Characterization of Blebbistatin Inhibition of Smooth Muscle Myosin

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Characterization of Blebbistatin Inhibition of Smooth Muscle Myosin and Nonmuscle Myosin-2 Hai-Man Zhang, Huan-Hong Ji, Tong Ni, Rong-Na Ma, Aibing Wang, and Xiang-dong Li Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00311 • Publication Date (Web): 17 Jul 2017 Downloaded from http://pubs.acs.org on July 18, 2017

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Characterization of Blebbistatin Inhibition of Smooth Muscle Myosin and Nonmuscle Myosin-2 Hai-Man Zhang1,2, Huan-Hong Ji1, Tong Ni1,2, Rong-Na Ma1,a, Aibing Wang3, and Xiang-dong Li1,2,* 1

Group of Cell Motility and Muscle Contraction, State Key Laboratory of Integrated Management of Pest Insects

and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China, 100101 2

University of Chinese Academy of Sciences, Beijing, China, 100049

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Laboratory of Animal Models & Functional Genomics (LAMFG), Research Center of Reverse Vaccinology

(RCRV), College of Veterinary Medicine, Hunan Agricultural University, Changsha, Hunan, China, 410128 a

Current address: Hongqiao International Institute of Medicine, Shanghai Tongren Hospital, Faculty of Public

Health, Shanghai Jiao Tong University School of Medicine, Shanghai, China, 200025 * To whom correspondence should be addressed: Xiang-dong Li, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China; Tel: +86-10-6480-6015; Email: [email protected] Running title: Blebbistatin inhibition of myosin-2

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Abbreviations: NM2, nonmuscle myosin-2; SmM, smooth muscle myosin; Myo5a-HMM, Myosin-5a HMM; MLCK, myosin light chain kinase; CaM, calmodulin.

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ABSTRACT Blebbistatin is a potent and specific inhibitor of the motor functions of class II myosins, including striated muscle myosin and nonmuscle myosin-2 (NM2). However, the blebbistatin inhibition of NM2c has not been determined and it remains controversial on the efficacy of blebbistatin inhibition of smooth muscle myosin (SmM), which is highly homologous to NM2. To clarify these issues, we analyzed the effects of blebbistatin on the motor activities of the recombinant SmM and three NM2s (NM2a, 2b, and 2c). We found that blebbistatin potently inhibits the actin-activated ATPase activities of SmM and NM2s with following IC50 values: 6.47 µM (SmM), 3.58 µM (NM2a), 2.30 µM (NM2b), and 1.57 µM (NM2c). To identify the blebbistatin-resistant myosin-2 mutant, we performed mutagenesis analysis of the conserved residues in the blebbistatin-binding site of SmM and NM2s. We found that the A456F mutation renders SmM and NM2s to be blebbistatin resistant without greatly altering their motor activities and phosphorylation-dependent regulation, making A456F a useful mutant for investigating the cellular function of NM2s. Key words: ATPase, blebbistatin, in vitro actin-gliding assay, nonmuscle myosin, smooth muscle myosin

