Muscle myosins form folded monomers, dimers, and tetramers during filament polymerization in vitro.

Muscle contraction depends on the cyclical interaction of myosin and actin filaments. Therefore, it is important to understand the mechanisms of polymerization and depolymerization of muscle myosins. Muscle myosin 2 monomers exist in two states: one with a folded tail that interacts with the heads (10S) and one with an unfolded tail (6S). It has been thought that only unfolded monomers assemble into bipolar and side-polar (smooth muscle myosin) filaments. We now show by electron microscopy that, after 4 s of polymerization in vitro in both the presence (smooth muscle myosin) and absence of ATP, skeletal, cardiac, and smooth muscle myosins form tail-folded monomers without tail-head interaction, tail-folded antiparallel dimers, tail-folded antiparallel tetramers, unfolded bipolar tetramers, and small filaments. After 4 h, the myosins form thick bipolar and, for smooth muscle myosin, side-polar filaments. Nonphosphorylated smooth muscle myosin polymerizes in the presence of ATP but with a higher critical concentration than in the absence of ATP and forms only bipolar filaments with bare zones. Partial depolymerization in vitro of nonphosphorylated smooth muscle myosin filaments by the addition of MgATP is the reverse of polymerization.

The central role of the tail in switching off 10S myosin II activity.

Myosin II is a motor protein with two heads and an extended tail that plays an essential role in cell motility. Its active form is a polymer (myosin filament) that pulls on actin to generate motion. Its inactive form is a monomer with a compact structure (10S sedimentation coefficient), in which the tail is folded and the two heads interact with each other, inhibiting activity. This conformation is thought to function in cells as an energy-conserving form of the molecule suitable for storage as well as transport to sites of filament assembly. The mechanism of inhibition of the compact molecule is not fully understood. We have performed a 3-D reconstruction of negatively stained 10S myosin from smooth muscle in the inhibited state using single-particle analysis. The reconstruction reveals multiple interactions between the tail and the two heads that appear to trap ATP hydrolysis products, block actin binding, hinder head phosphorylation, and prevent filament formation. Blocking these essential features of myosin function could explain the high degree of inhibition of the folded form of myosin thought to underlie its energy-conserving function in cells. The reconstruction also suggests a mechanism for unfolding when myosin is activated by phosphorylation.

The expression and functional activities of smooth muscle myosin and non-muscle myosin isoforms in rat prostate.

Benign prostatic hyperplasia (BPH) is mainly caused by increased prostatic smooth muscle (SM) tone and volume. SM myosin (SMM) and non-muscle myosin (NMM) play important roles in mediating SM tone and cell proliferation, but these molecules have been less studied in the prostate. Rat prostate and cultured primary human prostate SM and epithelial cells were utilized. In vitro organ bath studies were performed to explore contractility of rat prostate. SMM isoforms, including SM myosin heavy chain (MHC) isoforms (SM1/2 and SM-A/B) and myosin light chain 17 isoforms (LC17a/b ), and isoform ratios were determined via competitive RT-PCR. SM MHC and NM MHC isoforms (NMMHC-A, NMMHC-B and NMMHC-C) were further analysed via Western blotting and immunofluorescence microscopy. Prostatic SM generated significant force induced by phenylephrine with an intermediate tonicity between phasic bladder and tonic aorta type contractility. Correlating with this kind of intermediate tonicity, rat prostate mainly expressed LC17a and SM1 but with relatively equal expression of SM-A/SM-B at the mRNA level. Meanwhile, isoforms of NMMHC-A, B, C were also abundantly present in rat prostate with SMM present only in the stroma, while NMMHC-A, B, C were present both in the stroma and endothelial. Additionally, the SMM selective inhibitor blebbistatin could potently relax phenylephrine pre-contracted prostate SM. In conclusion, our novel data demonstrated the expression and functional activities of SMM and NMM isoforms in the rat prostate. It is suggested that the isoforms of SMM and NMM could play important roles in BPH development and bladder outlet obstruction.

The structure of the actin-smooth muscle myosin motor domain complex in the rigor state.

