. a The data for nucleotide-binding of the chicken smooth muscle (GgSmS1) and the rabbit skeletal muscle myosins were taken from Cremo and Geeves (1998). Values for actin binding were taken from Van Dijk et al. (1999b). Overall structure Purified MyoE was crystallized in complex with MgADP·VO4, and a data set was collected to 3.0 Å (Table II). The structure of the MyoE motor domain was solved by molecular replacement using a polyalanine model of amino acids 80–759 of the closed myosin-II structure in complex with MgADP·BeF3 (F.J.Kull and K.C.Holmes, manuscript in preparation). The search process revealed four molecules in the asymmetric unit. Because some subdomains of all four molecules were in the wrong position, they were deleted and the remaining model was refined using strict non-crystallographic symmetry. After several rounds of model building and refinement, these subdomains were re-included and the structure was refined further, excluding regions that were not visible or differed significantly among the four molecules. Table 2. Data collection, refinement and model quality statistics Data collection Temperature of data collection (K) 100 Crystal space group P21 Unit cell parameters a = 51.82 Å, b = 143.67 Å, c = 236.05 Å; β = 94.86° Resolution range (Å)a 50.0–3.0 (3.2–3.0) Data completeness (%) 79.7 (57.4) Unique reflections 54 968 Data redundancy 3.8 (2.0) Rsym (%)b 19.5 (28.0) I/σ 7.02 (2.87) Model refinement statistics Resolution range (Å) 50.0–3.0 Reflections (work set/test set) 49 376/5579 Number of protein/ligandc atoms 21 069/132 Sigma cutoff None Model quality Rwork (%)d 22.8 Rfree (%)e 27.3 Average B factor (Å2) 57.4 Ramachandran outliers None R.m.s. bond length (Å) 0.008 R.m.s. bond angles (°) 1.376 R.m.s. dihedrals (°) 21.220 R.m.s. improper (°) 0.863 Cross-validated σ coordinate error (Å) 0.57 aValues in parentheses correspond to the highest resolution shell. bRsym = ΣhΣi|I(h) − Ii(h)|/ΣhΣiIi(h), where Ii(h) and I(h) are the ith and mean measurements of the intensity of reflection, h. cThe ligand atoms include four molecules of ADP, four Mg2+ ions and a vanadate group. dRwork = Σh|Fo − Fc|/ΣhFo, where Fo and Fc are the observed and calculated structure factor amplitudes of reflection, h. eRfree is the same as Rwork, but calculated on the ∼9% of the data excluded from refinement. The final MyoE model consists of four molecules in complex with MgADP·VO4, containing a total of 2641 residues and 64 water molecules. The four molecules in the asymmetric unit are grouped around a solvent pore, which extends through the crystal. The overall fold of the structure is similar to that of the conventional myosins (Figure 1A). Superposition of the four crystallographically independent molecules shows that the positions of the converter domain, SH1 helix and the relay-loop/helix differ, while the core structures are very similar, with a root mean squared deviation (r.m.s.d.) of 1.2 Å for 574 common α-carbon atoms (Figure 1B). The converter domains and adjacent relay region in the four molecules vary substantially more, due to a rigid body rotation of ∼10° directed both parallel to and perpendicular to the long axis of the actin filament (Figure 1B). Figure 1.Structure of the motor momain of the class-I myosin MyoE from D.discoideum. (A) Ribbon diagram of MyoE, colored according to structural domains. The N-terminus (green) lacks the SH3-like domain present in conventional myosin, but otherwise has a similar fold. It contains part of the nucleotide-binding site and leads into the upper 50 kDa domain (red). The lower 50 kDa domain (white) contains the main actin-binding motifs, switch-2 and the relay region. The C-terminal domain (cyan) consists of the converter domain, the beginning of the lever arm helix (magenta), and a long α-helix connecting the converter to loop-2 in the actin-binding site. The transition state analog MgADP·VO4 is shown in orange, and loop-1, loop-2 and the strut loop are shown in yellow. (B) Superposition of the four molecules in the asymmetric unit shows good overlay in the core regions. The relay helices, adjacent relay loops and converter domains are in different positions. Colored lines indicate the trajectory of the lever arm helix as it leaves the converter domain for each molecule. (C) Superposition of MyoE and the Dictyostelium myosin II structures shows the differences in the actin-binding loops (MyoE, cyan and yellow; DdMyoII, red and green). All figures were produced using Molscript (Kraulis, 1991) and Raster3d (Merritt and Bacon, 1997). Download figure Download PowerPoint Comparison of MyoE to the myosin II structures The MyoE structure is very similar to that of the Dictyostelium motor domain in complex with MgADP·VO4 (Smith and