Modulation of Actin Affinity and Actomyosin Adenosine Triphosphatase by Charge Changes in the Myosin Motor Domain

Furch, Marcus; Geeves, Michael A.; Manstein, Dietmar J.

doi:10.1021/bi972851y

Cited by 174 publications

(197 citation statements)

References 40 publications

(60 reference statements)

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“…Previously, we have shown that the presence of R-actinin repeats does not alter the kinetic characteristics of the motor domain (11). Thus we can compare the results obtained with constructs E531Q and Q532E with those obtained for Q532E (20/+12) and (20/ +12) (10).…”

Section: Resultsmentioning

confidence: 73%

“…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: 99%

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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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Section: Resultsmentioning

confidence: 73%

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Stabilization of the Actomyosin Complex by Negative Charges on Myosin

et al. 2000

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“…Herein, the loop 3 region composed by residues 559-565 of wild-type M765 (EEPRFSK) was replaced by the corresponding sequence of human skeletal muscle myosin (QKPVVKGKAE) in M765(Sk) or human nonmuscle myosin (QKPKQLKDK) in M765(NM) ( Figure 1). These three recombinant constructs were overexpressed in D. discoideum with a C-terminal His tag and purified by Ni-chelate chromatography (20).…”

Section: Resultsmentioning

confidence: 99%

“…The amount of P i liberated was evaluated colorimetrically (50). Alternatively, the amount of ADP liberated was estimated by the pyruvate kinase-lactate deshydrogenase assay (20). Very comparable results were obtained independently of the method used.…”

Section: Methodsmentioning

confidence: 99%

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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“…The N-terminal finger of actin is negatively charged across the phylogenic tree (Vandekerckhove and Weber 1978), and in muscles, this finger is known to make the weak (ionic) interaction with loop 2 of myosin (Sutoh 1982a, b;DasGupta and Reisler 1989;Furch et al 1998); the loop 2 joins 50K and 20K domains and has 5 Lys residues. There is a line of evidence that the N-terminal finger activates S1 ATP hydrolysis rate in solution (Sutoh et al 1991;Cook et al 1993), which may suggest that the N-terminal finger also activates the strong (hydrophobic) interaction.…”

Section: Negative Charges Of Actin's N-terminusmentioning

confidence: 99%

Use of thin filament reconstituted muscle fibres to probe the mechanism of force generation

Kawai

Ishiwata

2006

J Muscle Res Cell Motil

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The technique of selective removal of the thin filament by gelsolin in bovine cardiac muscle fibres, and reconstitution of the thin filament from isolated proteins is reviewed, and papers that used reconstituted preparations are discussed. By comparing the results obtained in the absence/presence of regulatory proteins tropomyosin (Tm) and troponin (Tn), it is concluded that the role of Tm and Tn in force generation is not only to expose the binding site of actin to myosin, but also to modify actin for better stereospecific and hydrophobic interaction with myosin. This conclusion is further supported by experiments that used a truncated Tm mutant and the temperature study of reconstituted fibres. The conclusion is consistent with the hypothesis that there are three states in the thin filament: blocked state, closed state, and open state. Tm is the major player to produce these effects, with Tn playing the role of Ca 2+ sensing and signal transmission mechanism. Experiments that changed the number of negative charges at the N-terminal finger of actin demonstrates that this part of actin is essential to promote the strong interaction between actin and myosin molecules, in addition to the well-known weak interaction that positions the myosin head at the active site of actin prior to force generation. KeywordsActin; Regulatory proteins; Tropomyosin; Troponin; Myosin; Gelsolin; Cross-bridge kinetics; Sinusoidal analysis; BDM Thin filament reconstituted muscle fibre systemFor the purpose of understanding the cross-bridge interaction with actin and the role of regulatory proteins in this interaction, we reconstituted the thin filament in cardiac muscle fibres. This method is schematically represented in Fig. 1. The thin filament is first removed by gelsolin (Fig. 1A to 1B), followed by sequential reconstitution with G-actin (Fig. 1C), and regulatory proteins, tropomyosin (Tm) and troponin (Tn) (Fig. 1D). Gelsolin is an actin and thin filament severing protein, hence its action is specific and it does not disrupt other sarcomeric structure. However, overtreatment with gelsolin would disrupt the Z-line structure, because actin is one of the main constituents in the Z-line. The thin filament reconstitution method was initially developed at Ishiwata's laboratory and the key properties of the Correspondence to: Masataka Kawai, masataka-kawai@uiowa.edu. reconstituted fibres, such as the recovery of isometric tension, the tension-pCa relationship, and the function of spontaneous oscillatory contraction, were examined on skeletal fibres (Funatsu et al. 1994), followed by cardiac fibres (Fujita et al. 1996; Ishiwata 1998, 1999; for brief review see Ishiwata et al. 1998). In these works, it was demonstrated that bovine cardiac fibres were successful as the reconstituted system, in which actin, Tm and Tn can be replaced with those prepared from other muscles, for example, rabbit skeletal muscle proteins (Fujita et al. 1996;Fujita and Ishiwata 1999). The reconstitution method in cardiac fibres was successfully applied at ...

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Modulation of Actin Affinity and Actomyosin Adenosine Triphosphatase by Charge Changes in the Myosin Motor Domain

Cited by 174 publications

References 40 publications

Stabilization of the Actomyosin Complex by Negative Charges on Myosin

Stabilization of the Actomyosin Complex by Negative Charges on Myosin

Functional Characterization of the Secondary Actin Binding Site of Myosin II

Use of thin filament reconstituted muscle fibres to probe the mechanism of force generation

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