Myosin-based motility utilizes catalysis of ATP to drive the relative sliding of F-actin and myosin. The earliest detailed model based on cryo-electron microscopy (cryoEM) and X-ray crystallography postulated that higher actin affinity and lever arm movement were coupled to closure of a feature of the myosin head dubbed the actin-binding cleft. Several studies since then using crystallography of myosin-V and cryoEM structures of F-actin bound myosin-I, -II and -V have provided details of this model. The smooth muscle myosin II interaction with F-actin may differ from those for striated and non-muscle myosin II due in part to different lengths of important surface loops. Here we report a ∼6 Šresolution reconstruction of F-actin decorated with the nucleotide-free recombinant smooth muscle myosin-II motor domain (MD) from images recorded using a direct electron detector. Resolution is highest for F-actin and the actin-myosin interface (3.5-4 Å) and lowest (∼6-7 Å) for those parts of the MD at the highest radius. Atomic models built into the F-actin density are quite comparable to those previously reported for rabbit muscle actin and show density from the bound ADP. The atomic model of the MD, is quite similar to a recently published structure of vertebrate non-muscle myosin II bound to F-actin and a crystal structure of nucleotide free myosin-V. Larger differences are observed when compared to the cryoEM structure of F-actin decorated with rabbit skeletal muscle myosin subfragment 1. The differences suggest less closure of the 50 kDa domain in the actin bound skeletal muscle myosin structure.

Characterization of Blebbistatin Inhibition of Smooth Muscle Myosin and Nonmuscle Myosin-2.

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 assessed and remains controversial with respect to its efficacy with 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 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 for SmM, 3.58 µM for NM2a, 2.30 µM for NM2b, and 1.57 µM for 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 resistant to blebbistatin without greatly altering their motor activities or phosphorylation-dependent regulation, making A456F a useful mutant for investigating the cellular function of NM2s.

Highly selective inhibition of myosin motors provides the basis of potential therapeutic application.

Direct inhibition of smooth muscle myosin (SMM) is a potential means to treat hypercontractile smooth muscle diseases. The selective inhibitor CK-2018571 prevents strong binding to actin and promotes muscle relaxation in vitro and in vivo. The crystal structure of the SMM/drug complex reveals that CK-2018571 binds to a novel allosteric pocket that opens up during the "recovery stroke" transition necessary to reprime the motor. Trapped in an intermediate of this fast transition, SMM is inhibited with high selectivity compared with skeletal muscle myosin (IC50 = 9 nM and 11,300 nM, respectively), although all of the binding site residues are identical in these motors. This structure provides a starting point from which to design highly specific myosin modulators to treat several human diseases. It further illustrates the potential of targeting transition intermediates of molecular machines to develop exquisitely selective pharmacological agents.

Helical model of smooth muscle myosin filament and the ribbons made of caldesmon: history revisited.

In early studies on smooth muscle, I described a crude myosin fraction (CMF) in which self-assembly of myosin filaments was observed. For the first time, the 14-nm periodicity stemming from regular arrangement of myosin heads on the filament surface was observed (Sobieszek in J Mol Biol 70:741-744, 1972). In this fraction, we also observed formation of long ribbon-shaped aggregates exhibiting a 5.6-nm periodicity, characteristic of tropomyosin (TM) paracrystals (Sobieszek and Small in Phil Trans R Soc Lond B 265:203-212, 1973). We therefore concluded that these ribbons were made of TM and they might be related to the myosin ribbons observed in electron micrographs (EM) of intact smooth muscle (Lowy and Small in Nature 227:46-51, 1970; Small and Squire in Mol Biol 67:117-149, 1972). Subsequently, Small (J Cell Sci 24:327-349, 1977) concluded that the ribbons observed in the EM sections were an artifact, but their observation in the CMF was not addressed. I have now revisited two aspects of the above studies. Firstly, based on my new multi-angle laser-scattering data and considering the length and stability of the building unit for the filament, a myosin trimer fit better to the previously proposed helical structure. Secondly, after two decades of systematic examinations of protein compositions in multiple smooth muscle extracts and isolated filaments, I concluded that the ribbons were made of caldesmon and not TM. Thirdly, actin-activated ATPase activity measurements indicated that modulation of this activity (by CaD and TM) was synergistic, cooperative and depended on myosin to actin ratio.

Myosin light chain kinase steady-state kinetics: comparison of smooth muscle myosin II and nonmuscle myosin IIB as substrates.

