The Actin-Myosin Interface

The actin-myosin interface

Michael Lorenz1 and Kenneth C. Holmes

Biophysics Group, Max-Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany

Edited* by James A. Spudich, Stanford University School of Medicine, Stanford, CA, and approved April 30, 2010 (received for review March 19, 2010)

In order to understand the mechanism of muscle contraction at the Results atomic level, it is necessary to understand how myosin binds to The First Run with Unconstrained Coordinates. As starting coordi- actin in a reversible way. We have used a novel molecular dynamics nates for f-actin we used PDB ID code 2ZWH derived from technique constrained by an EM map of the actin-myosin complex X-ray fiber diffraction data (7). For myosin II we used the crystal- at 13-Å resolution to obtain an atomic model of the strong-binding lographic coordinates 2MYS (8). MD trajectories are often more (rigor) actin-myosin interface. The constraining force resulting from stable with crystallographically derived coordinates. Since the the EM map during the molecular dynamics simulation was suffi- coordinates of f-actin were the output from an MD process, cient to convert the myosin head from the initial weak-binding we replaced each of the two main domains of the actin monomer state to the strong-binding (rigor) state. Our actin-myosin model coordinates with the best overlaid coordinates from 1J6Z for suggests extensive contacts between actin and the myosin head each domain (9) taking over just the DNAse I binding loop (S1). S1 binds to two actin monomers. The contact surface between and C terminus from 2ZWH. This ensures good secondary struc- actin and S1 has increased dramatically compared with previous ture for the starting coordinates. Moreover, the 2MYS coordi- models. A number of loops in S1 and actin are involved in establish- nates needed to be modified by removing the added methyl ing the interface. Our model also suggests how the loop carrying groups from the surface lysines [2MYS crystal structure was the critical Arg 405 Glu mutation in S1 found in a familial cardio- solved with methylated surface lysines (8)] and by adding the myopathy might be functionally involved. missing side chain coordinates to the light chain Cα coordinates (this has no effect on the actin-myosin interface). Initially, the cryoelectron microscopy ∣ myosin II ∣ myosin V atomic coordinates were fitted to the EM density as rigid bodies by least squares. Subsequently an MD trajectory was run for 5 ns. he binding of myosin to actin can be weak or strong. The The 2MYS coordinates of myosin II are in the postpower stroke Taffinity, which changes over 5 orders of magnitude, is con- weak-binding form, whereas the EM density data are derived trolled by ATP binding to the myosin head at a position from the rigor-like strong-binding form (4). Therefore, we antici- remote from the actin binding site. ATP binding produces “weak pated quite big changes on running an MD simulation. binding.” In the absence of ATP the binding is “strong.” The Indeed, under the influence of the strong-binding (rigor) EM mechanism of this interaction and its control by ATP is central density the myosin II coordinates deform to become very much to an understanding of muscle contraction (1). Thus we need like 1W8J of myosin V (10), which is already in the strong-binding to know the structure of the strong-binding state of myosin to form. The actin binding cleft closes and the β-sheet (Fig. 1B) actin in atomic detail. Attempts to crystallize the complex have takes on the more twisted configuration shown by myosin V. been unsuccessful. However, incubating myosin heads (subfrag- In addition, part of loop 2 (see Fig. 1B) initially inserted as ment 1, S1) with filamentous actin (f-actin) produces “decorated random coil was moved into the actin-myosin interface (residues actin” in which each myosin head binds to the actin filament in 633–640 and 643–646). the strong-binding or “rigor” configuration. Decorated actin may The final MDFF calculations were carried out in the absence be used as a model of the actin-myosin “strong-binding” mode. of water because embedding the acto-S1 complex in a water box