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Class II myosins (myosin-2) comprises skeletal, cardiac, smooth, and nonmuscle isoforms, forming the largest subfamily of the myosin superfamily (1-2). Myosin-2 is a hexamer molecule composed of two heavy chains and two pairs of light chains (20-kDa regulatory light chain and 17-kDa essential light chain, referred as RLC and ELC, respectively). The heavy chain contains an N-terminal catalytic motor domain, a light chain binding lever arm and a long coiled-coil rod terminated by a short nonhelical tailpiece. Multiple myosin molecules can polymerize into bipolar filaments via the long coiled-coil rod. Nonmuscle myosin-2s (NM2s) are ubiquitously distributed in eukaryotes, participating in a variety of cellular processes, including cell migration, cell shape changes, cytokinesis, endocytosis and exocytosis (3-5). Vertebrates express three NM2 isoforms, named NM2a, NM2b, and NM2c, which are encoded by three distinct genes in human, i.e., MHY9, MHY10, and MHY14, respectively (4, 6-7). While a few cells express only a single NM2 isoform, most express more than one. Although sharing overall similar structural and biochemical properties, each isoforms of vertebrate NM2 has distinct enzymatic property and cellular distribution (8-11). The biological functions of NM2s have been investigated using a variety of techniques, including gene knockout, siRNA knockdown, and pharmacological inhibitors (4-5). Both gene knockout and siRNA knockdown are very useful in revealing the biological functions of a specific gene. However, both techniques are time-consuming and less powerful in determining the specific function of a protein. On the other hand, the use of pharmacological inhibitor is time-efficient and only blocks the specific function of a protein but the protein is still present. The most widely used pharmacological inhibitor of NM2 is blebbistatin. Blebbistatin is a cell permeable small molecule that specifically inhibits the motor function of myosin-2 but has little effect on other types of myosin (12-13). However, the blebbistatin inhibition of NM2c has not been determined and it remains controversial on the efficacy of blebbistatin inhibition of smooth muscle myosin (SmM), which is highly homologous to NM2. An earlier study, which was performed using chicken gizzard SmM-HMM prepared by chymotrypsin digestion, showed that blebbistatin poorly inhibits the actin-activated ATPase activity of SmM-HMM with an IC50 value ~80 µM, about 16-fold weaker than NM2-HMM (13). However, later studies showed that blebbistatin is a potent inhibitor of SmM. Eddinger et al. (14) found that blebbistatin potently inhibits recombinant rabbit SmM-HMM (IC50 ~3 µM). Wang et al. (15) reported an intermediate value (IC50 ~15 µM) for the blebbistatin inhibition of chicken gizzard SmM-HMM prepared by chymotrypsin digestion. The blebbistatin-binding site is located at the apex of the 50-kDa cleft (16). The residues with which blebbistatin binds are highly conserved among various myosin-2 isoforms, including smooth muscle myosin (SmM) and NM2s. Kinetics analyses show that blebbistatin inhibits myosin ATPase cycle by binding to myosin-ADP-Pi state and inhibiting the phosphate release step (17). Therefore, blebbistatin inhibits myosin in an actin-detached state, thus preventing artifacts arising from the formation of rigid actomyosin cross-linking. This

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property makes blebbistatin highly suitable for studing the cellular functions of NM2. However, blebbistatin cannot distinguish the cellular functions of NM2 isoforms, because both NM2a and NM2b, and likely NM2c, are potently inhibited by blebbistatin (13). This problem can be solved by expressing blebbistatin-resistant NM2 mutants. In the present study, we compared the blebbistatin inhibition of recombinant SmM and three isoforms of NM2. We found that blebbistatin potently inhibits the motor activities of both SmM and NM2c with an IC50 values similar to those of NM2a and NM2b. Moreover, we find that, replacing A456 (a conserved residue in the blebbistatin-binding pocket) with a bulky residue abolishes the blebbistatin inhibition of SmM and NM2s without greatly affecting their motor activity and phosphorylation-dependent regulation.

EXPERIMENTAL PROCEDURES Materials Restriction enzymes and modifying enzymes were purchased from New England Biolabs (Beverly, MA), unless indicated otherwise. Anti-FLAG M2 affinity agarose, trypsin inhibitor (Egg), phosphoenol pyruvate (PEP), 2, 4-Dinitrophenyl-hydrazine, glucose oxidase, N, N-Dimethylformamide (DMF) and pyruvate kinase (PK) were from Sigma Co. (St. Louis, MO). ATP, dithiothreitol (DTT) and dimethylsulfoxide (DMSO) were from Amersco. Blebbistatin (-) was from Sigma Co. (St. Louis, MO) or Selleck (Houston, USA). Catalase was from Worthington Biomedical Co. FLAG peptide (DYKDDDDK) was synthesized by Augct Co. (Beijing, China). Rhodamine-phalloidin were from Invitrogen. Oligonucleotides were synthesized by Sunbiotech Co. (Beijing, China). AccuScript Reverse Transcriptase (Stratagene). Myosin-5a HMM (Myo5a-HMM), myosin light chain kinase (MLCK), calmodulin (CaM), rabbit skeletal muscle, and actin were prepared as described previously (18-20). SmM and NM2 constructs The cDNA of chicken gizzard SmM (Genbank accession number: XM_015294140.1) was obtained from the two cDNAs encoding residues 1-1112 and 1103-1943 of SmM, respectively. The cDNA encoding residues 659-1112 was amplified by PCR using the plasmid of SmM-1105/pFastHFTc (19) as template. The cDNA encoding residues 1103-1943 was obtained by RT-PCR of chicken gizzard total RNA. The cDNA encoding residues 659-1943 was obtained by overlapping PCR using the above two cDNAs as template, and was then subcloned into SmM-1105/pFastHFTc using the sites of EcoRI and XhoI to produce SmM-FL/pFastHFTc. The cDNAs of NM2a-FL (residues 1-1960), NM2b-FL (residues 1-1976), and NM2c-FL (residues 1-2000) were amplified by PCR with the templates GFP-human NMHC II-A, GFP-human NMHC II-B, and mouse