Rayment, 1996), MgADP·AlF4 (Fisher et al., 1995) or MgADP·BeF3 (F.J.Kull and K.C.Holmes, manuscript in preparation), and to the chicken smooth muscle motor domain/motor domain essential light chain complex with bound MgADP·AlF4 or MgADP·BeF3 (Dominguez et al., 1998). MyoE shares ∼49% sequence identity with DdMyoII. Because the C-terminal regions of the Dictyostelium structures in complex with MgADP·AlF4 or MgADP·VO4 are considerably disordered, our comparison will focus on the closed DdMyoII·MgADP·BeF3 and the SmMD·MgADP·AlF4 structures. These two structures are very similar, with an r.m.s.d. of 1.2 Å for α-carbon atoms in 659 residues. N-terminus Myosin-Is lack the N-terminal SH3-like subdomain found in myosin-II. A functional myosin-II construct lacking the N-terminal 80 amino acids can be expressed in Dictyostelium, but displays greatly reduced stability and motile activity. In contrast, a mutant lacking only the SH3-like domain, but containing the first 15 amino acids shows wild-type-like properties (S.Fujita-Becker, D.J.Manstein and K.Sutoh, manuscript in preparation). Although the sequence of the N-terminus is not conserved among myosin-IIs, the SH3-like domain is held in place by contacts between aromatic residues in the N-terminus and hydrophobic pockets on the core domain. In order to stabilize this region in the absence of the SH3-like domain, MyoE has polar or charged amino acids at positions equivalent to the hydrophobic pockets. Actin-binding region The actin-binding region of myosin consists of a number of structures and loops (Milligan, 1996) including the helix–loop–helix motif of the lower 50 kDa domain, loop-2, the CM loop, loop-3 and loop-4 (Figure 1C). The helix–loop–helix motif forms part of the primary actin binding site, and its structure in MyoE is similar to that observed in conventional myosin. A notable feature of this site is the presence of a number of hydrophobic residues flanked by potentially complementary ionic and polar groups. Loop-2, which has a completely different conformation in MyoE, has been shown to be involved in both weak and the strong binding interactions with actin. It also plays an important role in controlling the rate of product release (Furch et al., 1998; Van Dijk et al., 1999a; Joel et al., 2001). Loop-3, which contains positively charged residues and is involved in the secondary binding actin site (Van Dijk et al., 1999b), is more extended and has a different conformation. Loop-4 is five residues longer in MyoE, and contains many charged amino acids (Figure 1C). The CM loop lies at the front of the motor domain and is important for normal myosin activity. It was shown that a point mutation at position Arg403 in human β-cardiac myosin (the equivalent of Arg397 in Dictyostelium myosin-II) is associated with familial hypertrophic cardiomyopathy (Geisterfer-Lowrance et al., 1990). The CM loop in MyoE is of special interest as it contains a serine at the TEDS-site position (Ser334). Phosphorylation of this serine stimulates actin-activated ATPase and motor activity. In myosin-II, aspartic or glutamic acid residues are found at the equivalent position. Motors without a negative charge at this position display low motility (Wang et al., 1998; De La Cruz et al., 2001; Liu et al., 2001). In the MyoE structure, this loop is disordered and the phosphorylation site is not visible. Based on the purification procedure and kinetic data, it is unlikely that the TEDS-site serine is phosphorylated. Because this loop is well defined in most structures of myosin II motor domains, its disorder in MyoE suggests that phosphorylation may be required in order to constrain its orientation. Nucleotide-binding site The nucleotide-binding site, which consists of the purine-binding loop (NPxxxxxxY), the P-loop (GESGAGKT), switch-1 [AKTxxN(N/D)NSSR] and switch-2 [DI(S/A/Y/F)GFE], is conserved among all myosins. It is therefore not surprising that only minor structural differences are observed in the polypeptide backbone atom positions of MyoE and myosin-II (Figure 2A). Differences, therefore, are restricted to the size and nature of non-conserved residues in the binding motifs. A noticeable difference between conventional and unconventional myosins is the presence of aromatic side chains at position three of the switch-2 motif (Figure 2B). It has been speculated that having a small side chain at this position is important for efficient release of phosphate (Yount et al., 1995). In a mutational study of this region (Murphy et al., 2001), DdSer456 was mutated to leucine, resulting in the ATPase being uncoupled from motility and a shorter step size. Introduction of a larger side chain was predicted to sterically interfere with formation of the closed