Myosin light chain kinase (MLCK) phosphorylates S19 of the myosin regulatory light chain (RLC), which is required to activate myosin's ATPase activity and contraction. Smooth muscles are known to display plasticity in response to factors such as inflammation, developmental stage, or stress, which lead to differential expression of nonmuscle and smooth muscle isoforms. Here, we compare steady-state kinetics parameters for phosphorylation of different MLCK substrates: (1) nonmuscle RLC, (2) smooth muscle RLC, and heavy meromyosin subfragments of (3) nonmuscle myosin IIB, and (4) smooth muscle myosin II. We show that MLCK has a ~2-fold higher kcat for both smooth muscle myosin II substrates compared with nonmuscle myosin IIB substrates, whereas Km values were very similar. Myosin light chain kinase has a 1.6-fold and 1.5-fold higher specificity (kcat /Km ) for smooth versus nonmuscle-free RLC and heavy meromyosin, respectively, suggesting that differences in specificity are dictated by RLC sequences. Of the 10 non-identical RLC residues, we ruled out 7 as possible underlying causes of different MLCK kinetics. The remaining 3 residues were found to be surface exposed in the N-terminal half of the RLC, consistent with their importance in substrate recognition. These data are consistent with prior deletion/chimera studies and significantly add to understanding of MLCK myosin interactions. SIGNIFICANCE OF THE STUDY: Phosphorylation of nonmuscle and smooth muscle myosin by myosin light chain kinase (MLCK) is required for activation of myosin's ATPase activity. In smooth muscles, nonmuscle myosin coexists with smooth muscle myosin, but the two myosins have very different chemo-mechanical properties relating to their ability to maintain force. Differences in specificity of MLCK for different myosin isoforms had not been previously investigated. We show that the MLCK prefers smooth muscle myosin by a significant factor. These data suggest that nonmuscle myosin is phosphorylated more slowly than smooth muscle myosin during a contraction cycle.

Role of the essential light chain in the activation of smooth muscle myosin by regulatory light chain phosphorylation.

The activity of smooth and non-muscle myosin II is regulated by phosphorylation of the regulatory light chain (RLC) at serine 19. The dephosphorylated state of full-length monomeric myosin is characterized by an asymmetric intramolecular head-head interaction that completely inhibits the ATPase activity, accompanied by a hairpin fold of the tail, which prevents filament assembly. Phosphorylation of serine 19 disrupts these head-head interactions by an unknown mechanism. Computational modeling (Tama et al., 2005. J. Mol. Biol. 345, 837-854) suggested that formation of the inhibited state is characterized by both torsional and bending motions about the myosin heavy chain (HC) at a location between the RLC and the essential light chain (ELC). Therefore, altering relative motions between the ELC and the RLC at this locus might disrupt the inhibited state. Based on this hypothesis we have derived an atomic model for the phosphorylated state of the smooth muscle myosin light chain domain (LCD). This model predicts a set of specific interactions between the N-terminal residues of the RLC with both the myosin HC and the ELC. Site directed mutagenesis was used to show that interactions between the phosphorylated N-terminus of the RLC and helix-A of the ELC are required for phosphorylation to activate smooth muscle myosin.

Avian synaptopodin 2 (fesselin) stabilizes myosin filaments and actomyosin in the presence of ATP.

Smooth muscle cells maintain filaments of actin and myosin in the presence of ATP, although dephosphorylated myosin filaments and actin-myosin interactions are unstable under those conditions in vitro. Several proteins that stabilize myosin filaments and that stabilize actin-myosin interactions have been identified. Fesselin or synaptopodin 2 appears to be another such protein. Rapid kinetic measurements and electron microscopy demonstrated that fesselin, isolated from turkey gizzard muscle, reduced the rate of dissociation of myosin filaments. Addition of fesselin increased both the length and thickness of myosin filaments. The rate of detachment of myosin, but not heavy meromyosin, from actin was also greatly reduced by fesselin. Data from this study suggest that fesselin stabilizes myosin filaments and tethers myosin to actin. These results support the view that one role of fesselin is to organize contractile units of myosin and actin.

Kinetics of myosin light chain kinase activation of smooth muscle myosin in an in vitro model system.

