Immunochemical Probing of the Functional Role of the 238–246 and 567–574 Sequences of Myosin Heavy Chain

Blotnick, Edna; Miller, Cora; Gröschel‐Stewart, Ute; Mühlrád, András

doi:10.1111/j.1432-1033.1995.tb20804.x

Cited by 6 publications

(4 citation statements)

References 34 publications

(33 reference statements)

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“…It looks like the binding energy utilized for this additional interface and present only in striated muscle myosin isoforms is not at all related to the actin-induced ATPase activation process. In agreement with the lack of a functional role of loop 3 in the actin-activated ATPase activity, no difference in the activity was found when the accessibility of loop 3 was hindered either by changing the degree of saturation of the actin filaments by myosin (73) or by antibodies directed against skeletal muscle myosin loop 3 (30). Furthermore, our results suggest that only the loop 3 region of striated muscle myosins has a modulating effect on the interaction with actin.…”

Section: Addition Of Positive Charges In Loop 3 Destabilizes the Over...supporting

confidence: 88%

“…This interaction is supported by the fact † Supported by the Centre National de la Recherche Scientifique, the Association Franc ¸aise contre les myopathies, the Max Planck Society, and by Grant MA 1081/5- that proteolytic cleavage of loop 3 inhibits the actin-activated ATPase activity of both rabbit skeletal and scallop muscle myosin (28,29). Moreover, an antipeptide antibody directed against loop 3 of skeletal muscle myosin has a strong inhibitory effect on the sliding velocity of actin filaments in an in vitro motility assay (30). On the other hand, crosslinking reactions with smooth muscle myosin (31) or Dictyostelium discoideum myosin (21) did not provide evidence for an interaction between myosin loop 3 and actin.…”

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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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Section: Addition Of Positive Charges In Loop 3 Destabilizes the Over...supporting

confidence: 88%

mentioning

confidence: 99%

Functional Characterization of the Secondary Actin Binding Site of Myosin II

et al. 1999

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“…In weak-binding complexes, on the other hand, the simultaneous cross-linking of S1 to two actin monomers early in the reaction time course together with a comparable salt effect on the cross-linking reaction and on the interaction show that these two subsites are important components of the weak-binding interface. This conclusion does not contradict the fact that the S1‚nucleotide complex may interact with subdomain 2 of the second actin monomer (37), and it is supported by the fact that an antibody directed against the S1 loop of residues 567-574 has a strong inhibitory effect on the movement of actin filaments in in vitro motility assays (77). The slight differences in the amount of S1 bound to actin observed in cosedimentation experiments in the presence of ATP analogues are probably due to differences in the binding constants of these analogues for S1 bound to actin, i.e., the tendency to form the ternary actin-S1‚nucleotide complexes (78,79).…”

Section: Discussionmentioning

confidence: 90%

Effect of ATP Analogues on the Actin−Myosin Interface

1998

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The interaction between skeletal myosin subfragment 1 (S1) and filamentous actin was examined at various intermediate states of the actomyosin ATPase cycle by chemical cross-linking experiments. Reaction of the actin-S1 complex with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and N-hydroxysuccinimide generated products with molecular masses of 165 and 175 kDa, in which S1 loops of residues 626-647 and 567-578 were cross-linked independently to the N-terminal segment of residues 1-12 of one actin monomer, and of 265 kDa, in which the two loops were bound to the N termini of two adjacent monomers. In strong-binding complexes, i.e., without nucleotide or with ADP, S1 was sequentially cross-linked to one and then to two actin monomers. In the weak-binding complexes, two types of cross-linking pattern were observed. First, during steady-state hydrolysis of ATP or ATPgammaS at 20 degreesC, the cross-linking reaction gave rise to a small amount of unknown 200 kDa product. Second, in the presence of AMPPNP, ADP.BeFx, ADP.AlF4-, or ADP.VO43- or with S1 internally cross-linked by N,N'-p-phenylenedimaleimide, only the 265 kDa product was obtained. The presence of 200 mM salt inhibited cross-linking reactions in both weak- and strong-binding states, while it dissociated only weak-binding complexes. These results indicate that, in the weak-binding state populated with the ADP.Pi analogues, skeletal S1 interacts predominantly and with an apparent equal affinity with the N termini of two adjacent actin monomers, while these ionic contacts are much less significant in stabilizing the rigor actin-S1 complexes. They also suggest that the electrostatic actin-S1 interface is not influenced by the type of ADP.Pi analogue bound to the active site.

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“…As a consequence, one would expect a high degree of solvation, which would minimize any difference in their microenvironment. One should also note that these two loop structures are equally accessible to peptide antibodies (Cheung and Reisler, 1992;Blotnick et al, 1995).…”

Section: Kinetics Of S1 Cross-linking To F-actinmentioning

confidence: 99%

Interaction of Myosin with F-Actin: Time-Dependent Changes at the Interface Are Not Slow

Dijk

Céline

Barman³

et al. 2000

Biophysical Journal

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The kinetics of formation of the actin-myosin complex have been reinvestigated on the minute and second time scales in sedimentation and chemical cross-linking experiments. With the sedimentation method, we found that the binding of the skeletal muscle myosin motor domain (S1) to actin filament always saturates at one S1 bound to one actin monomer (or two S1 per actin dimer), whether S1 was added slowly (17 min between additions) or rapidly (10 s between additions) to an excess of F-actin. The carbodiimide (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, EDC)-induced cross-linking of the actin-S1 complex was performed on the subsecond time scale by a new approach that combines a two-step cross-linking protocol with the rapid flow-quench technique. The results showed that the time courses of S1 cross-linking to either of the two actin monomers are identical: they are not dependent on the actin/S1 ratio in the 0.3-20-s time range. The overall data rule out a mechanism by which myosin rolls from one to the other actin monomer on the second or minute time scales. Rather, they suggest that more subtle changes occur at the actomyosin interface during the ATP cycle.

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Immunochemical Probing of the Functional Role of the 238–246 and 567–574 Sequences of Myosin Heavy Chain

Cited by 6 publications

References 34 publications

Functional Characterization of the Secondary Actin Binding Site of Myosin II

Functional Characterization of the Secondary Actin Binding Site of Myosin II

Effect of ATP Analogues on the Actin−Myosin Interface

Interaction of Myosin with F-Actin: Time-Dependent Changes at the Interface Are Not Slow

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