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NMHC IIC-GFP (5, 21), and subcloned into pFastHFTc. The cDNAs of NM2a-HMM (residues 1-1337), NM2b-HMM (residues 1-1231), and NM2c-HMM (residues 1-1365) were PCR-amplified from the corresponding full-length constructs and subcloned into pFastHFTc. The cDNA of the RLC of NM2 (Genbank accession number: BC099425.1) was obtained by RT-PCR of mouse kidney total RNA, and the cDNA of the ELC of NM2 (Genbank accession number: 314122163) was obtained by RT-PCR of human liver total RNA. The resulted cDNAs were subcloned into the baculovirus vector pFastBac (Invitrogen) at the sites of BamHI and XhoI. Site-directed mutations were introduced using standard techniques. All the constructs were confirmed by DNA sequencing. For clarity, the residue numbers of SmM and NM2 refer to Dictyostelium discoideum myosin-2 (Figure 1). The recombinant baculoviruses were prepared using the Bac-to-Bac system (Invitrogen) as described previously (22).

Figure 1. The blebbistatin-binding site in myosin-2 and the conservation of the contact residues in different myosins. (A) Selected residues in the blebbistatin-binding site in the Dictyostelium myosin-2 (PDB ID: 1YV3). (B) Identities of the residues in different myosins corresponding to the residues in the blebbistatin-binding site of Dictyostelium myosin-2. The inhibition constants (IC50) of blebbistatin for various myosins are cited from reference (13) (column A) or obtained from this study (column B). The number in parenthesis represents the number of independent measurements. n.d., not determined.

Expression and purification of SmM and NM 2 SmM-FL and SmM-HMM (Sm-1063) was expressed in sf9 cells and purified with Anti-FLAG M2 affinity chromatography as described previously (19), except that the extract solution and the dialysis solution for

