Structural and Kinetic Studies of Phosphorylation-dependent Regulation in Smooth Muscle Myosin

Rosenfeld, Steven S.; Xing, Jun; Cheung, Herbert C.; Brown, F.; Kar, S.; Sweeney, H. Lee

doi:10.1074/jbc.273.44.28682

Cited by 33 publications

(52 citation statements)

References 43 publications

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“…The interpretation of this titration was not as simple as for thioP- (12). Because ADP binds HMM with a much smaller fluorescence change than ATP, the displacement of ADP can be monitored from the net increase in fluorescence as ATP replaces ADP.…”

Section: Resultsmentioning

confidence: 99%

ADP Binding Induces an Asymmetry between the Heads of Unphosphorylated Myosin

Berger

Fagnant

Heizmann

et al. 2001

Journal of Biological Chemistry

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Light chain phosphorylation is the key event that regulates smooth and non-muscle myosin II ATPase activity. Here we show that both heads of smooth muscle heavy meromyosin (HMM) bind tightly to actin in the absence of nucleotide, irrespective of the state of light chain phosphorylation. In striking contrast, only one of the two heads of unphosphorylated HMM binds to actin in the presence of ADP, and the heads have different affinities for ADP. This asymmetry suggests that phosphorylation alters the mechanical coupling between the heads of HMM. A model that incorporates strain between the two heads is proposed to explain the data, which have implications for how one head of a motor protein can gate the response of the other.A common feature of the myosin II family is that all of its members contain two heads connected to an ␣-helical coiledcoil tail. Although the single-headed subfragment of myosin (S1) 1 has always been considered a good model system for studying myosin structure and function, it is clear that some important features of whole myosin may be overlooked by studying only this model species. A prominent example is that phosphorylation-dependent regulation of actin-activated ATPase activity of smooth and vertebrate non-muscle myosin II is manifested only in subfragments that have two heads as well as a minimal length of the coiled-coil tail (1). In contrast, single-headed myosin is unregulated and always active (2). Tethering of the two heads together via the coiled-coil tail can also impose steric and mechanical constraints on the interaction of the two heads with actin. This has been explored in the case of skeletal muscle heavy meromyosin (3, 4) (HMM) but not for the regulated HMMs, where light chain phosphorylation may also be expected to affect the coupling between the two heads.Structural differences between phosphorylated and unphosphorylated HMM in the presence of ATP were recently revealed through analysis of two-dimensional crystalline arrays by image reconstruction and docking of high resolution structures into the observed density maps (5). In the phosphorylated state the two heads showed no contact with each other. In contrast, the unphosphorylated HMM showed an interaction between the two heads, with the actin-binding interface of one head bound to the converter domain of the other. This asymmetrical interaction between the unphosphorylated heads suggested a mechanism for how phosphorylation-dependent regulation of activity is achieved. One head cannot interact with actin and therefore its ATPase activity will not be enhanced, whereas the other head cannot undergo the rotation of the converter domain that is necessary to achieve phosphate release (5). This unexpected structure provided evidence that the coupling between the heads of double-headed smooth muscle heavy meromyosin is influenced by the state of light chain phosphorylation. To further understand how the two heads of smooth muscle HMM interact with each other, we undertook a kinetic analysis of HMM in both the unphosphorylated ...

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Section: Resultsmentioning

confidence: 99%

ADP Binding Induces an Asymmetry between the Heads of Unphosphorylated Myosin

Berger

Fagnant

Heizmann

et al. 2001

Journal of Biological Chemistry

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“…Under these conditions UP-HMM has a higher sedimentation coefficient (57), a lower proteolytic susceptibility of the head-rod junction (58), a less mobile motor domain and RLC (59), and a lower basal ATPase activity (57) than P-HMM. UP-HMM appears to have heads flexed back toward the rod (60,61), and P-HMM appears to have the heads extended away from the rods (60).…”

Section: Changes In Acr Fluorescence Signal May Report the Heads-up (mentioning

confidence: 99%

Novel Sensors of the Regulatory Switch on the Regulatory Light Chain of Smooth Muscle Myosin