Cryoelectron microscopy and 3D reconstructions of actin deco- showed the same overall features such as the closing of the actin BIOPHYSICS AND rated with myosin II have produced a density map at 13-Å reso- binding cleft. We found that calculations in vacuum were suffi- COMPUTATIONAL BIOLOGY lution (2). Combining this with structural data from the head of cient in order to uncover the main structure segments and loops myosin V, which can be crystallized in the strong-binding myosin that participate in the acto-S1 interaction. The map force scaling conformation (3), gave a near-atomic view of the actin-myosin was set to 0.2 in order to avoid it dominating the MD simulation. rigor interaction (4). The actin binding site comprises parts of The final model used five actin monomers to allow the major the upper and lower 50-K domains that are separated by a cleft S1-binding actin monomer (AC3; see Fig. 1A for an explanation) in the weak-binding form. The cleft closes on strong binding. all possible interactions with neighboring actins. The EM density However, the interface appears incomplete: Some additional lo- force was not applied to the lever arm in S1 (residues 795–843), cal refolding of myosin and actin appears to be necessary. We the regulatory light chain and the essential light chain. The results have now used constrained molecular dynamics—molecular dy- are shown in Fig. 2. namics flexible fitting (MDFF) (5) to fit atomic models into The first runs displayed a number of problems including a the interface using the EM density map as a constraint. In MDFF tendency to shift the structure toward the actin helix axis caused the gradient of the density is used as an extra force field. It by a small but significant radial fall off of the EM density. Also appears that, in addition to the elements of the upper and lower the limited resolution of the EM density map resulted in strong 50-K domains recognized in the earlier study, part of the flexible gradients near the edges of the molecule, which caused some loop 2 between the upper and lower 50-K domains in S1 and the actin-DNase I binding loop in actin form essential parts of the – Author contributions: M.L. and K.C.H. designed research; M.L. performed research; M.L. interface. Furthermore, loop 4 in S1 (sequence 364 380) located and K.C.H. analyzed data; and M.L. and K.C.H. wrote the paper. – close to the cardiomyopathy loop (sequence 400 418) and loop 3 The authors declare no conflict of interest. – (sequence 567 577) move in toward the actin binding interface, *This Direct Submission article had a prearranged editor. suggesting contacts with two actin monomers. Similar molecular 1To whom correspondence should be addressed. E-mail: Michael.Lorenz@ dynamics studies were undertaken previously without EM mpimf-heidelberg.mpg.de. restraints (6). However, compared with the previous study our This article contains supporting information online at www.pnas.org/lookup/suppl/ studies indicate about twice the area of interaction. doi:10.1073/pnas.1003604107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1003604107 PNAS ∣ July 13, 2010 ∣ vol. 107 ∣ no. 28 ∣ 12529–12534 Downloaded by guest on October 6, 2021 Fig. 1. (A) Starting structure of the actin-S1 complex with five actin monomers (AC1–AC5) and S1. The col- ors in the S1 structure indicate the upper 50-K domain (red), the lower 50-K domain (orange), the remaining parts including the converter and the SH3-domain and the lever arm (yellow) and the two light chains (brown) for the unconstrained MD runs as described in the text. Also shown is the EM density (gray surface). (B) Important segments and loops of S1, which are often referred to in the text. ELC: essential light chain, RLC: regulatory light chain. Generated with Chimera (11).