During activation of smooth muscle contraction, one myosin light chain kinase (MLCK) molecule rapidly phosphorylates many smooth muscle myosin (SMM) molecules, suggesting that muscle activation rates are influenced by the kinetics of MLCK-SMM interactions. To determine the rate-limiting step underlying activation of SMM by MLCK, we measured the kinetics of calcium-calmodulin (Ca²âºCaM)-MLCK-mediated SMM phosphorylation and the corresponding initiation of SMM-based F-actin motility in an in vitro system with SMM attached to a coverslip surface. Fitting the time course of SMM phosphorylation to a kinetic model gave an initial phosphorylation rate, kp(o), of ~1.17 heads s⁻¹ MLCK⁻¹. Also, we measured the dwell time of single streptavidin-coated quantum dot-labeled MLCK molecules interacting with surface-attached SMM and phosphorylated SMM using total internal reflection fluorescence microscopy. From these data, the dissociation rate constant from phosphorylated SMM was 0.80 s⁻¹, which was similar to the kp(o) mentioned above and with rates measured in solution. This dissociation rate was essentially independent of the phosphorylation state of SMM. From calculations using our measured dissociation rates and Kd values, and estimates of SMM and MLCK concentrations in muscle, we predict that the dissociation of MLCK from phosphorylated SMM is rate-limiting and that the rate of the phosphorylation step is faster than this dissociation rate. Also, association with SMM (11-46 s⁻¹) would be much faster than with pSMM (<0.1-0.2 s⁻¹). This suggests that the probability of MLCK interacting with unphosphorylated versus phosphorylated SMM is 55-460 times greater. This would avoid sequestering MLCK to unproductive interactions with previously phosphorylated SMM, potentially leading to faster rates of phosphorylation in muscle.

Homology model of nonmuscle myosin heavy chain IIA and binding mode analysis with its inhibitor blebbistatin.

Nonmuscle myosin heavy chain IIA (NMMHC IIA, gene code: MYH9) plays a critical role in physiological and pathological functions. A homology model of NMMHC IIA was constructed based on the crystal structure of smooth muscle myosin II. Blebbistatin, a myosin II ATPase inhibitor, had been found to bind to NMMHC IIA with Leu228 as the important amino acid residue and van der Waals contacts as the main force of the interaction. The final complex demonstrated that the destruction of the salt bridge occurred between the Arg204 and Glu427 residues when blebbistatin was present. Molecular dynamic simulation of the complex showed that the binding affinity of blebbistatin to NMMHC IIA was strongly sensitive to the nucleotide binding region and actin binding region. The disturbance of the two regions increased the enhancement of the binding cavity with blebbistatin and resulted in a slightly more expanded conformation in the nucleotide binding region and actin binding region. A combined pharmacophore- and docking-based virtual screening was performed to identify several saponins as potential inhibitors for NMMHC IIA. These findings introduce new insights on the binding mode of blebbistatin and NMMHC IIA and novel leading compounds from natural products for NMMHC IIA-related diseases.

Structural dynamics of muscle protein phosphorylation.

We have used site-directed spectroscopic probes to detect structural changes, motions, and interactions due to phosphorylation of proteins involved in the regulation of muscle contraction and relaxation. Protein crystal structures provide static snapshots that provide clues to the conformations that are sampled dynamically by proteins in the cellular environment. Our site-directed spectroscopic experiments, combined with computational simulations, extend these studies into functional assemblies in solution, and reveal details of protein regions that are too dynamic or disordered for crystallographic approaches. Here, we discuss phosphorylation-mediated structural transitions in the smooth muscle myosin regulatory light chain, the striated muscle accessory protein myosin binding protein-C, and the cardiac membrane Ca(2+) pump modulator phospholamban. In each of these systems, phosphorylation near the N terminus of the regulatory protein relieves an inhibitory interaction between the phosphoprotein and its regulatory target. Several additional unifying themes emerge from our studies: (a) The effect of phosphorylation is not to change the affinity of the phosphoprotein for its regulated binding partner, but to change the structure of the bound complex without dissociation. (b) Phosphorylation induces transitions between order and dynamic disorder. (c) Structural states are only loosely coupled to phosphorylation; i.e., complete phosphorylation induces dramatic functional effects with only a partial shift in the equilibrium between ordered and disordered structural states. These studies, which offer atomic-resolution insight into the structural and functional dynamics of these phosphoproteins, were inspired in part by the ground-breaking work in this field by Michael and Kate Barany.

Modification of interface between regulatory and essential light chains hampers phosphorylation-dependent activation of smooth muscle myosin.