Mazhari

Selser

Cremo

2004

Journal of Biological Chemistry

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Here we used three different approaches to test this notion, which are reactivity of cysteine thiols, pyrene and acrylodan spectral analysis, and pyrene fluorescence quenching. All methods detected significant differences between the unphosphorylated and phosphorylated regulatory light chain N termini in heavy meromyosin, a double-headed subfragment with an intact regulatory switch. These differences were not observed for subfragment-1, a single-headed, unregulated subfragment. In the presence of either ATP or ADP, phosphorylation increased the solvent exposure and decreased the polarity of the environment about position 23 of the regulatory light chain of heavy meromyosin. These phosphorylation-induced structural changes were not as evident in the absence of nucleotides. Nucleotide binding to unphosphorylated heavy meromyosin caused a decrease in exposure and an increase in polarity of the N terminus, whereas the effects of nucleotide on phosphorylated heavy meromyosin were the opposite. We showed a direct correlation between the kinetics of nucleotide binding/turnover and the conformational change reported by acrylodan at position 23 of the regulatory light chain. Acrylodan-A23C also reports the heads up (extended) to flexed (folded) transition in unphosphorylated heavy meromyosin. This is the first demonstration of direct coupling of nucleotide binding to conformational changes in the N terminus of the regulatory light chain.Smooth muscle myosin (SMM) 1 and nonmuscle myosin are hexameric motor ATPases of ϳ500 kDa mass composed of pairs of HC, ELC, and RLC. A globular motor domain (N-terminal HC) and a light chain binding domain (HC ϩ ELC ϩ RLC) form a head (S1), and the two heads are dimerized by the HC C-terminal halves, which form a coiled-coil. The motor domain contains the catalytic site and the actin binding site (1). Only the head domain and its subfragments have been crystallized to date so there are no atomic resolution structures of a doubleheaded construct.The actin-activated ATPase activity and motor properties of SMM and nonmuscle myosin are regulated by phosphorylation of Ser-19 of the RLC, which is greater than 10 nm from the catalytic site (1-4). Phosphorylation enhances the ATPase activity by more than 1000-fold at saturating actin concentrations (5, 6). Domain requirements for regulation have been elucidated through studies of various proteolytic and expressed subfragments of SMM and nonmuscle myosin. HMM, which lacks the C-terminal two-thirds of the tail, is double-headed and regulated (5-7), but expressed HMM constructs with truncated tails too short to form stable double-headed structures are unregulated (8 -10) as is S1 (7,11,12) and single-headed myosin (13,14). Furthermore, a nonmuscle HMM construct without one of the motor domains but with both regulatory domains is not regulated (15). Therefore, two full heads connected together with enough coiled-coil to allow for dimerization are required for regulation.Removal of the RLC abolishes regulation (16), but the ELC can be removed with re...

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“…Interestingly, the overall structure of myosin proteins appears to be quite conserved, while their biochemical and kinetic properties are quite divergent. It has been proposed that sequence variability in the two surface loops of myosin, which are susceptible to proteolysis and divide myosin into three domains (25,50, and 20 kDa, Figure 1), may play a role in kinetically tuning a particular myosin to perform specific cellular functions (3)(4)(5)(6). In the current study, we examine the role of loop 2, a surface loop in the actin-binding region of myosin, in kinetically tuning myosin V, a nonmuscle myosin that functions as an organelle transporter.…”

mentioning

confidence: 99%

Functional Role of Loop 2 in Myosin V

Yengo

Sweeney

2004

Biochemistry

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Myosin V is molecular motor that is capable of moving processively along actin filaments. The kinetics of monomeric myosin V containing a single IQ domain (MV 1IQ) differ from nonprocessive myosin II in that actin affinity is higher, phosphate release is extremely rapid, and ADP release is ratelimiting. We generated two mutants of myosin V by altering loop 2, a surface loop in the actin-binding region thought to alter actin affinity and phosphate release in myosin II, to determine the role that this loop plays in the kinetic tuning of myosin V. The loop 2 mutants altered the apparent affinity for actin (K ATPase ) without altering the maximum ATPase rate (V MAX ). Transient kinetic analysis determined that the rate of binding to actin, as well as the affinity for actin, was dependent on the net positive charge of loop 2, while other steps in the ATPase cycle were unchanged. The maximum rate of phosphate release was unchanged, but the affinity for actin in the M‚ADP‚Pi-state was dramatically altered by the mutations in loop 2. Thus, loop 2 is important for allowing myosin V to bind to actin with a relatively high affinity in the weak binding states but does not play a direct role in the product release steps. The ability to maintain a high affinity for actin in the weak binding states may prevent diffusion away from the actin filament and increase the degree of processive motion of myosin V.Myosins make up a large superfamily of motor proteins that are capable of using the chemical energy from ATP hydrolysis to power the directed movement on actin filaments and function in a wide variety of cellular processes from muscle contraction to organelle transport (1). During the actomyosin ATPase cycle myosin shifts between actindetached (weak-binding) and actin-attached states (strongbinding), and force generation occurs through a conformational change in myosin during the transition from the weakto the strong-actin binding states (reviewed in ref 2). Interestingly, the overall structure of myosin proteins appears to be quite conserved, while their biochemical and kinetic properties are quite divergent. It has been proposed that sequence variability in the two surface loops of myosin, which are susceptible to proteolysis and divide myosin into three domains (25, 50, and 20 kDa, Figure 1), may play a role in kinetically tuning a particular myosin to perform specific cellular functions (3-6). In the current study, we examine the role of loop 2, a surface loop in the actin-binding region of myosin, in kinetically tuning myosin V, a nonmuscle myosin that functions as an organelle transporter.Myosin V has several unique biochemical features that allow it to move processively along actin (7), take multiple steps along actin without diffusing away, and function as an organelle transporter (8). The kinetics of myosin V are different from nonprocessive muscle myosin II in that ADP release in myosin V is slow and rate-limiting, while phosphate release is fast (9-11). This allows myosin V to populate the strong-binding s...

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Structural and Kinetic Studies of Phosphorylation-dependent Regulation in Smooth Muscle Myosin

Cited by 33 publications

References 43 publications

ADP Binding Induces an Asymmetry between the Heads of Unphosphorylated Myosin

ADP Binding Induces an Asymmetry between the Heads of Unphosphorylated Myosin

Novel Sensors of the Regulatory Switch on the Regulatory Light Chain of Smooth Muscle Myosin

Functional Role of Loop 2 in Myosin V

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