Conformational changes in subdomain I of actin induced by proteolytic cleavage within the DNase I‐binding loop: energy transfer from tryptophan to AEDANS

Kuznetsova, Irina M.; Antropova, Olga; Turoverov, Konstantin K.; Khaitlina, Sofia

doi:10.1016/0014-5793(96)00238-4

Cited by 37 publications

(23 citation statements)

References 19 publications

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“…The results summarized in Table 1 confirm that the effect of the D-loop modifications on various motile properties of both myosins II and V is indeed more pronounced in the subtilisin-cleaved actin compared with the M47A mutant actin. Moreover, the similarity of the trends, in which the motile properties are affected by both modifications, suggests that although in the cleaved actin the effects of the modification may propagate to the more distant elements of actin structure (25), in the experiments described here, the contribution of these allosteric effects to the interaction with myosin is negligible, if present at all, compared with the impact of the modifications within the D-loop itself.…”

Section: Comparison Of the Effects Of Subtilisin Cleavage And The M47amentioning

confidence: 83%

D-loop of Actin Differently Regulates the Motor Function of Myosins II and V

Kubota

Mikhailenko

Okabe

et al. 2009

Journal of Biological Chemistry

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To gain more information on the manner of actin-myosin interaction, we examined how the motile properties of myosins II and V are affected by the modifications of the DNase I binding loop (D-loop) of actin, performed in two different ways, namely, the proteolytic digestion with subtilisin and the M47A point mutation. In an in vitro motility assay, both modifications significantly decreased the gliding velocity on myosin II-heavy meromyosin due to a weaker generated force but increased it on myosin V. On the other hand, single molecules of myosin V "walked" with the same velocity on both the wild-type and modified actins; however, the run lengths decreased sharply, correlating with a lower affinity of myosin for actin due to the D-loop modifications. The difference between the single-molecule and the ensemble measurements with myosin V indicates that in an in vitro motility assay the non-coordinated multiple myosin V molecules impose internal friction on each other via binding to the same actin filament, which is reduced by the weaker binding to the modified actins. These results show that the D-loop strongly modulates the force generation by myosin II and the processivity of myosin V, presumably affecting actin-myosin interaction in the actomyosin-ADP⅐P i state of both myosins. Subdomain 2 of actin, which contains the D-loop (residues 38 -52), slightly changes its conformation during actin polymerization and interacts with the C terminus of the adjacent subunit in actin filament (1, 2). This region is suggested to be important for actin-myosin interaction; it was found that binding of myosin II induces conformational changes in subdomain 2 (3, 4), whereas the proteolytic digestion of the D-loop inhibits actin-activated ATPase of myosin II-subfragment 1 (S1) 3 and decreases the velocity of actin filaments on myosin II-HMM in an in vitro motility assay (5, 6).However, although as many as 24 classes of myosin have already been found (7), the contribution of the D-loop to actinmyosin interaction has so far been studied only for myosin II. Although all myosins carry out their motor functions by interacting with actin filaments, each class has a different role in vivo. For example, myosin V is an intracellular transporter, which is different from the role of myosin II, engaged mainly in muscle contraction or the formation of contractile ring. The rate-limiting step in the ATPase cycle in the presence of actin is the phosphate release in the case of myosin II but ADP release in myosin V, which allows this motor to spend most of its ATPase cycle (Ͼ90%) strongly bound to actin. Furthermore, similarly to kinesin (8), two heads of myosin V work cooperatively, coordinating their biochemical cycles via internal load (9 -11). These characteristics enable myosin V to move processively on actin filaments (12), and recent studies indicate that, similarly to myosin II, the attachment of myosin V also induces conformational changes in the D-loop region of actin (13).Here, to determine whether the D-loop contributes to the interact...

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Section: Comparison Of the Effects Of Subtilisin Cleavage And The M47amentioning

confidence: 83%

D-loop of Actin Differently Regulates the Motor Function of Myosins II and V

Kubota

Mikhailenko

Okabe

et al. 2009

Journal of Biological Chemistry

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“…Therefore, the quantum yield of these red spectrum tryptophan residues must be small. Hence, the fluorescence spectrum of G-actin must mainly be formed by the buried residues Trp-340 and Trp-356 (51). If Trp-340 and Trp-356 determine actin fluorescence, we can conclude that the t-BH treatment induces structural alterations in helix 338-348 and/or in loop 355-359 of the actin molecule.…”