We examined the regulatory importance of interactions between regulatory light chain (RLC), essential light chain (ELC), and adjacent heavy chain (HC) in the regulatory domain of smooth muscle heavy meromyosin. After mutating the HC, RLC, and/or ELC to disrupt their predicted interactions (using scallop myosin coordinates), we measured basal ATPase, V(max), and K(ATPase) of actin-activated ATPase, actin-sliding velocities, rigor binding to actin, and kinetics of ATP binding and ADP release. If unphosphorylated, all mutants were similar to wild type showing turned-off behaviors. In contrast, if phosphorylated, mutation of RLC residues smM129Q and smG130C in the F-G helix linker, which interact with the ELC (Ca(2+) binding in scallop), was sufficient to abolish motility and diminish ATPase activity, without altering other parameters. ELC mutations within this interacting ELC loop (smR20M and smK25A) were normal, but smM129Q/G130C-R20M or -K25A showed a partially recovered phenotype suggesting that interaction between the RLC and ELC is important. A molecular dynamics study suggested that breaking the RLC/ELC interface leads to increased flexibility at the interface and ELC-binding site of the HC. We hypothesize that this leads to hampered activation by allowing a pre-existing equilibrium between activated and inhibited structural distributions (Vileno, B., Chamoun, J., Liang, H., Brewer, P., Haldeman, B. D., Facemyer, K. C., Salzameda, B., Song, L., Li, H. C., Cremo, C. R., and Fajer, P. G. (2011) Broad disorder and the allosteric mechanism of myosin II regulation by phosphorylation. Proc. Natl. Acad. Sci. U.S.A. 108, 8218-8223) to be biased strongly toward the inhibited distribution even when the RLC is phosphorylated. We propose that an important structural function of RLC phosphorylation is to promote or assist in the maintenance of an intact RLC/ELC interface. If the RLC/ELC interface is broken, the off-state structures are no longer destabilized by phosphorylation.

Crystal structure of a phosphorylated light chain domain of scallop smooth-muscle myosin.

We have determined the crystal structure of a phosphorylated smooth-muscle myosin light chain domain (LCD). This reconstituted LCD is of a sea scallop catch muscle myosin with its phosphorylatable regulatory light chain (RLC SmoA). In the crystal structure, Arg(16), an arginine residue that is present in this isoform but not in vertebrate smooth-muscle RLC, stabilizes the phosphorylation site. This arginine interacts with the carbonyl group of the phosphorylation-site serine in the unphosphorylated LCD (determined previously), and with the phosphate group when the serine is phosphorylated. However, the overall conformation of the LCD is essentially unchanged upon phosphorylation. This result provides additional evidence that phosphorylation of the RLC is unlikely to act as an on-switch in regulation of scallop catch muscle myosin.

Broad disorder and the allosteric mechanism of myosin II regulation by phosphorylation.

Double electron electron resonance EPR methods was used to measure the effects of the allosteric modulators, phosphorylation, and ATP, on the distances and distance distributions between the two regulatory light chain of myosin (RLC). Three different states of smooth muscle myosin (SMM) were studied: monomers, the short-tailed subfragment heavy meromyosin, and SMM filaments. We reconstituted myosin with nine single cysteine spin-labeled RLC. For all mutants we found a broad distribution of distances that could not be explained by spin-label rotamer diversity. For SMM and heavy meromyosin, several sites showed two heterogeneous populations in the unphosphorylated samples, whereas only one was observed after phosphorylation. The data were consistent with the presence of two coexisting heterogeneous populations of structures in the unphosphorylated samples. The two populations were attributed to an on and off state by comparing data from unphosphorylated and phosphorylated samples. Models of these two states were generated using a rigid body docking approach derived from EM [Wendt T, Taylor D, Trybus KM, Taylor K (2001) Proc Natl Acad Sci USA 98:4361-4366] (PNAS, 2001, 98:4361-4366), but our data revealed a new feature of the off-state, which is heterogeneity in the orientation of the two RLC. Our average off-state structure was very similar to the Wendt model reveal a new feature of the off state, which is heterogeneity in the orientations of the two RLC. As found previously in the EM study, our on-state structure was completely different from the off-state structure. The heads are splayed out and there is even more heterogeneity in the orientations of the two RLC.

Role of the tail in the regulated state of myosin 2.