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SmM-FL contained 500 mM KCl. To express NM2-FL and NM2-HMMs, the sf9c cells were co-infected with recombinant baculoviruses encoding the heavy chain, the RLC, and the ELC of NM2. The expressed NM2-FL and NM2-HMM were purified as described for SmM-FL and SmM-HMM, respectively. The purified proteins were aliquoted and quick-frozen in liquid nitrogen and stored at -80°C. The protein concentration of SmM and NM 2 was determined by absorbance at OD280nm. ATPase assay The ATPase activity was measured at 25°C using an ATP regeneration system as described previously (18) with the following modifications. The ATPase activities of SmM and NM2 under phosphorylated conditions were assayed in a solution containing ~0.5 µM myosin head, 30 mM Tris-HCl pH 7.5, 32 mM KCl, 1 mM MgCl2, 0.2 mM CaCl2, 15 µg/ ml MLCK, 1 µM CaM, 1mM DTT, 0.3 mM ATP, 20 units/ ml PK, 2.5 mM PEP, 0-100 µM actin. The ATPase activities of SmM and NM2 under unphosphorylated conditions were assayed in a solution containing ~1 µM myosin head, 30 mM Tris-HCl pH 7.5, 32 mM KCl, 1 mM MgCl2, 1 mM EGTA, 1mM DTT, 0.3 mM ATP, 20 units/ ml PK, 2.5 mM PEP, 0-100 µM actin. The blebbistatin inhibitions on the ATPase activities of SmM-HMM and NM2-HMM were only analyzed under phosphorylated conditions. The ATPase activity of Myo5a-HMM was measured in a solution containing ~20 µM Myo5a-HMM, 20 mM MOPS-KOH, pH 7.0, 100 mM KCl, 1 mM MgCl2, 0.25 mg/ ml BSA, 1 mM EGTA, 1mM DTT, 12 µM CaM, 0.5 mM ATP, 2.5 mM PEP, 20 units/ ml PK, 40 µM actin. In vitro actin-gliding assay The in vitro actin-gliding assays of SmM and NM2a were performed using full-length myosins which were phosphorylated in the flow chamber. About 20 µl 0.4 mg/ ml SmM-FL in Rigor solution (25 mM Imidazole-HCl, pH7.5, 25 mM KCl, 5 mM MgCl2, and 1 mM EGTA) was introduced into a nitrocellulose-coated flow chamber and incubated for 10 min on ice. After blocked with 20 µl 1 mg/ ml BSA in Rigor solution on ice for 5 min, the flow chamber was incubated with 20 µl phosphorylation buffer (5.5 µM CaM, 1.3 µM MLCK, 0.2 mM CaCl2, 5 mM ATP, 1 mM DTT, 5 nM unlabeled F-actin in Rigor solution) at 25°C for 10 min. The flow chamber was washed with 20 µl 1 mg/ ml BSA in Rigor solution to remove the unbound proteins and then incubated with 20 µl 5 nM rhodamine-phalloidin-labeled F-actin in Actin-gliding buffer I (2.5 mg/ ml of glucose, 2 units/ml of catalase, 40 units/ ml of glucose oxidase and indicated concentration of blebbistatin in Rigor solution) on ice for 5 min. The unbound F-actin was washed away with 20 µl Motility buffer I (with indicated concentration of blebbistatin). Before observation, the flow chamber was perfused with 20 µl Motility buffer II (0.5 % methylcellulose, 5 mM ATP, and indicated concentration of blebbistatin in Motility buffer I). The movements of fluorescent actin filaments were recorded under an inverted microscope (Olympus IX7.1) equipped with a home-made temperature control system at 25°C. The velocity of F-actin movement was determined using a house-written Matlab

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(Mathworks, Inc.) program. Blebbistatin was dissolved in DMSO with the final concentration of 5% in the assay solution. The in vitro actin-gliding assay of NM2a-FL was performed similarly as that of SmM-FL except that the assay was performed at 30°C. The in vitro actin-gliding assay of Myo5a-HMM was performed similarly as that of NM2a-FL, except omitting the phosphorylation step and using 20~50 nM Myo5a-HMM.

RESULTS SmM and three NM2 isoforms are potently inhibited by blebbistatin. To determine the specificity and potency of blebbistatin inhibition on the motor functions of SmM and NM2s, we analyzed the effects of blebbistatin on their actin-activated ATPase activities and in vitro actin-gliding activities. The recombinant proteins of full-length and HMM-like versions of SmM and NM2s, including NM2a, NM2b, and NM2c, were expressed in Sf9 insect cells and purified using Anti-FLAG M2 affinity gel. SDS-PAGE showed that the purified SmM and NM2s constructs contain the heavy chain with expected molecular masses, and stoichiometric ELC and RLC (Figure 2A).

Figure 2. The effects of blebbistation on the ATPase activity of SmM-HMM, NM2-HMM, and Myo5a-HMM. (A) SDS-PAGE of purified SmM and NM2 full-length and HMM constructs. MHC, the heavy chain of myosin full-length (FL) or HMM; RLC, regulatory light chain; ELC, essential light chain. (B) Effects of blebbistatin on the ATPase activity of SmM-HMM, NM2-HMM and Myo5a-HMM. The ATPase activities of SmM-HMM and NM2-HMMs were measured in the presence of 100 µM actin under phosphorylated condition. The ATPase activities of Myo5a-HMM were measured in the presence of 40 µM actin. The activity in the absence of blebbistatin were 0.48 s-1 (SmM-HMM), 0.35 s-1 (NM2a-HMM), 0.30 s-1 (NM2b-HMM), 0.31 s-1 (NM2c-HMM), and 3.81 s-1 (Myo5a-HMM). Data sets were fitted to a hyperbolic equation to determine IC50. The IC50 values from multiple assays are reported in Figure 1B.

