Differences in the ionic interaction of actin with the motor domains of nonmuscle and muscle myosin II

Dijk, Juliette van; Furch, Marcus; Derancourt, Jean; Batra, Renu; Knetsch, Menno L. W.; Manstein, Dietmar J.; Chaussepied, Patrick

doi:10.1046/j.1432-1327.1999.00172.x

Cited by 18 publications

(20 citation statements)

References 64 publications

(88 reference statements)

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“…We conclude that the coupling between biochemical and structural states (as revealed by the coupling between the nucleotide-binding site and the force-generating region) is different for the two myosins. This difference in coupling is presumably related to the observed differences in ATPase kinetics between muscle and nonmuscle myosins (21,(26)(27)(28).…”

Section: Quantitative Analysis Of Epr Spectra: Resolved Structural Stmentioning

confidence: 99%

Muscle and nonmuscle myosins probed by a spin label at equivalent sites in the force-generating domain

Agafonov

Nesmelov

Titus

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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We have engineered a mutant of Dictyostelium discoideum (Dicty) myosin II that contains the same fast-reacting ''SH1'' thiol as in muscle myosin, spin-labeled it, and performed electron paramagnetic resonance (EPR) to compare the structure of the forcegenerating region of the two myosins. Dicty myosin serves as a model system for muscle myosin because of greater ease of mutagenesis, expression, and crystallization. The catalytic domains of these myosins have nearly identical crystal structures in the apo state, but there are significant differences in ATPase kinetics, and there are no crystal structures of skeletal muscle myosin with bound nucleotides, so another structural technique is needed. Previous EPR studies, with a spin label attached to SH1 in muscle myosin, have resolved the key structural states of this region. Therefore, we have performed identical experiments on both myosins spin-labeled at equivalent sites. Spectra were identical for the two myosins in the apo and ADP-bound states. With bound ADP and phosphate analogs, (i) both proteins exhibit two resolved structural states (prepowerstroke, postpowerstroke) in a single biochemical state (defined by the bound nucleotide), and (ii) these structural states are essentially identical in the two myosins but (iii) are occupied to different extents as a function of the biochemical state. We conclude that (i) myosin structural and biochemical states do not have a one-to-one correspondence, and (ii) Dicty myosin can serve as a good analog for structural studies of muscle myosin only if differences in the coupling between biochemical and structural states are taken into account.Dictyostelium ͉ electron paramagnetic resonance ͉ EPR ͉ site-directed spin labeling ͉ SDSL M yosin II is a motor enzyme involved in muscle contraction and cell motility through a cycle of actin-activated ATP hydrolysis. The coupling of ATP hydrolysis and force generation is determined by myosin's head domain, subfragment 1 (S1). The myosin ATPase cycle can be described to a first approximation by the following scheme:

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Section: Quantitative Analysis Of Epr Spectra: Resolved Structural Stmentioning

confidence: 99%

Muscle and nonmuscle myosins probed by a spin label at equivalent sites in the force-generating domain

Agafonov

Nesmelov

Titus

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…The removal of negative charge in position 531 reduces K A 10-fold, while addition of negative charge in position 532 increases K A 5-fold. These effects are much larger than single or double charge changes in other regions of the actin-binding site of myosin II (9,10,27,28). It is striking that the introduction of a single negative charge at position 532 has the same effect on k -A as the introduction of 12 positive charges in the loop 2 region (see Table 2).…”

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confidence: 98%

Stabilization of the Actomyosin Complex by Negative Charges on Myosin

et al. 2000

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Sequence comparisons of members of the myosin superfamily show a high degree of charge conservation in a surface exposed helix (Dictyostelium discoideum myosin II heavy chain residues S510 to K546). Most myosins display a triplet of acidic residues at the equivalent positions to D. discoideum myosin II residues D530, E531, and Q532. The high degree of charge conservation suggests strong evolutionary constrain and that this region is important for myosin function. Mutations at position E531 were shown to strongly affect actin binding [Giese, K. C., and Spudich, J. A. (1997) Biochemistry 36, 8465-8473]. Here, we used steady-state and transient kinetics to characterize the enzymatic competence of mutant constructs E531Q and Q532E, and their properties were compared with those of a loop 2 mutant with a 20 amino acid insertion containing 12 positive charges (20/+12) [Furch et al. (1998) Biochemistry 37, 6317-6326], double mutant Q532E(20/+12), and the native motor domain constructs. Our results confirm that charge changes at residues 531 and 532 primarily affect actin binding with little change being communicated to the nucleotide pocket. Mutation D531Q reduces actin affinity (K A ) 10-fold, while Q532E leads to a 5-fold increase. The observed changes in K A stem almost exclusively from variations in the dissociation rate constant (k -A ), with the introduction of a single negative charge at position 532 having the same effect on k -A as the introduction of 12 positive charges in the loop 2 region.Myosin II drives muscle contraction and motile processes such as cytokinesis and cell motility. The ATP-dependent 1 interaction of the myosin head with actin is central to myosin driven motile processes. Atomic models of the actomyosin rigor complex show the interface between myosin and actin to consist of four major contact regions, suggesting a sequential mechanism of binding (1, 2). A first contact involves a highly charged, lysine-rich loop on myosin (loop 2) formed by residues S619 to V630 in the case of Dictyostelium discoideum myosin II and a cluster of acidic residues at the N-terminus of actin (3-6). A second contact region involves a helix-loop-helix structure formed by residues S510 to K546 (unless otherwise stated amino acid residues are numbered according to the D. discoideum myosin II sequence). A loop formed by residues L399 to V411 makes a third contact. These three contacts involve a single actin monomer and form the primary actin-binding site of myosin. In addition a loop protruding between residues L547 and H572 may reach the neighboring actin monomer one actin helix turn below (Figure 1).Previously, we have shown that the light-chain-binding domain (LCBD) plays no major role in the biochemical behavior of the myosin (7-9). Recombinant constructs without LCBD can be produced and purified in large amounts and are ideally suited for systematic studies of the structure, kinetics and function of the myosin motor. Therefore, we used construct M765, corresponding to the first 765 residues of D. disc...