unrealistic bending of structures. Moreover, parts of loop 2 were by a 22° rotation of the upper 50-K domain around an axis that pushed into the actin-myosin interface rather indiscriminately. runs approximately along the β-sheet and is roughly at right an- Nevertheless, the final model we obtained showed a good gles to the helix axis. match with the myosin V motor domain structure (10). The over- The N-C-terminal domain may be treated in the same way. lay shown (Fig. 3) was calculated in a highly extensible program Superposition of the N-C-terminal domain from myosin II (yel- for interactive visualization and analysis of molecular structures, low, Fig. S3, SI Text) on myosin V (apple green, Fig. S3, SI Text)is called Chimera (11), based on secondary structure elements, achieved by a rotation of 8.1° around an axis that runs close to showing the converged myosin II structure in blue and the myosin strand 3 of the β-sheet. Because this axis lies close to the domain V structure in red. The actin binding cleft in myosin II closed and boundary, the rotation amounts to an 8.1° bending of the β-sheet. the β-sheet took on the more twisted conformation found in The converter domain carries the long light chain bearing lever myosin V that is thought to represent the strong-binding confor- arm of myosin. The orientation of the converter domain achieved mation of S1 (12). by the process outlined above leads to a positioning of the lever The rmsd between the starting model and the converged mod- arm that is in excellent agreement with the EM density map. el was slightly more than 7 Å after 5 ns of MD simulation, using The two rotations described above produced a model of backbone atoms of five actin monomers and S1 for the RMSD myosin 2 in the strong-binding form (Figs. S4 and S5, SI Text). calculation (Fig. S1, SI Text). Even though the MDFF runs – converged after 1 2 ns, the refinements were extended to over Adding the Missing Loops. Loop 1. Loop 1 in myosin II (199–219) is 5 ns to ensure convergence. disordered as in most myosin structures. The residues from 205– 215 are missing in 2MYS (8) and thus were model built and Modeling Myosin II in the Strong-Binding Form. To counter the pro- inserted as a random coil and were not exposed to the map force blems described in the previous section we first generated starting or harmonic constraints. coordinates of myosin II in the strong-binding form based as far as possible on crystallographically derived coordinates. Then Loop 2. Loop 2, between the upper and lower 50-K domains, is harmonic constraints were applied to areas not close to the inter- disordered in most myosin S1 structures. It is missing in face to prevent unwarranted movement. – Previous studies (12) showed that myosin V without bound 2MYS from residues 627 646. However, initial studies described nucleotide is in the strong-binding form. As described above, above showed that at least some of loop 2 is involved in the actin- unconstrained refinement of myosin II starting in the weak-bind- myosin interface. The resolution of the map is too low to build the ing state transforms into a myosin V strong-binding (rigor-like) missing structure without initial modeling. We have constructed state when exposed to the map force (see Fig. 3). Thus myosin the part of loop 2 that appears to be involved in actin binding by dictyos- V provides a good template for modeling myosin II in its hand using the EM map and crystallographic data from telium strong-binding state. The head of myosin II can be divided into myosin II as a guide. three domains (upper 50-K, lower 50-K, and N-C domain) each Loop 2 is of variable length and is rich in lysine and glycine of which is very similar in structure to the corresponding domain residues. At the C terminus of the loop (647 in skeletal myosin in myosin V. The 50-K upper, 50-K lower, and N-C-terminal II; 635 in myosin V) is an invariant threonine at the N terminus of domain of myosin II weak binding and myosin V strong binding helix W (649–667). The eight residues on the N-terminal side of can be matched separately onto one another with high coincidence the threonine are conserved in myosin II. They are quite different (SI Text). It transpires that two rotational transformations of the in myosin V (loop 2 is also much longer) and indeed in all other individual domains suffice to bring the whole structure of myosin II myosins. The N terminus of loop 2 is marked by an invariant phe- into good coincidence with myosin V. By using these transforma- nylalanine. Dictyostelium discoideum myosin II (Dd) is unusual tions to generate starting coordinates we are able to preserve the because the loop 2 region is short—it has only 16 residues. crystallographic quality of the data. The whole of loop 2 is visible in 2AKA (13) and shows a β-hairpin. For myosin V we used chain A of 1W8J (10). The two mole- The form taken may be indicative of a preferred fold of the poly- cules were brought to similar orientations by overlaying the lower peptide chain. Therefore, we have built an extended polypeptide 50-K domains (Fig. S2, SI Text). Then the upper 50-K domain of chain (working from C to N) toward the actin surface from the myosin II weak binding may be fitted to myosin V strong binding beginning of the W helix into a type-2 β-bend. After the bend an

Fig. 2. (A) The starting structure of the acto-S1 complex as described in the text, embedded in the EM map. (B) The converged acto-S1 complex after 1-ns MD run in MDFF. It can be seen that the upper 50-K domain closes. Both pictures show the main S1-binding actin monomer as surface representation in dark green, and S1 is shown as a ribbon. Only the motor domain and the beginning of the lever arm are shown—color code as in Fig. 1B. The orientation is looking along the helix axis from the + end of decorated actin. Pictures generated with Chimera (11).