conformation. However, the kinetic properties and structure of MyoE, which has a tyrosine in this position, indicate this is not the case (Table I; Figure 2B). Figure 2.Comparison of the nucleotide-binding site of MyoE and myosin-II from Dictyostelium and smooth muscle. (A) The nucleotide-binding sites of MyoE (cyan), DdM754 (red) and Gg smII (green) are shown with the MgADP·VO4 from MyoE. (B) Although the sequences of the nucleotide binding regions, including the P-loop, switch-1 and switch-2 are almost entirely conserved, a significant difference occurs in switch-2, where a conserved alanine (red) or serine in amoeboid class-II myosins (green), is replaced by tyrosine (cyan). The introduction of a bulkier side chain pushes a conserved leucine away. (C) Major differences are seen in the length and conformation of loop-1 in MyoE (cyan), DdM754 (red) and Gg smII (green). The green dotted line connects the part of loop-1 that is not visible in the crystal structure of Gg smII. Download figure Download PowerPoint Loop-1 has been implicated in influencing access to the nucleotide-binding site. It has been speculated that the mobility of this loop correlates to the rate of ADP release (Kurzawa-Goertz et al., 1998; Sweeney et al., 1998), and our results lend support to this view. MyoE has a short, conformationally restrained loop and high ADP affinity (Figure 2C; Table I). The higher ADP affinity of MyoE when compared with that of DdMyoII is caused by differences in the rate of ADP release, as nucleotide binding to both types of myosin occurs at similar rates. Relay region and converter domain As its name implies, the relay region functions in communicating conformational information between the actin binding site, nucleotide binding site and converter domain. Major sequence differences between myosin-IIs and myosin-Is in the relay region occur at the point in the relay helix where a kink forms when switch-2 is in the 'closed' position (Figure 3A). Myosin is in the 'closed' state when ATP or transition state analogues are bound, and in the 'open' state when ADP is bound or nucleotide is absent from the binding pocket. In MyoE, this kink is slightly more pronounced due to Asn616 forming a hydrogen bond with Thr418 at the position of the kink of the relay helix, which allows the Cα of Thr418 to move 1.5 Å further into the core of myosin (Figure 3B). Figure 3.Contacts between the relay region, converter domain and lever arm helix allow these structural elements to move together. (A) The relay region and SH1 helix of MyoE are shown in cyan. In MyoE and other class-I myosins, there is a hydrogen bond between Thr418 in the relay helix and Asn618 from the SH1 helix. (B) Close-up view of this region, viewed along the relay helix. The kink forms at Thr418. (C) Highly conserved residues form a hydrophobic core, and polar residues further stabilize the link via conserved hydrogen bonds (dashed lines). This core interaction is further supported by a small, hydrophobic, highly conserved extension into the converter formed by residues Tyr630 and Val677 (DdTyr699 and DdIle744). At the tip of the relay loop (cyan), conserved Glu429 (DdGlu497) forms hydrogen bonds to residue Thr675 of the converter domain (brown) (DdThr742; at this position there is always a threonine or a serine) and to the backbone nitrogen atoms of converter residues Lys674 and Lys676. (D) Hydrophobic interactions between the lever arm helix (cyan) and core domain (white). All class-I myosins contain an aromatic residue at the positions of Tyr69 and/or Tyr71 (red) in close contact with the conserved Phe686 (red) in the lever arm helix. Either a glycine or an alanine is found at the equivalent position to Phe686 in class-II myosins. Download figure Download PowerPoint As the C-terminal end of the relay helix is tightly coupled to the converter domain, this kinking leads to a rotational movement of the converter domain. Highly conserved hydrophobic residues are responsible for the coupling, which occurs primarily in the area between the tip of the relay loop and the β-core of the converter domain (Figure 3C). Hydrophobic contacts between the first turn of the lever arm helix and the N-terminal domain further stabilize the 'up' conformation. In MyoE, contacts are formed between Phe686, Tyr69 and Tyr71 (Figure 3D). Although these residues are completely conserved in myosin-Is, similar hydrophobic contacts are absent in myosin-IIs. Discussion Our results show the core structural elements and topology of MyoE to be essentially identical to those in myosin-II. Given the high sequence conservation between the two motors, this is not unexpected. The differences that are observed are subtle, and occur in loop regions and residues mediating