scholarly journals Selective Perturbation of the Myosin Recovery Stroke by Point Mutations at the Base of the Lever Arm Affects ATP Hydrolysis and Phosphate Release

2007 ◽  
Vol 282 (24) ◽  
pp. 17658-17664 ◽  
Author(s):  
András Málnási-Csizmadia ◽  
Judit Tóth ◽  
David S. Pearson ◽  
Csaba Hetényi ◽  
László Nyitray ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Point Mutations ◽  
Phosphate Release ◽  
Lever Arm ◽  
Recovery Stroke

scholarly journals Structural Basis for Actin Assembly, Activation of ATP Hydrolysis, and Delayed Phosphate Release

Cell ◽  
2010 ◽  
Vol 143 (2) ◽  
pp. 275-287 ◽  
Author(s):  
Kenji Murakami ◽  
Takuo Yasunaga ◽  
Taro Q.P. Noguchi ◽  
Yuki Gomibuchi ◽  
Kien X. Ngo ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Structural Basis ◽  
Actin Assembly ◽  
Phosphate Release

scholarly journals Mechanochemical Function of Myosin II: Investigation into the Recovery Stroke and ATP Hydrolysis

2020 ◽  
Vol 124 (45) ◽  
pp. 10014-10023
Author(s):  
Anthony P. Baldo ◽  
Jil C. Tardiff ◽  
Steven D. Schwartz
Keyword(s):  
Atp Hydrolysis ◽  
Myosin Ii ◽  
Recovery Stroke

scholarly journals Two Essential Light Chains Regulate the MyoA Lever Arm To Promote Toxoplasma Gliding Motility

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Melanie J. Williams ◽  
Hernan Alonso ◽  
Marta Enciso ◽  
Saskia Egarter ◽  
Lilach Sheiner ◽  
...  
Keyword(s):  
Light Chain ◽  
Therapeutic Potential ◽  
Point Mutations ◽  
Gliding Motility ◽  
Host Cells ◽  
Light Chains ◽  
Apicomplexan Parasites ◽  
Essential Light Chain ◽  
Lever Arm ◽  
Parasite Motility

ABSTRACT Key to the virulence of apicomplexan parasites is their ability to move through tissue and to invade and egress from host cells. Apicomplexan motility requires the activity of the glideosome, a multicomponent molecular motor composed of a type XIV myosin, MyoA. Here we identify a novel glideosome component, essential light chain 2 (ELC2), and functionally characterize the two essential light chains (ELC1 and ELC2) of MyoA in Toxoplasma. We show that these proteins are functionally redundant but are important for invasion, egress, and motility. Molecular simulations of the MyoA lever arm identify a role for Ca2+ in promoting intermolecular contacts between the ELCs and the adjacent MLC1 light chain to stabilize this domain. Using point mutations predicted to ablate either the interaction with Ca2+ or the interface between the two light chains, we demonstrate their contribution to the quality, displacement, and speed of gliding Toxoplasma parasites. Our work therefore delineates the importance of the MyoA lever arm and highlights a mechanism by which this domain could be stabilized in order to promote invasion, egress, and gliding motility in apicomplexan parasites. IMPORTANCE Tissue dissemination and host cell invasion by apicomplexan parasites such as Toxoplasma are pivotal to their pathogenesis. Central to these processes is gliding motility, which is driven by an actomyosin motor, the MyoA glideosome. Others have demonstrated the importance of the MyoA glideosome for parasite motility and virulence in mice. Disruption of its function may therefore have therapeutic potential, and yet a deeper mechanistic understanding of how it works is required. Ca2+-dependent and -independent phosphorylation and the direct binding of Ca2+ to the essential light chain have been implicated in the regulation of MyoA activity. Here we identify a second essential light chain of MyoA and demonstrate the importance of both to Toxoplasma motility. We also investigate the role of Ca2+ and the MyoA regulatory site in parasite motility and identify a potential mechanism whereby binding of a divalent cation to the essential light chains could stabilize the myosin to allow productive movement.

scholarly journals Genetic and biochemical analysis of Msh2p-Msh6p: role of ATP hydrolysis and Msh2p-Msh6p subunit interactions in mismatch base pair recognition.

1997 ◽  
Vol 17 (5) ◽  
pp. 2436-2447 ◽  
Author(s):  
E Alani ◽  
T Sokolsky ◽  
B Studamire ◽  
J J Miret ◽  
R S Lahue
Keyword(s):  
Base Pair ◽  
Biochemical Analysis ◽  
Atp Hydrolysis ◽  
Point Mutations ◽  
Binding Domain ◽  
Dominant Negative ◽  
Mutant Protein ◽  
Atp Binding ◽  
Mutant Proteins ◽  
Mismatch Recognition

