Crystal structure of the rigor‐like human non‐muscle myosin‐2 motor domain

Münnich, Stefan; Pathan-Chhatbar, Salma; Manstein, Dietmar J.

doi:10.1016/j.febslet.2014.11.007

Cited by 19 publications

(11 citation statements)

References 32 publications

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“…Fitting analysis produced distributions that were not statistically distinct from their nucleotide-free analogs ( Table 1), although the average primary distance derived from the Relay-to-HW construct does exhibit a slight negative shift relative to Apo conditions, consistent with a similar construct studied previously without actin present (7). Overall, this result is not surprising, as evidence from previous structural studies indicates that the effects of MgADP within the myosin CD probably manifest as subtle rotations of individual structural elements, with minimal relative spatial displacement (29)(30)(31)(32)(33). We therefore hold these inter-helix distance centers constant in the constrained modeling of the MgADP-bound rigor complex in the following section.…”

Section: No Significant Changes In Inter-helix Distance Distributionssupporting

confidence: 83%

High-resolution models of actin-bound myosin from EPR of a bifunctional spin label

Binder

Thompson

Thomas

2018

Preprint

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We have employed two complementary high-resolution electron paramagnetic resonance (EPR) techniques with a bifunctional spin label (BSL) to test and refine protein structural models based on crystal structures and cryo-EM. We demonstrate this approach by investigating the effects of nucleotide binding on the structure of myosin's catalytic domain (CD), while myosin is in complex with actin. Unlike conventional spin labels attached to single Cys, BSL reacts with a pair of Cys; in this study, we thoroughly characterize BSL's rigid, highly stereoselective attachment to protein α-helices, which permits accurate measurements of orientation and distance. Distance constraints were obtained from double electron-electron resonance (DEER) on myosin constructs labeled with BSL specifically at two sites. Constraints for orientation of individual helices were obtained previously from continuous-wave EPR (CW-EPR) of myosin labeled at specific sites with BSL in oriented muscle fibers. We have shown previously that CW-EPR of BSL quantifies helix orientation within actin-bound myosin; here we show that the addition of high-resolution distance constraints by DEER alleviates remaining spatial ambiguity, allowing for direct testing and refinement of atomic structural models. This approach is applicable to any orientable complex (e.g., membranes or filaments) in which site-specific di-Cys mutation is feasible.

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Section: No Significant Changes In Inter-helix Distance Distributionssupporting

confidence: 83%

High-resolution models of actin-bound myosin from EPR of a bifunctional spin label

Binder

Thompson

Thomas

2018

Preprint

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“…See also Supplementary Movie 1 . f Surface representation of myosin IIb in the rigor state (4PD3 34 ). The OM-PPS site is completely open and unable to bind OM strongly since the converter is unprimed.…”

Section: Introductionmentioning

confidence: 99%

Mechanistic and structural basis for activation of cardiac myosin force production by omecamtiv mecarbil

et al. 2017

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Omecamtiv mecarbil is a selective, small-molecule activator of cardiac myosin that is being developed as a potential treatment for heart failure with reduced ejection fraction. Here we determine the crystal structure of cardiac myosin in the pre-powerstroke state, the most relevant state suggested by kinetic studies, both with (2.45 Å) and without (3.10 Å) omecamtiv mecarbil bound. Omecamtiv mecarbil does not change the motor mechanism nor does it influence myosin structure. Instead, omecamtiv mecarbil binds to an allosteric site that stabilizes the lever arm in a primed position resulting in accumulation of cardiac myosin in the primed state prior to onset of cardiac contraction, thus increasing the number of heads that can bind to the actin filament and undergo a powerstroke once the cardiac cycle starts. The mechanism of action of omecamtiv mecarbil also provides insights into uncovering how force is generated by molecular motors.

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“…Middle , For kinetic studies of NM2C, R788K, and R788E, the motor domain was directly fused to spectrin repeats 1 and 2 from Dictyostelium α-actinin (light grey) which serves as an artificial lever arm. The concept of the artificial lever arm has been successfully used in structural and kinetic studies of myosins-2 ( Heissler and Manstein, 2011 ; Furch et al, 1999 ; Kliche et al, 2001 ; Münnich et al, 2014 ). Bottom : For structural studies, the N-terminal 45 amino acids were deleted.…”

Section: Introductionmentioning

confidence: 99%

Mechanistic insights into the active site and allosteric communication pathways in human nonmuscle myosin-2C

Chinthalapudi

Heissler

Preller

et al. 2017

eLife

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Despite a generic, highly conserved motor domain, ATP turnover kinetics and their activation by F-actin vary greatly between myosin-2 isoforms. Here, we present a 2.25 Å pre-powerstroke state (ADP⋅VO4) crystal structure of the human nonmuscle myosin-2C motor domain, one of the slowest myosins characterized. In combination with integrated mutagenesis, ensemble-solution kinetics, and molecular dynamics simulation approaches, the structure reveals an allosteric communication pathway that connects the distal end of the motor domain with the active site. Disruption of this pathway by mutation of hub residue R788, which forms the center of a cluster of interactions connecting the converter, the SH1-SH2 helix, the relay helix, and the lever, abolishes nonmuscle myosin-2 specific kinetic signatures. Our results provide insights into structural changes in the myosin motor domain that are triggered upon F-actin binding and contribute critically to the mechanochemical behavior of stress fibers, actin arcs, and cortical actin-based structures.

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Crystal structure of the rigor‐like human non‐muscle myosin‐2 motor domain

Cited by 19 publications

References 32 publications

High-resolution models of actin-bound myosin from EPR of a bifunctional spin label

High-resolution models of actin-bound myosin from EPR of a bifunctional spin label

Mechanistic and structural basis for activation of cardiac myosin force production by omecamtiv mecarbil

Mechanistic insights into the active site and allosteric communication pathways in human nonmuscle myosin-2C

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