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We measured the actin-activated ATPase activity of SmM-HMM and NM2-HMM under phosphorylated conditions in the presence of blebbistatin (Figure 2B). The actin-activated ATPase activity of SmM-HMM was strongly inhibited by blebbistatin with the IC50 of 6.47 ± 1.95 µM, which is slightly higher than those of the HMM of NM2s (3.58 ± 0.92 µM for NM2a, 2.30 ± 0.24 µM for NM2b, 1.57 ± 0.09 µM for NM2c) (Figure 1B). We used Myo5a-HMM, a blebbistatin-resistant myosin as a negative control. Consistent with previous report (13), the actin-activated ATPase activity of Myo5a-HMM is not inhibited by blebbistatin. Those results indicate that blebbistatin is an effective inhibitor of SmM and all three isoforms of NM2. We also examined the blebbistatin inhibition on the in vitro actin-gliding activity of SmM-FL. For better quantification of the actin-sliding velocity, we first set up an optimal conditions for in vitro actin-gliding assay of SmM. We found that when phosphorylated by MLCK in the flow chamber rather than in solution, SmM-FL produced the most robust movements of actin filaments. We then measured the in vitro actin-gliding activity of SmM-FL in the presence of blebbistatin. Consistent with the results of ATPase assay, the in vitro actin-gliding activity of SmM-FL is potently inhibited by 100 µM blebbistatin (Figure S1 and 3). Under similar conditions, 100 µM blebbistatin strongly inhibits the in vitro actin-gliding activity of NM2a-FL and NM2b-FL, but not that of Myo5a-HMM (Figure S1 and 3). In case of NM2a-FL, we could not obtained consistent data for its in vitro actin-gliding activity, and we therefore did not examine its sensitivity to blebbistatin. Taken together, our results indicate that blebbistatin is a potent inhibitor of SmM and all three isoforms of NM2.

Figure 3. The effects of blebbistatin on the in vitro actin-gliding activities of SmM, NM2a, NM2b-FL and Myo5a. The in vitro actin-gliding activities of SmM-FL, NM2a, NM2b-FL, and Myo5a-HMM were measured in the absence or presence of 100 µM blebbistatin. The velocity of the moving actin filaments were plotted as a histogram and fitted to a Gaussian curve, defining the average velocity (see Figure S1). These experiments were repeated for three times for each conditions. The data shown are the mean ± std of the average velocities of three independent experiments. Asterisks denote a statistically significant difference (*, P > 0.05; **, 0.05>P>0.01; ***, P200 µM (A456F), and 19.54 ± 4.00 µM (T474A). (B) The ATPase activities of SmM-HMM-WT and -A467F under phosphorylated or unphosphorylated conditions. The data are the mean ± std of 3-4 independent assays.

We next introduced a number of point mutations in the blebbistatin-binding site in SmM. In Dictyostelium myosin-2, blebbistatin binds in a hydrophobic pocket at the apex of the 50-kDa cleft, mainly mediated by hydrophobic interactions. Among the blebbistatin contact residues, Ser456 and Thr474 are highly conserved in myosin-2, but not in other types myosin, including myosin-1, -5, and -10 (Figure 1B). The residue homologous to Ser456 in SmM and NM2 is Ala, which has small side chain, but those in myosin-1, -5, and -10 have bulky