contacts between elements essential for motor function. Spudich has proposed that motor activity of myosin is fine-tuned by changes in the length and flexibility of surface loops (Spudich, 1994), and the MyoE structure certainly lends support to this paradigm. The greatest variation in the loops occurs in the actin binding site and the loop-1 region, and is reflected in differences in the kinetics of actin and nucleotide binding. An important difference between myosin-I and conventional myosin is that myosin-I has its motor activity regulated by phosphorylation at the TEDS site on the CM loop. Kinetics show that phosphorylation stabilizes the actomyosin complex by reducing the koff rate without greatly affecting kon (U.Dürrwang and D.J.Manstein, manuscript in preparation) and consistent effects have been observed after changes in a single negatively charged residue at the actin binding site of myosin-II (Furch et al., 2000). These data indicate that the CM loop is primarily involved in stabilizing electrostatic interactions with actin. The greatest changes in loop length are observed in loop-4 (Figure 1C). Based on actomyosin models derived from electron microscopy, loop-4 is the loop that is furthest removed from the actin surface. Its extended length in myosin-Is suggests it may be involved in either interactions with actin or regulatory proteins that are bound to actin. It has been shown for rat myr-1 that a head fragment localizes to the same actin structures as the full-length construct (Tang and Ostap, 2001). According to this study, the head of myr-1 localizes to the highly dynamic actin structures at the cell cortex and not to the actin filaments that are regulated by tropomyosin. An extended loop-4 might be responsible for this localization due to steric interference with tropomyosin, which is bound to less dynamic actin filaments as they occur in stress fibers. While changes in the loop structures are mainly involved in regulating kinetics and interactions with binding partners, changes in amino acid residues mediating contacts between elements essential for motor function act to fine-tune the mechanical properties of myosin-Is. Examples of this fine-tuning include the more highly kinked relay helix and the hydrophobic interactions between the lever arm and N-terminal domain. These changes result in MyoE being able to undergo a greater lever arm rotation than conventional myosin-IIs. As the overall shapes of the catalytic domains are so similar and image reconstructions of actin filaments decorated with myosin-I in the rigor state showed the geometry of actin binding by the catalytic domain to be virtually indistinguishable from that of myosin-IIs (Jontes et al., 1995, 1998; Jontes and Milligan, 1997; Carragher et al., 1998), we superposed the MyoE structure with a model of myosin S1 attached to an actin filament in order to visualize how MyoE is oriented when bound to an actin filament (Figure 4). We found that the MyoE converter extends ∼30° further up than the converter of myosin-II in the closed conformation. Based on this, we predict that MyoE could produce a power stroke 20–30° greater than the 60–70° rotation of the myosin-II lever arm. Recent single molecule step size measurements support the presence of a larger power stroke in myosin-I, and indicate its lever arm undergoes a rotation of at least 100° during force generation (D.Köhler, C.Ruff, E.Meyhöfer and M.Bähler, manuscript in preparation). Figure 4.A model of chicken skeletal muscle myosin motor core (white), converter domain and lever arm (yellow) in the near-rigor state attached to the actin filament (dark gray). Dictyostelium myosin-II in complex with ADP-BeF3 with a modeled extended lever arm in the 'up' or transition state position is shown in red. The MyoE converter domain and modeled extended lever arm (cyan) is in an ∼30° higher position. Download figure Download PowerPoint The structure also reveals detailed information about the contacts that persist during movement of the converter domain and relay region. Tight coupling of these structural elements is an essential part of the pathway linking small conformational changes in the nucleotide binding site to larger movements of the converter domain and lever arm. As described above, the converter domains and adjacent relay region in the four molecules in the asymmetric unit vary substantially in their orientation due to a rigid body rotation of up to 10°. In contrast, myosin-II crystal structures have indicated only translations along the actin long axis. Thus, the four independent molecules observed in the MyoE crystal visualize three important structural features at atomic resolution: (i) the converter domain and relay regions are rigid