Myosin 2 from vertebrate smooth muscle or non-muscle sources is in equilibrium between compact, inactive monomers and thick filaments under physiological conditions. In the inactive monomer, the two heads pack compactly together, and the long tail is folded into three closely packed segments that are associated chiefly with one of the heads. The molecular basis of the folding of the tail remains unexplained. By using electron microscopy, we show that compact monomers of smooth muscle myosin 2 have the same structure in both the native state and following specific, intramolecular photo-cross-linking between Cys109 of the regulatory light chain (RLC) and segment 3 of the tail. Nonspecific cross-linking between lysine residues of the folded monomer by glutaraldehyde also does not perturb the compact conformation and stabilizes it against unfolding at high ionic strength. Sequence comparisons across phyla and myosin 2 isoforms suggest that the folding of the tail is stabilized by ionic interactions between the positively charged N-terminal sequence of the RLC and a negatively charged region near the start of tail segment 3 and that phosphorylation of the RLC could perturb these interactions. Our results support the view that interactions between the heads and the distal tail perform a critical role in regulating activity of myosin 2 molecules through stabilizing the compact monomer conformation.

Modeling smooth muscle myosin's two heads: long-lived enzymatic roles and phosphorylation-dependent equilibria.

Smooth muscle myosin has two heads, each capable of interacting with actin to generate force and/or motion as it hydrolyzes ATP. These heads are inhibited when their associated regulatory light chain is unphosphorylated (0P), becoming active and hydrolyzing ATP maximally when phosphorylated (2P). Interestingly, with only one of the two regulatory light chains phosphorylated (1P), smooth muscle myosin is active but its ATPase rate is <2P. To explain published 1P single ATP turnover and steady-state ATPase activities, we propose a kinetic model in which 1P myosin exists in an equilibrium between being fully active (2P) and inhibited (0P). Based on the single ATP turnover data, we also propose that each 2P head adopts a hydrolytic role distinct from its partner at any point in time, i.e., one head strongly binds actin and hydrolyzes ATP at its actin-activated rate while the other weakly binds actin. Surprisingly, the heads switch roles slowly (<0.1 s(-1)), suggesting that their activities are not independent. The phosphorylation-dependent equilibrium between active and inhibited states and the hydrolytic role that each head adopts during its interaction with actin may have implications for understanding regulation and mechanical performance of other members of the myosin family of molecular motors.

Phosphorylation-induced structural changes in smooth muscle myosin regulatory light chain.

We have performed complementary time-resolved fluorescence resonance energy transfer (TR-FRET) experiments and molecular dynamics (MD) simulations to elucidate structural changes in the phosphorylation domain (PD) of smooth muscle regulatory light chain (RLC) bound to myosin. PD is absent in crystal structures, leaving uncertainty about the mechanism of regulation. Donor-acceptor pairs of probes were attached to three site-directed di-Cys mutants of RLC, each having one Cys at position 129 in the C-terminal lobe and the other at position 2, 3, or 7 in the N-terminal PD. Labeled RLC was reconstituted onto myosin subfragment 1 (S1). TR-FRET resolved two simultaneously populated structural states of RLC, closed and open, in both unphosphorylated and phosphorylated biochemical states. All three FRET pairs show that phosphorylation shifts the equilibrium toward the open state, increasing its mol fraction by approximately 20%. MD simulations agree with experiments in remarkable detail, confirming the coexistence of two structural states, with phosphorylation shifting the system toward the more dynamic open structural state. This agreement between experiment and simulation validates the additional structural details provided by MD simulations: In the closed state, PD is bent onto the surface of the C-terminal lobe, stabilized by interdomain salt bridges. In the open state, PD is more helical and straight, resides farther from the C-terminal lobe, and is stabilized by an intradomain salt bridge. The result is a vivid atomic-resolution visualization of the first step in the molecular mechanism by which phosphorylation activates smooth muscle.

Allosteric inhibition of the protein-protein interaction between the leukemia-associated proteins Runx1 and CBFbeta.

The two subunits of core binding factor (Runx1 and CBFbeta) play critical roles in hematopoiesis and are frequent targets of chromosomal translocations found in leukemia. The binding of the CBFbeta-smooth muscle myosin heavy chain (SMMHC) fusion protein to Runx1 is essential for leukemogenesis, making this a viable target for treatment. We have developed inhibitors with low micromolar affinity which effectively block binding of Runx1 to CBFbeta. NMR-based docking shows that these compounds bind to CBFbeta at a site displaced from the binding interface for Runx1, that is, these compounds function as allosteric inhibitors of this protein-protein interaction, a potentially generalizable approach. Treatment of the human leukemia cell line ME-1 with these compounds shows decreased proliferation, indicating these are good candidates for further development.