Section: Discussionmentioning

confidence: 96%

Thetert-Butyl Hydroperoxide-Induced Oxidation of Actin Cys-374 Is Coupled with Structural Changes in Distant Regions of the Protein

1999

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The susceptibility of monomeric actin to both methionine and cysteine oxidation when treated with the oxidizing agent tert-butyl hydroperoxide (t-BH) was investigated. The results show that no methionine residue was susceptible to oxidation by t-BH at concentrations of 1-20 mM, while Cys-374, one of the five cysteine residues of the actin molecule, was found to be the site of the oxidative modification. Perturbations in the intrinsic tryptophan fluorescence and the decreased susceptibility to limited proteolysis by alpha-chymotrypsin and subtilisin of oxidized actin give an indication of some alterations in protein conformation in subdomain 1, and in the central segment of surface loop 39-51, in subdomain 2. Urea denaturation curves indicate a lower conformational stability for the oxidized actin. G-actin structural alterations due to Cys-374 oxidation produced by t-BH result in a decrease in the maximum rate of polymerization, an increase in both the delay time and the time required for half-maximum assembly, a decrease in the elongation rate, and enhancement of the critical monomer concentration for polymerization. The results suggest that oxidation of actin Cys-374 induces structural alterations in the conformation of at least two different distant regions of the molecule. The involvement of both the C-terminus of the actin polypeptide chain and the DNase-I-binding loop in the intermonomer interactions in the polymer could account for the altered kinetics of polymerization shown by the oxidized actin.

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“…This effect too is reversed by phalloidin binding to the modified F-actin (36). These data point to a possible role of segment [61][62][63][64][65][66][67][68][69] showing that replacements of residues 60 -69 with alanine abolish the ability of actin to bind DNase I (71), provides more evidence for conformational coupling between this stretch of residues and 38 -52 loop on the top of subdomain 2.…”

Section: Discussionmentioning

confidence: 96%

“…However, the insensitivity of S1 interaction with dansyl-labeled actin to antidansyl IgG binding has led to interpretation of these data in terms of allosteric effects of S1 binding to the other sites in actin (63). Evidence for intramolecular signal transmission in the reverse direction, from loop 38 -52 to subdomain 1, has been provided by studies on G-actin (64,65). An intra-or intermolecular conformational coupling between loop 38 -52 and myosin binding sites in subdomain 1 of actin is also a likely explanation for the suppression of acto-S1 ATPase activity when this loop is disrupted by subtilisin or ECP cleavage.…”

Section: Discussionmentioning

confidence: 99%

The DNase-I Binding Loop of Actin May Play a Role in the Regulation of Actin-Myosin Interaction by Tropomyosin/Troponin

Moraczewska

Gruszczynska-Biegala

Redowicz

et al. 2004

Journal of Biological Chemistry

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, on its interaction with myosin S1 in the presence and absence of tropomyosin or tropomyosin-troponin. Both individual modifications reduced activation of S1 ATPase by actin to a similar extent. The effect of ECP cleavage, but not of subtilisin cleavage, was partially reversed by stabilization of interprotomer contacts with phalloidin, indicating different pathways of signal transmission from the N-and C-terminal parts of loop 38 -52 to myosin binding sites. ECP cleavage diminished the affinity to tropomyosin and reduced its inhibition of acto-S1 ATPase at low S1 concentrations, but increased the tropomyosin-mediated cooperative enhancement of the ATPase by S1 binding to actin. These effects were reversed by phalloidin. Subtilisin-cleaved actin more closely resembled unmodified actin than the ECP-modified actin. Limited proteolysis of the modified and unmodified F-actins revealed an allosteric effect of ECP cleavage on the conformation of the actin subdomain 4 region that is presumably involved in tropomyosin binding. Our results point to a possible role of the N-terminal part of loop 38 -52 of actin in communication between tropomyosin and myosin through changes in actin structure.

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Conformational changes in subdomain I of actin induced by proteolytic cleavage within the DNase I‐binding loop: energy transfer from tryptophan to AEDANS

Cited by 37 publications

References 19 publications

D-loop of Actin Differently Regulates the Motor Function of Myosins II and V

D-loop of Actin Differently Regulates the Motor Function of Myosins II and V

Thetert-Butyl Hydroperoxide-Induced Oxidation of Actin Cys-374 Is Coupled with Structural Changes in Distant Regions of the Protein

The DNase-I Binding Loop of Actin May Play a Role in the Regulation of Actin-Myosin Interaction by Tropomyosin/Troponin

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