12530 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1003604107 Lorenz and Holmes Downloaded by guest on October 6, 2021 The MD runs in MDFF were carried out for 5 ns for models 1 and 2 where the density force was set to 0.1 and for 3 ns for model 3 where the density force was set to 0.2. For all three models we calculated the acto-S1 contact surface and the potential intermolecular H bonds and potential electrostatic interaction. The surface areas are shown in Table 1. S1 not only makes major contacts with the actin monomer (AC3) but also with the monomer (AC1) below the major actin monomer in the actin helix (see Fig. 1A). The measurements of the contact surface area are in good agreement with previous results (19). The solvent accessible surface areas were calculated with visual molecular dynamics (VMD) (20) using the “measure sasa” routine (20). The sasa-parameter “sradius,” which is the additional value in Å added to the atomic radius in order to scan the solvent accessible Fig. 3. Comparison of the converged structure of myosin II in blue and myo- area, was set to 1.4 Å. sin V (chain A of 1W8J) in red shown as secondary structure cartoons. The Furthermore, to regularize the result produced by the uncon- actin binding cleft in myosin II has closed during the refinement and the strained refinement of the DNase I binding loop, the rather β-sheet has become more twisted. Generated with Chimera (11). similar crystallographic coordinates of the loop from the actin: DNase I crystal structure 1ATN (21) were inserted into the actin extended polypeptide runs away from the actin surface toward coordinates. This enhanced the interface with S1 by moving the the invariant phenylalanine that marks the N terminus of loop DNase I binding loop into the S1 contact area as is described in 2. This configuration lies entirely within that part of the EM den- the following section. All three models converged to a similar sity that was empty in earlier fits. With the given build, the Phe result with the same loops and segments being involved in the 646 that is invariant in myosin II interacts with the Pro 602 of the actin-S1 interface. “strut” (14), which is common to all myosin II (see Fig. 4). This The details of the residues involved in contacts between actin may be a stabilizing interaction. Furthermore, on strong binding and S1 varied with the altered loop and harmonic constraint con- of skeletal myosin II to actin, the sequence between Gly 634 and ditions, thereby providing an estimate of the accuracy of the Gln 647 would become an ordered part of the actin binding site, method. The rms deviations of the three models from each other in which case all the trypsin-sensitive lysine residues would be- ranged between 1.0 and 1.1 Å. From the initial MYS2 coordinates come unavailable for tryptic cleavage. This would provide a nice of myosin II used in the unconstrained first runs, the values ran- explanation of the protection of loop 2 from tryptic attack when ged between 4.7 and 4.9 Å (the rmsd values were determined by myosin is bound to actin (15). Murphy and Spudich (16) also selecting the backbone atoms of significant secondary structure found that changes in loop 2 altered the affinity of S1 for actin elements of the 50-K upper and 50-K lower domains, resulting both in the absence and presence of nucleotide. Chaussepied (17) in 165 residues). In the next section we describe the details of proposed that residues 633–642 in S1 are already essential for the model 1 where the nucleotide binding pocket of S1 was preserved weak-binding state of the acto-S1 complex. by applying strong harmonic constraints to the P loop and switch 1 (these already had the geometry of the strong-binding state because myosin II had been matched onto myosin V). Loop 3. Loop 3 was completed by inserting the three missing re- The rmsd of the actin structure from the initial starting coor- sidues 572–574. It is located in the lower 50-K domain of S1 from dinates was 1.3 Å, mainly resulting from the regions that make residues 567–577 and moves toward AC1 (first actin monomer; actin-S1 contacts and actin-actin contacts. The rmsd of the heavy see Fig. 1A) during the MDFF refinement. This possible contact chain of S1 from the initial starting coordinates was 2.6 Å. was already suggested by an earlier EM reconstruction (18).