Recent studies have shown that Saccharomyces cerevisiae Msh2p and Msh6p form a complex that specifically binds to DNA containing base pair mismatches. In this study, we performed a genetic and biochemical analysis of the Msh2p-Msh6p complex by introducing point mutations in the ATP binding and putative helix-turn-helix domains of MSH2. The effects of these mutations were analyzed genetically by measuring mutation frequency and biochemically by measuring the stability, mismatch binding activity, and ATPase activity of msh2p (mutant msh2p)-Msh6p complexes. A mutation in the ATP binding domain of MSH2 did not affect the mismatch binding specificity of the msh2p-Msh6p complex; however, this mutation conferred a dominant negative phenotype when the mutant gene was overexpressed in a wild-type strain, and the mutant protein displayed biochemical defects consistent with defects in mismatch repair downstream of mismatch recognition. Helix-turn-helix domain mutant proteins displayed two different properties. One class of mutant proteins was defective in forming complexes with Msh6p and also failed to recognize base pair mismatches. A second class of mutant proteins displayed properties similar to those observed for the ATP binding domain mutant protein. Taken together, these data suggested that the proposed helix-turn-helix domain of Msh2p was unlikely to be involved in mismatch recognition. We propose that the MSH2 helix-turn-helix domain mediates changes in Msh2p-Msh6p interactions that are induced by ATP hydrolysis; the net result of these changes is a modulation of mismatch recognition.

Swing of the lever arm of a myosin motor at the isomerization and phosphate-release steps

Nature ◽  
10.1038/24640 ◽  
1998 ◽  
Vol 396 (6709) ◽  
pp. 380-383 ◽  
Author(s):  
Yoshikazu Suzuki ◽  
Takuo Yasunaga ◽  
Reiko Ohkura ◽  
Takeyuki Wakabayashi ◽  
Kazuo Sutoh
Keyword(s):  
Phosphate Release ◽  
Myosin Motor ◽  
Lever Arm

scholarly journals Structures of the substrate-engaged 26S proteasome reveal the mechanisms for ATP hydrolysis-driven translocation

2018 ◽  
Author(s):  
Andres H. de la Peña ◽  
Ellen A. Goodall ◽  
Stephanie N. Gates ◽  
Gabriel C. Lander ◽  
Andreas Martin
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
26S Proteasome ◽  
Atp Binding ◽  
Conformational States ◽  
Phosphate Release ◽  
Protein Substrates ◽  
Cellular Processes ◽  
Hexameric Atpase ◽  
Central Pore

AbstractThe 26S proteasome is the primary eukaryotic degradation machine and thus critically involved in numerous cellular processes. The hetero-hexameric ATPase motor of the proteasome unfolds and translocates targeted protein substrates into the open gate of a proteolytic core, while a proteasomal deubiquitinase concomitantly removes substrate-attached ubiquitin chains. However, the mechanisms by which ATP hydrolysis drives the conformational changes responsible for these processes have remained elusive. Here we present the cryo-EM structures of four distinct conformational states of the actively ATP-hydrolyzing, substrate-engaged 26S proteasome. These structures reveal how mechanical substrate translocation accelerates deubiquitination, and how ATP-binding, hydrolysis, and phosphate-release events are coordinated within the AAA+ motor to induce conformational changes and propel the substrate through the central pore.

scholarly journals FRET and optical trapping reveal mechanisms of actin activation of the power stroke and phosphate release in myosin V

2020 ◽  
Vol 295 (51) ◽  
pp. 17383-17397 ◽  
Author(s):  
Laura K. Gunther ◽  
John A. Rohde ◽  
Wanjian Tang ◽  
Joseph A. Cirilo ◽  
Christopher P. Marang ◽  
...  
Keyword(s):  
Active Site ◽  
Maximum Rate ◽  
Optical Trapping ◽  
Structural Changes ◽  
Nucleotide Binding ◽  
Power Stroke ◽  
Myosin V ◽  
Phosphate Release ◽  
Lever Arm ◽  
Binding Regions

Myosins generate force and motion by precisely coordinating their mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. We utilized a FRET approach to examine the kinetics of structural changes in the force-generating lever arm in myosin V. We directly compared the FRET results with single-molecule mechanical events examined by optical trapping. We introduced a mutation (S217A) in the conserved switch I region of the active site to examine how myosin couples structural changes in the actin- and nucleotide-binding regions with force generation. Specifically, S217A enhanced the maximum rate of lever arm priming (recovery stroke) while slowing ATP hydrolysis, demonstrating that it uncouples these two steps. We determined that the mutation dramatically slows both actin-induced rotation of the lever arm (power stroke) and phosphate release (≥10-fold), whereas our simulations suggest that the maximum rate of both steps is unchanged by the mutation. Time-resolved FRET revealed that the structure of the pre– and post–power stroke conformations and mole fractions of these conformations were not altered by the mutation. Optical trapping results demonstrated that S217A does not dramatically alter unitary displacements or slow the working stroke rate constant, consistent with the mutation disrupting an actin-induced conformational change prior to the power stroke. We propose that communication between the actin- and nucleotide-binding regions of myosin assures a proper actin-binding interface and active site have formed before producing a power stroke. Variability in this coupling is likely crucial for mediating motor-based functions such as muscle contraction and intracellular transport.

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