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Molecular characterization of linker and loop-mediated structural modulation and hinge motion in the C4-C5 domains of
Chang Yoon Doh1, Nikhil Bharambe1, Joshua B Holmes1
1Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, OH, USA.
Insights
The cardiac myosin binding protein C (cMyBPC) central domains utilize a flexible linker and loop to enable large-scale bending, revealing a novel "hinge-and-latch" mechanism for protein regulation.
Area of Science:
- Biochemistry
- Structural Biology
- Molecular Dynamics
Background:
- Cardiac myosin binding protein C (cMyBPC) is crucial for cardiac muscle function.
- The central C4 and C5 domains (C4C5) of cMyBPC possess a flexible linker and a cardiac-isoform specific loop.
- The functional significance of these structural elements in cMyBPC regulation remains under-explored.
Purpose of the Study:
- To investigate the structural and dynamic roles of the linker and loop regions within the C4C5 domains of cMyBPC.
- To elucidate the contribution of these regions to the protein's conformational flexibility and stability.
Main Methods:
- Expression of recombinant C4C5 proteins with modified linker and loop regions.
- Biophysical experiments to assess structural and thermal properties.
- Extended molecular dynamics simulations and principal component analyses.
Main Results:
- The linker and C5 loop regions significantly influence the secondary structure and thermal stability of C4C5.
- Molecular dynamics simulations revealed that C4C5 can adopt a fully bent or latched conformation.
- Analysis identified specific residue interactions characterizing the bent conformation, supporting a "hinge-and-latch" mechanism.
Conclusions:
- A "hinge-and-latch" mechanism involving the linker and loop regulates cMyBPC's large-scale conformational changes.
- These dynamics modulate cMyBPC's motion and global conformation, potentially impacting actomyosin activity.
- Findings offer insights into muscle isoform-specific regulation and cMyBPC's role in signal propagation.
Introduction:
The central C4 and C5 domains (C4C5) of cardiac myosin binding protein C (cMyBPC) contain a flexible interdomain linker and a cardiac-isoform specific loop. However, their importance in the functional regulation of cMyBPC has not been extensively studied.
Methods And Results:
We expressed recombinant C4C5 proteins with deleted linker and loop regions and performed biophysical experiments to determine each of their structural and dynamic roles. We show that the linker and C5 loop regions modulate the secondary structure and thermal stability of C4C5. Furthermore, we provide evidence through extended molecular dynamics simulations and principle component analyses that C4C5 can adopt a completely bent or latched conformation. The simulation trajectory and interaction network analyses reveal that the completely bent conformation of C4C5 exhibits a specific pattern of residue-level interactions. Therefore, we propose a "hinge-and-latch" mechanism where the linker allows a great degree of flexibility and bending, while the loop aids in achieving a completely bent and latched conformation. Although this may be one of many bent positions that C4C5 can adopt, we illustrate for the first time in molecular detail that this type of large scale conformational change can occur in the central domains of cMyBPC.
Conclusions:
Our hinge-and-latch mechanism demonstrates that the linker and loop regions participate in dynamic modulation of cMyBPC's motion and global conformation. These structural and dynamic features may contribute to muscle isoform-specific regulation of actomyosin activity, and have potential implications regarding its ability to propagate or retract cMyBPC's regulatory N-terminal domains.
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