We refer to myosin II matched onto myosin V and with loops BIOPHYSICS AND The Actin-Myosin Interface of Model 1. The refinement of the acto- rebuilt as described as “myo2-5.” S1 complex using MDFF (5) shows strong interactions not only COMPUTATIONAL BIOLOGY from S1 to AC3 (the major actin binding site) but also from S1 to Refinement of Myo2-5. We used myo2-5 as a starting model for AC1, which increases the contact surface and probably stabilizes several MD runs in MDFF with slightly altered parameters. In the strong-binding state. Fig. 5 shows the binding site of the acto- addition to the secondary structure preserving energy function S1 complex in stereo. described in the Materials and Methods, we added harmonic constraints to several areas of actin and S1. The harmonic constraints Contacts of S1 with AC3. The binding of S1 to AC3 (see Fig. 1A) were applied to the Cα atoms of the designated areas in the 2 shows a stable contact surface with S1 of 1;385 Å as shown in molecules and were slightly altered for the three different runs Table 1. The contact surface indicates several potential electro- in MDFF. The scaling of the density force from the EM map static interactions and H bonds. The interaction between the was also changed. Parameters are shown in Table S1 (SI Text). lower 50-K domain and actin described in ref. 4—helix and bulge

Table 1. Contact surfaces of the actin-myosin models Myosin2-5 Model 1 Model 2 Model 3 Contact surface 945.0 1,385.0 1,429.0 1,295.0 Fig. 4. Loop 2 sequences (after ref. 25). Three selected myosin II sequences 2 AC3-S1 (Å ) and one myosin V are shown: Gg Sk—skeletal myosin II, Dd—Dictyostelium Contact surface 242.0 595.0 587.0 358.0 myosin II, squid myosin II (26), and Gg m5—chicken brain myosin V. The ends 2 AC1-S1 (Å ) of loop 2 are shown in bold type. The C terminus of loop 2 ends in helix W. Total contact 1,187.0 1,980.0 2,016.0 1,653.0 The sequence numbers of the last listed amino acid are shown on the right. 2 surface (Å ) Note that loop 2 in myosin V is considerably longer than in myosin II. Myosin IIs have a conserved phenylalanine (red) not present in myosin V. Loop 2 is Surface areas are listed separately for interactions between S1 and the generally not visible in crystal structures of myosin S1. The exception is Dd, major actin (AC3) and the actin located underneath the major actin (AC1). 2 which is short and ordered in two crystal structures. Surface is given in Å .

Lorenz and Holmes PNAS ∣ July 13, 2010 ∣ vol. 107 ∣ no. 28 ∣ 12531 Downloaded by guest on October 6, 2021 Fig. 5. Stereo view of the contacts of the acto-S1 complex of model 1. AC1 is shown in dark green, AC3 in light green, and S1 in yellow. Loop 2 is highlighted in dark blue, cardiomyopathy loop in red, loop 4 in pink, and loop 3 in orange. The DNase I binding loop in actin is highlighted in cyan. The orientation is approximately at right angle with the actin filament axis. The labels show residue type, residue number, and chain identifier (A for actin, H for S1). Generated with Chimera (11).