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aromatic side chain, which might sterically prevent blebbistatin from binding. Thr474 is absolute conserved in myosin-2, and the residue homologous to Thr474 in other myosins is Cys (in myosin-1) or Ala (in myosin-5a and -10). The side chain of Thr474 forms hydrogen bond with Y634, which forms strong hydrophobic interaction with blebbistatin (Figure 1A), suggesting that the side chain of Thr474 might be essential for blebbistatin binding. We therefore produced two SmM-HMM mutants, i.e., SmM-HMM-A456F and -T474A, and examined their sensitivity to blebbistatin. While T474A does not greatly affect the blebbistatin inhibition of SmM-HMM, the A456F mutation nearly abolishes the blebbistatin inhibition (Figure 4A). The motor activity of SmM is regulated by the MLCK phosphorylation of RLC. To investigate whether the phosphorylation-dependent regulation SmM is altered by A467F mutation, we measured the actin-activated ATPase activities of the SmM-HMM-A467F under phosphorylated or unphosphorylated conditions (Figure 4B). Similar to the wild-type, SmM-HMM-A467F has low basal and actin-activated ATPase activity under unphosphorylated conditions, and the actin-activated activity is significantly stimulated under phosphorylated conditions. The regulation level (the ratio between the actin-activated ATPase activities under phosphorylated conditions versus unphosphorylated conditions) of SmM-HMM-A467F is similar to the wild-type. These results indicate that A467F mutation renders SmM to be blebbistatin-resistant without altering its phosphorylation-dependent regulation.

The A456F mutatation of NM2 strongly dampenes the blebbistain inhibition. Ala456 is conserved in SmM and NM2s. We expected that, similar to SmM-A456F, the A456F mutants of NM2s are also resistant to blebbistatin inhibition. We introduced the A456F mutation in NM2a-HMM, NM2b-HMM, and NM2c-HMM. Similar to the effects on SmM-HMM, the A456F mutation does not greatly affect the phosphorylation-dependent regulation of NM2-HMMs (Figure 5A). The A456F mutation does not alter the actin-activated ATPase activity of NM2a-HMM (Figure 5B). However, the A456F mutation substantially alters the actin-activated ATPase activity of NM2b-HMM and NM2c. The A456F mutation substantially enhances the actin-activated ATPase activity of NM2b-HMM (Figure 5C). The Vmax and Kactin values of NM2b-HMM-A456F were about twice as those of the wild-type. On the other hand, The A456F mutation clearly decreased the actin-activated ATPase activity of NM2c-HMM (Figure 5D). Noticeably, the basal ATPase activity of NM2c-HMM-A456F was substantially higher than that of the wild-type. Nevertheless, similar to that of SmM-HMM-A456F, the actin-activated ATPase activities of all these NM2-HMM-A456F mutants are only marginally inhibited by blebbistatin (Figure 6).

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Figure 5. Effects of the A456F mutation on the ATPase activities of NM2s. (A) The ATPase activities of NM2-HMM-WT and -A454F under phosphorylated or unphosphorylated conditions. The ATPase assay was conducted in the presence of 80 µM actin. The data are the mean ± std of three independent measurements. (B-D) The actin-activated ATPase activities of NM2s-HMM-WT and -A467F under phosphorylated conditions. Curves are the least-squares fits of the data points based upon the equation: V = (Vmax X [actin])/(Kactin + [actin]) + V0, where Vmax is the maximal activity; Kactin is the apparent dissociation constant for actin, and V0 is the activity in the absence of actin. Note: Since the ATPase activity of NM2c-HMM increased almost linearly without saturation under the assay conditions, i.e., 0 -120 actin, no attempts were made to obtain Vmax and Kactin.

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Figure 6. The ATPase activities of NM2 A456F mutants are resistant to blebbistatin inhibition. The relative ATPase activities of the WT and the A456F mutant of NM2a-HMM (A), NM2b-HMM (B), and NM2c-HMM (C) in the presence of blebbistatin. The ATPase assay was conducted in the presence of 100 µM actin under phosphorylated conditions. The ATPase activities in the absence of blebbistatin were 0.35 s-1 (NM2a-HMM-WT), 0.35 s-1 (NM2a-HMM-A456F), 0.30 s-1 (NM2b-HMM-WT), 0.37 s-1 (NM2b-HMM-A456F), 0.31 s-1 (NM2c-HMM-WT), and 0.20 s-1 (NM2c-HMM-A456F). The data are the mean ± std of 3-4 independent assays.