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“…Doublet bands of 145-155 kDa in the case of M765(Sk) and 165-175 kDa in the case of Sk-S1 were obtained. The faster migrating 145 and 165 kDa bands contained actin cross-linked to myosin loop 2 (21,53,54). The fact that the M765(Sk)-derived 155 kDa band contains actin cross-linked to myosin loop 3 is consistent with previous work that located the actin cross-linking site of the 175 kDa product within the 8 kDa stretch of residues (containing loop 3) at 27-35 kDa from the C-terminus of Sk-S1 (25,55).…”

Section: Resultsmentioning

confidence: 99%

“…Thrombin treatment does not cleave M765 but degrades monomeric actin independently of the presence of myosin at residues 28, 39, and 113, leading to actin peptides 40-375 (A37), 114-375 (A27), 40-113 (A10), 1-28, and 29-39 (21,25,56,57). skeletal muscle myosin) with the actin-peptide 1-28 bound to the motor domain (21,58).…”

Section: Resultsmentioning

confidence: 99%

“…Biochemical and molecular genetic studies with chimeric proteins have clearly demonstrated that loop 2 tunes the catalytic efficiency of the different myosin isotypes (17)(18)(19)(20)(21)(22). Similar to the docking process of other protein-protein interfaces (23,24), the association rate of myosin and actin is governed by the number of charges present in loop 2 (20).…”

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confidence: 99%

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Functional Characterization of the Secondary Actin Binding Site of Myosin II

et al. 1999

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The role of the interaction between actin and the secondary actin binding site of myosin (segment 565-579 of rabbit skeletal muscle myosin, referred to as loop 3 in this work) has been studied with proteolytically generated smooth and skeletal muscle myosin subfragment 1 and recombinant Dictyostelium discoideum myosin II motor domain constructs. Carbodiimide-induced cross-linking between filamentous actin and myosin loop 3 took place only with the motor domain of skeletal muscle myosin and not with those of smooth muscle or D. discoideum myosin II. Chimeric constructs of the D. discoideum myosin motor domain containing loop 3 of either human skeletal muscle or nonmuscle myosin were generated. Significant actin cross-linking to the loop 3 region was obtained only with the skeletal muscle chimera both in the rigor and in the weak binding states, i.e., in the absence and in the presence of ATP analogues. Thrombin degradation of the cross-linked products was used to confirm the cross-linking site of myosin loop 3 within the actin segment 1-28. The skeletal muscle and nonmuscle myosin chimera showed a 4-6-fold increase in their actin dissociation constant, due to a significant increase in the rate for actin dissociation (k -A ) with no significant change in the rate for actin binding (k +A ). The actin-activated ATPase activity was not affected by the substitutions in the chimeric constructs. These results suggest that actin interaction with the secondary actin binding site of myosin is specific for the loop 3 sequence of striated muscle myosin isoforms but is apparently not essential either for the formation of a high affinity actinmyosin interface or for the modulation of actomyosin ATPase activity.Myosin molecules are actin-based molecular motors that use the energy supplied by the hydrolysis of ATP to perform various cell motility processes. The catalytic activity of myosin resides in its conserved motor domain, which interacts with actin, binds to and hydrolyzes ATP, and produces the force necessary for movement along actin filaments. Although the 3-D structure of the motor domain is highly conserved within the myosin family (1, 2), the enzymatic and motile activities of different myosin isoforms show a high degree of divergence (3). These functional variations are probably not due to fundamental differences in the molecular mechanism used by each isotype to convert the chemical energy into mechanical force but rather reflect a fine-tuning to specific functional requirements that is brought about by variations in the primary sequence of the motor domain itself. In agreement with this idea, sequence alignments reveal large differences in primary sequence at the proposed actin-myosin interface (4). Within this interface, one can clearly distinguish electrostatic and hydrophobic contacts (5, 6). The formation of the initial collision complex formed in the presence of ATP and ADP‚P i is largely an ionic interaction, whereas the transition from the weak to the strong binding complex has both ionic and hydro...

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Differences in the ionic interaction of actin with the motor domains of nonmuscle and muscle myosin II

Cited by 18 publications

References 64 publications

Muscle and nonmuscle myosins probed by a spin label at equivalent sites in the force-generating domain

Muscle and nonmuscle myosins probed by a spin label at equivalent sites in the force-generating domain

Stabilization of the Actomyosin Complex by Negative Charges on Myosin

Functional Characterization of the Secondary Actin Binding Site of Myosin II

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