Molecular modeling of the myosin-S1(A1) isoform

Aydt, Ewald M.; Wolff, Gerhard; Morano, Ingo

doi:10.1016/j.jsb.2007.04.002

Cited by 42 publications

(41 citation statements)

References 38 publications

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“…5). This result is consistent with the previously published results of electron microscopy (19), functional mutations (37), cross-linking (30), and molecular modeling (38). In contrast to the ELC-dependent distances in the strongly bound complex, in the weakly bound complex the distance within acto-S1A1 (9.6 ± 0.2 nm) was essentially the same as in acto-S1A2 (9.7 ± 0.2 nm).…”

Section: Discussionsupporting

confidence: 92%

Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain

Guhathakurta

Próchniewicz

Thomas

2015

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

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We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to determine the role of myosin essential light chains (ELCs) in structural transitions within the actomyosin complex. Skeletal muscle myosins have two ELC isoforms, A1 and A2, which differ by an additional 40-45 residues at the N terminus of A1, and subfragment 1 (S1) containing A1 (S1A1) has higher catalytic efficiency and higher affinity for actin than S1A2. ELC's location at the junction between the catalytic and light-chain domains gives it the potential to play a central role in the force-generating power stroke. Therefore, we measured site-directed TR-FRET between a donor on actin and an acceptor near the C terminus of ELC, detecting directly the rotation of the light-chain domain (lever arm) relative to actin (power stroke), induced by the interaction of ATPbound myosin with actin. TR-FRET resolved the weakly bound (W) and strongly bound (S) states of actomyosin during the W-to-S transition (power stroke). We found that the W states are essentially the same for the two isoenzymes, but the S states are quite different, indicating a much larger movement of S1A1. FRET from actin to a probe on the N-terminal extension of A1 showed close proximity to actin. We conclude that the N-terminal extension of A1-ELC modulates the W-to-S structural transition of acto-S1, so that the light-chain domain undergoes a much larger power stroke in S1A1 than in S1A2. These results have profound implications for understanding the contractile function of actomyosin, as needed in therapeutic design for muscle disorders.muscle | actomyosin | light chains | fluorescence resonance energy transfer A central challenge in the biophysics of motility is to understand the molecular mechanisms of structural transitions in the actomyosin ATPase (enzyme code 3.6.4.1) cycle, in which myosin alters its affinity for actin in a nucleotide-dependent manner from weak (W) actin-binding states (with ATP or ADP-P i bound to the myosin active site) to strong (S) actin-binding states (with ADP or no nucleotide, i.e., rigor), resulting in the generation of force. It is particularly important to obtain direct information about the structural dynamics of the whole actomyosin complex, because there is evidence that each protein, when studied separately, undergoes changes in structure and dynamics during transitions from the W to S states (1-5). S states are relatively stable and easy to study, but the W actomyosin complex is less accessible to study, due to its transient and structurally dynamic nature.The myosin heavy chain starts with the N-terminal catalytic domain (CD), which contains the nucleotide-binding pocket and the actin-binding site. A small converter region connects the CD to an extended α-helical segment that forms the core of the lightchain domain (LCD) (6, 7). The LCD contains two IQ motifs that serve as binding sites for one essential light chain (ELC) and one regulatory light chain (RLC) and is proposed to function as a lever arm that amplifies small structural c...

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Section: Discussionsupporting

confidence: 92%

Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain

Guhathakurta

Próchniewicz

Thomas

2015

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

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“…3A, red to green). Such sensitivity to the ionic strength specifies the electrostatic nature of this interaction and mirrors the model-based proposal that a positively charged NTE binds to a cluster of negatively charged residues in the C-terminal region of actin (19). These results clearly show that actin-ANT FRET is sensitive to the association of S1 NTEs with actin and also to ionic strength; both factors are known to be strong modulators of the actinmyosin interaction (19).…”

Section: Actin-ant Fret Biosensorsupporting

confidence: 77%

“…On the other hand, FRET was not affected in the presence of ATP, where S1 binds weakly and is rotationally disordered, and the distance between A1NTE and actin significantly increases compared with that in the strongly bound complex (25,26). FRET of actin-ANT complex was also significantly decreased by an increase in ionic strength (0.1 M KCl), further supporting the similarity between ANT-actin and actin-A1NTE interaction; a structural model of the acto-S1A1 complex (19) shows that the positively charged N terminus of A1 binds to a cluster of negatively charged residues in the C-terminal region of actin (19). Thus, we conclude that our actin- ANT FRET sensor (a) mimics well actin-A1NTE binding, (b) has potential as a platform for the discovery of allosteric modulators of the actin-myosin interaction, and (c) can ultimately help in developing therapies to treat muscle disorders.…”

Section: Validity Of Actin-ant Fret Sensorsupporting

confidence: 55%

High-throughput screen, using time-resolved FRET, yields actin-binding compounds that modulate actin–myosin structure and function

Guhathakurta

Próchniewicz

Grant

et al. 2018

Journal of Biological Chemistry

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We have used a novel time-resolved FRET (TR-FRET) assay to detect small-molecule modulators of actin-myosin structure and function. Actin-myosin interactions play crucial roles in the generation of cellular force and movement. Numerous mutations and post-translational modifications of actin or myosin disrupt muscle function and cause life-threatening syndromes. Here, we used a FRET biosensor to identify modulators that bind to the actin-myosin interface and alter the structural dynamics of this complex. We attached a fluorescent donor to actin at Cys-374 and a nonfluorescent acceptor to a peptide containing the 12 N-terminal amino acids of the long isoform of skeletal muscle myosin's essential light chain. The binding site on actin of this acceptor-labeled peptide (ANT) overlaps with that of myosin, as indicated by () a similar distance observed in the actin-ANT complex as in the actin-myosin complex and () a significant decrease in actin-ANT FRET upon binding myosin. A high-throughput FRET screen of a small-molecule library (NCC, 727 compounds), using a unique fluorescence lifetime readout with unprecedented speed and precision, showed that FRET is significantly affected by 10 compounds in the micromolar range. Most of these "hits" alter actin-activated myosin ATPase and affect the microsecond dynamics of actin detected by transient phosphorescence anisotropy. We conclude that the actin-ANT TR-FRET assay enables detection of pharmacologically active compounds that affect actin structural dynamics and actomyosin function. This assay establishes feasibility for the discovery of allosteric modulators of the actin-myosin interaction, with the ultimate goal of developing therapies for muscle disorders.

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“…[38][39][40][41][42] A structural model for this sequence has recently been proposed. 43 For a fairly extended chain with 3.5 Å per residue, there would be a total length of around 95 Å for 27 residues, whereas for a compact α-helical structure the length would be about 40 Å. Taking a plausible structure to be nearer to the extended end of these extremes, as in Ref.…”

Section: Discussionmentioning

confidence: 99%

Crystal Structure of the C1 domain of Cardiac Myosin Binding Protein-C: Implications for Hypertrophic Cardiomyopathy

Govada

Carpenter

Fonseca

et al. 2008

Journal of Molecular Biology

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Molecular modeling of the myosin-S1(A1) isoform

Cited by 42 publications

References 38 publications

Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain

Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain

High-throughput screen, using time-resolved FRET, yields actin-binding compounds that modulate actin–myosin structure and function

Crystal Structure of the C1 domain of Cardiac Myosin Binding Protein-C: Implications for Hypertrophic Cardiomyopathy

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