528–544 (S1) and segments 349–353 and 146–149 in AC3–is other. Instead we found a H bond from Asn 410 of S1 to Tyr maintained. Potential candidates for H bonds between S1 and 337 in AC3. They also found possible contacts of loop 4 to actin AC3 are mainly found in the loops of S1, namely, loop 2, loop (they called it “C-Loop”). Our results are in broad agreement 4, and the cardiomyopathy loop (see Fig. 1B). Furthermore, with the findings of the earlier MD simulation (6). the loop 541–545 (S1) shows possible H bonds to AC3. Furthermore, Onishi et al. (23) performed mutations of three Fig. 6A shows the major potential H bonds between S1 and regions in heavy meromyosin (HMM) by phosphorylation of re- AC3. In addition, two lysines in the modeled loop 2 show possible sidues 546–548 (hydrophobic region), residues 407 and 409 and interactions from Lys 640 (S1) to Asp 25 (AC3) and from Lys 642 412–414 in the cardiomyopathy loop, and finally residues 652 and (S1) to Glu 334 (AC3). Loop 4 running from 366 to 377 in S1 in 653 in loop 2. They all have significantly lower actin-activated AT- β the form of a -hairpin also shows a number of potential electro- Pase in HMM or even completely extinguish it. We found all B static interactions with residues in AC3. Fig. 6 displays the con- those mutations in the contact surface of acto-S1 with possible tact area of AC3 and S1 with potential electrostatic interactions. contacts to actin residues (see Table 2). The cardiomyopathy loop makes a direct electrostatic interaction via Glu 411 (S1) to Lys 336 (AC3). Other hydrophobic interactions also occur. The critical Arg 405 Gln mutation (identical Contacts of S1 with AC1. S1 also makes significant contacts with AC1 (see Fig. 1A). Milligan (18) suggested that there might be with the Arg 403 Gln mutation in human cardiac myosin), which – occurs in a severe familial hypertrophic cardiomyopathy and re- an interaction between loop 3 (567 577 in S1) and actin. This sults in a decreased actin-activated myosin ATPase (22), is not in was also suggested by Mornet et al. (15) by zero-cross-linking the interface: It faces away from actin. However, it may make a experiments. Furthermore, Blanchoin et al. (24) also favored salt bridge with Glu 631 in the now structured part of loop 2. the binding of S1 to two actin monomers in their kinetic studies. Therefore, its affect on the actin-myosin association is probably The EM map (2) used in the present paper shows the same features via a stabilization of the folded conformation of loop 2. Liu and as the Milligan map: a hole in the map and a “finger” below inter- co-workers (6) found in a MD simulation of the acto-S1 structure acting with actin. Fig. 7 shows model 1 with five actin monomers of Holmes et al. (2) that Lys 415 in S1 forms a stable H bond with and S1 in the color code as in Fig. 1B in a surface representation Glu 334 in AC3. In our refinement we did not find this salt bridge. plus the EM map. The hole in the surface representation matches However, the participating atoms are only 6 Å away from each the surface of the model rather well. We refer to this feature as the

Fig. 6. (A) Possible H bonds between S1 (yellow) and AC3 (green) with loop 4, the cardiomyopathy loop, and at the lower part of loop 2. The position of Arg 405 is shown in cyan as van der Waals surface pointing away from actin (see text). (B) Electrostatic interactions between S1 (yellow) and the major S1 binding actin monomer (AC3) in green. The main interacting partners in S1 come from loop 2, loop 4, and the cardiomyopathy loop (CM-Loop). The labels show residue type, residue number, and segment (A for actin, H for S1). Generated with Chimera (11).