We then examined the effects of the A456F mutation on the in vitro actin-gliding activities of NM2s and on their sensitivities to blebbistatin inhibition. NM2a-FL-A456F displayed robust in vitro actin-gliding activity with the average velocity slightly higher than the wild-type (Figure S1B and 7A). As expected, in vitro actin-gliding activity of NM2a-FL-A456F was only slightly inhibited by 100 µM blebbistatin (Figure 7A). On the other hand, NM2b-FL-A456F moved actin filaments at a velocity of 141 ± 24 nm/s, about 2.6 times as the wild-type (Figure S1C and 7B). This results is consistent with the ATPase assay that the A456F mutation significantly enhanced the actin-activated ATPase activity of NM2b-HMM (Figure 5C). Similar to the A456F mutants of SmM and NM2a, the in vitro actin-gliding activity of NM2a-FL-A456F was only slightly inhibited by 100 µM blebbistatin (Figure 7B).

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Figure 7. The in vitro actin-gliding activities of NM2-A456F mutants are resistant to blebbistatin inhibition. (A, B) The distributions of in vitro actin-gliding activities of NM2-FL-A456F (A) and NM2b-FL-A456F (B) in the absence or presence of 100 µM blebbistatin. The solid line shows a fit to a single Gaussian curve, defining the average velocity. All data shown are from a single experiment, but is representative of three independent experiments. The data in right panel are the mean ± std of the average velocities of three independent experiments. Asterisks denote a statistically significant difference (*, P > 0.05).

Substitution of bulky residues in A456 renders blebbistatin-resisitence of NM2a-HMM. To clarify the structural basis for the blebbistatin-resistance of the A456F mutant, we produced several additional Ala456 mutants of NM2a-HMM. Similar to the A456F mutation, mutating Ala456 to bulky residues, including Tyr, Trp, Arg, and Glu, greatly weakened the blebbistatin inhibition of NM2a-HMM. Of note is that all those mutations substantially decrease the actin-activated ATPase activity of NM2a-HMM in the absence of blebbistatin. On the other hand, the A456G mutation had little effect on the blebbistatin inhibition of NM2a-HMM (Figure 8A). Those results suggest that introduce of a bulky residue at 456 site sterically hinders the blebbistatin binding in NM2a. Seller and colleagues recently reported that the large side chain of M466 (corresponding to I455 in D. discoideum Myosin-2) is responsible for the blebbistatin resistance of Drosophila myosin-2 (25). They showed

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that the M466I mutation renders Drosophila myosin-2 to be sensitive to blebbistation inhibition. Conversely, we expected that the I455M mutation will render NM2a-HMM to resist blebbistatin inhibition. As expected, NM2a-HMM-I455M is only weakly inhibited by blebbistatin with the IC50 more than 200 µM (Figure 8B). Nevertheless, NM2a-HMM-A456F is more resistant to blebbistatin inhibition than I455M mutant (Figure 8B).

Figure 8. The effects of theA456 mutations on the blebbistatin inhibition of the ATPase activities of NM2a-HMM. (A) The ATPase activities of NM2a-HMM-WT and -A456 mutants in the absence or presence of 200 µM blebbistatin. (B) The effect of I455M mutation on the blebbistatin inhibition of the ATPase activity of NM2a-HMM. The ATPase activities of NM2a-HMM in the absence of blebbistatin were 0.35 s-1 (WT), 0.35 s-1 (A456F), and 0.34 s-1 (I455M). The ATPase assay was conducted in the presence of 100 µM actin under phosphorylated conditions. The data are the mean ± std of three independent measurements.