12532 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1003604107 Lorenz and Holmes Downloaded by guest on October 6, 2021 Table 2. Possible electrostatic interactions and H bonds between S1 and AC3 and AC1 (see Fig. 1 for explanation) Potential electrostatic interactions Potential H bonds AC3 – S1 AC1 – S1 AC3 – S1 AC1 – S1 ASP24 – GLU629 LYS50 – GLU576 GLY23 N – LYS637 O LYS50 NZ – ASN552 OD1 ASP25 – LYS640 ARG95 – GLU576 ASP24 N – GLY635 O ARG95 NE – PRO570 O ARG28 – GLU629 GLU99 – LYS569 LYS328 NZ – ARG371 O ARG95 NH1 – ALA575 O ARG147 – GLU373 TYR337 OH – ASN410 OD1 ARG95 NH2 – LYS572 O GLU167 – LYS544 SER348 OG – LYS637 O GLY46 O – LYS553 NZ ASP311 – ARG371 THR351 N – PRO529 O GLY48 O – LYS553 N LYS328 – GLU372 THR351 OG1 – PRO529 O GLN49 OE1 – SER549 OG GLU334 – LYS642 GLY23 O – GLY638 N TYR91 O – ALA571 N ARG335 – GLU372 ARG28 O – ASN410 ND2 LYS336 – GLU411 SER145 O – GLY643 N GLU167 O – LYS544 NZ GLN314 OE1 – ARG371 NH1 ILE329 O – ARG371 NH2 GLU334 O – LYS642 NZ

“Milligan contact.” The model of Liu and collegues (6) could not disordered loop 2 was involved in the actin-myosin interface. The account for the Milligan contact. MDFF method in the unconstrained run showed that the actin The initial MD trajectory moved the DNase I binding loop binding cleft, which is open in myosin II, has to close into a myosin away from the configuration given in 2ZWH to a configuration V-like structure in order to account for the strong-binding state. similar to that found in the crystal structure of the actin-DNase I However, the procedure distorted some regions near the bound- complex 1ATN (21). Replacing the DNase I binding loop in actin aries of the map and a falloff of radial density in the EM map with the original loop from the crystal structure revealed an produced a force pushing all structures 1–2 Å toward the helix axis. interaction of the DNase I binding loop with S1. During the sub- Therefore details of the unconstrained run are uncertain and are sequent MD trajectory the crystal configuration was modified, not further discussed. In order to rectify these deficiencies, we took but it retained the general shape of the DNase I binding loop. as a starting structure a modified myosin 2 modeled on the crystal- It showed strong interactions with S1 particularly through lographic structure of myosin 5. In addition, loop 2 in S1 and the 44–49 (AC1) with S1 543–554 (S1). DNase-binding loop in actin (AC1) were rebuilt as described in the We also found candidate H bonds between AC1 and S1 in the text. For stability, harmonic constraints were applied to selected S1 helix 543–554 to the DNase I binding loop in actin and from areas not involved in the interface. Three MDFF trajectories with loop 3 in S1 to residues Tyr 91 and Arg 95 in actin. Possible H varying starting parameters led to similar structures for the inter- bonds are shown in Fig. 8A. face. The generated interface is extensive and stereochemically Fig. 8B shows the potential electrostatic interactions of AC1 plausible. with S1. Lys 50 located in the DNase I binding loop in actin shows We found that myosin S1 interacts with two adjacent actin a weak interaction with Glu 576 from loop 3 in S1. However, the molecules. In addition to the established interactions between major contacts are established by the Milligan contact from Glu the lower 50-K domain and actin described in ref. 4, most of loop 576 (S1) to Arg 95 (AC1) and Lys 569 (S1) to Glu 99 (AC1). 2 is involved in establishing the association of S1 and AC3. Table 2 shows all potential electrostatic interactions and poten- Furthermore, the cardiomyopathy loop participates in the acto- tial H bonds between AC1 and S1 and AC3 and S1 during the S1 interaction. MDFF refinement. We also found substantial contacts between AC1 and S1. The DNase-binding loop showed strong contacts with S1 residues. BIOPHYSICS AND