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DISCUSSION In this paper, we report that blebbistatin potently inhibits the actin-activated ATPase activities of SmM and NM2s with following IC50 values: 6.47 µM (SmM), 3.58 µM (NM2a), 2.30 µM (NM2b), and 1.57 µM (NM2c). These results indicate that blebbistatin can be used for investigating the cellular functions of SmM and all three isoforms of NM2. So far four groups have investigated the blebbistatin inhibition on the ATPase activity of SmM-HMM. Both Sellers group (13) and Kohama group (15) characterized the blebbistatin inhibition using the SmM-HMM prepared from turkey or chicken gizzard SmM by chymotryptic digestion, and reported IC50 values of 79.6 µM and 15 µM, respectively. On the other hand, Ratz group (14) used the recombinant rabbit SmM-HMM, obtaining an IC50 of ~3 µM, and we used the recombinant chicken gizzard SmM-HMM, obtaining an IC50 of 6.59 µM. We reason that the difference in the IC50 values reported in those studies is largely due to different preparation of SmM-HMM. The SmM-HMM prepared by proteolytic cleavage treatment of naturally isolated SmM contains several undesired nicks in the heavy chain. Those undesired nicks in the heavy chain might decrease the blebbistatin sensitivity. The extent of undesired nicks in the heavy chain is dependent on the degree of protease treatment. It is possible that the SmM-HMM samples used by Sellers group (13) and Kohama group (15) contain different amount of SmM-HMM having the undesired nicks in the heavy chain, and therefore they obtained quite different IC50 values for blebbistatin inhibition. NM2 is ubiquitously distributed in almost all eukaryotic cells. The function of NM2 has been studied at cellular and animal levels using genetic manipulation and pharmacological inhibitor blebbistatin. However, vertebrate expresses three NM2 isoforms and all of them are sensitive to blebbistatin inhibition, complicating the in vivo cell biological application of blebbistatin. One solution is engineering a vertebrate cell line or animal expressing blebbistatin-resistant mutant of an NM2 isoform. The blebbistatin-resistant A456F mutant identified in this study is a useful candidate for this purpose. The A456F mutation renders all three NM2 isoforms to resist blebbistatin inhibition without greatly altering their motor activities and phosphorylation-dependent regulation. The motor function of NM2a is practically unchanged by the A456F mutation. On the other hand, the A456F mutation substantially enhances the actin-activated ATPase activity and in vitro actin-gliding activity of NM2b and decreases the actin-activated ATPase activity of NM2c. Nevertheless, both of NM2b-A456F and NM2c-A456F mutants still maintain their motor activities, although at abnormal levels. The different effects of the A456F mutation on different NM2s indicate an isoform-specific effect of this mutation. Therefore, the application of the A456F mutation in other myosin-2 isoforms requires prior characterization. Because all the residues in contact with the side chain of A456, including R238, F239 and I471,

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are absolute conserved among three NM2 isoforms, the isoform-specific effect of the A456F mutation is likely caused by structural difference in other part of the motor domain. It should be emphasized that we have not analyzed the behavior of the NM2s A456F mutant under loaded conditions. Load frequently distinguishes native and mutant myosin function at least in cardiac myosin (26-27) and we expect that NM2 in the cell is sometimes subjected to load during normal functioning. Further experiments are needed to clarify this issue.

ACKNOWLEDGEMENTS The authors thank Dr. Mei Shen for cloning the cDNA of NM2 light chains.

FUNDING INFORMATION This work was supported by the National Basic Research Program of China (2013CB932802) and the National Natural Science Foundation of China (31470791, 31672359) to XdL, and the National Natural Science Foundation of China (31571432) and Hunan Provincial Natural Science Foundation of China (2015JC3097) to AW.

CONFLICT OF INTEREST The authors declare that they have no conflicts of interest with the contents of this article.

AUTHOR CONTRIBUTIONS Hai-Man Zhang is the primary person responsible for performing the experiments of the present study. Huan-Hong Ji and Tong Ni contributed to in vitro actin-gliding assay, Rong-Na Ma and Aibing Wang contributed to create the cDNA constructs. Hai-Man Zhang and Xiang-dong Li designed research, analyzed the data, and wrote the manuscript. All authors reviewed the manuscript.

SUPPORTING INFORMATION The effects of blebbistatin on the in vitro actin-gliding activities of SmM-FL, NM2a-FL, NM2b-FL and Myo5a-HMM (Figure S1).

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For Table of Contents Use Only Characterization of Blebbistatin Inhibition of Smooth Muscle Myosin and Nonmuscle Myosin-2 Hai-Man Zhang1,2, Huan-Hong Ji1, Tong Ni1,2, Rong-Na Ma1,a, Aibing Wang3, and Xiang-dong Li1,2,*

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Characterization of Blebbistatin Inhibition of Smooth Muscle Myosin

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