Discussion Moreover, the MDFF refinement moved loop 3 toward the actin COMPUTATIONAL BIOLOGY MDFF (5) trajectories run on an actin-myosin complex using the helix axis resulting in substantial support of an additional site EM map at 13-Å resolution (2) as a constraint showed the cleft- predicted by Milligan (18). open weak-binding state (myosin 2 postrigor) changing into a Our results are also in good agreement with the findings by Liu structure very similar to the cleft-closed strong-binding state (myo- and colleagues (6). However, the contacts from loop 2 to actin sin 5 rigor) (Fig. 3). Moreover, the results showed that part of the differ as the part of loop 2 that makes contacts with AC3 in our acto-S1 structure was modeled in a different way to be buried in the actin-S1 contact surface. There is also a discrepancy of the buried solvent accessible surface area. Whereas in our structure 2 the total contact surface of AC1-S1 and AC3-S1 is 1980 Å , Liu 2 and colleagues measure only 463 Å . But they also favor loop 2, loop 4, and the cardiomyopathy loop in S1 as important segments to establish contacts with actin. Their MD simulations could not take account for the Milligan contact as shown in our acto-S1 structure that was driven by the map force during the MDFF refinement. The resulting acto-S1 model was achieved by using harmonic constraints in the regions of actin and S1, which do not contribute to the acto-S1 and also actin-actin interface. The reason was that the resolution is too low to let all of the structure move in the Fig. 7. Surface representation of the model 1 and the EM map at 13-Å resolution (orientation: at right angles to the helix axis). The cutoff level molecular dynamics process using MDFF. However, it seems of the map is chosen in order to show the hole in the density surrounded to be very likely that actin would not behave like a rigid body by regions of AC1 that bind to S1. Color code as in Fig. 1B, ELC in light brown, upon S1 binding, resulting in at least small movements of loops RLC in dark brown. Generated with Chimera (11). and segments in order to enhance the interface. Our actin-S1

Lorenz and Holmes PNAS ∣ July 13, 2010 ∣ vol. 107 ∣ no. 28 ∣ 12533 Downloaded by guest on October 6, 2021 Fig. 8. (A) Potential H-bond contacts between S1 (yellow) and AC1 (green). (B) Potential electrostatic contacts between AC1 (green) and S1 (yellow). The labels show residue type, residue number, and chain identifier (A for actin, H for S1). Generated with Chimera (11).

U model also shows some small movements of actin segments in the and SS the energy function used to preserve the secondary structure interface region. Further details would require higher resolution elements. A scaling factor adjusted by trial and error was applied locally U of the EM map. to EM in order to avoid dominance of the force resulting from the EM Our results reveal additional acto-S1 interactions that provide a density map. plausible model for the strong-binding state of S1 to actin. Since Calculations were carried out both in vacuum and in a water box in which the EM map has a resolution of only 13 Å, which is too low to show the protein complex had been embedded. The box was set up to have per- atomic structure, the details of the present model need to be iodic boundary conditions. The MD runs were carried out until the rmsd refuted or substantiated by additional experiments. Fortunately, between the refined structures and the initial coordinate set reached a stable the additional potential contacts suggest numerous biochemical value. We carried out 12 MD runs in vacuum and 3 MD runs in a water box. experiments. Convergence was generally reached after 1 to 2 ns of simulation depending on the parameter settings. The following parameters were changed during Materials and Methods the refinements: MDFF (5) is a MD simulation with each atom being subjected to an additional • force derived from the gradient of the EM density. The EM density map from The map force scaling, varying between 0.1 and 0.5, Holmes (2) was used to provide the constraining force field. This procedure • simulation using 2 actin monomers or 5 actin monomers, results in atoms being moved into higher density areas of the EM map. In • different start configurations of loop 2, addition, we added another energy term to preserve secondary structure • strength of constraints used to preserve secondary structure elements, elements as an option in MDFF. Thus the total energy function we used is • harmonic constraints—applied to selected areas. U ¼ U þ U þ U ; TOTAL MD EM SS ACKNOWLEDGMENTS. We thank Dr. Elizabeth Villa for her support to set up a preliminary version of MDFF and the fruitful discussions with her on the U U where MD is the MD energy function, EM is the energy function corre- manuscript. We also thank Dr. Anne Houdusse for her valuable comments sponding to a force derived from the gradient of the EM density, on the manuscript.

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12534 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1003604107 Lorenz and Holmes Downloaded by guest on October 6, 2021