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Dissertation
Roles of the N-terminal region of leiomodin in thin filament formation
Washington State University
Doctor of Philosophy (PhD), Washington State University
2022
DOI:
https://doi.org/10.7273/000005063
Abstract
Leiomodin (Lmod), a muscle protein, regulates thin filament formation, either by regulating the thin filament length, by nucleating actin to form new thin filaments, or both. Lmod allows controlled elongation of thin filaments at the pointed by binding to both tropomyosin (Tpm) and actin, with a single Tpm binding site (TpmBS), and a total of three actin-binding sites (ABSs). The disordered N-terminal part of Lmod contains the TpmBS, one of the ABSs, and a flexible linker with previously unknown function, connected to the second ABS, a leucine-rich-repeat (LRR) domain. Another disordered linker connects the LRR to the third ABS, a Wiskott Aldrich Homology (WH2) domain. Whether Lmod is a thin filament elongator, an actin nucleator, or both, in cells has been debated, due in part to a lack of structural data of its Tpm- and actin-binding interfaces in the disordered N-terminal region of the protein. Using nuclear magnetic resonance (NMR) and molecular dynamics simulations (MDS), we solved the structure of the Lmod TpmBS and Tpm binding interface. The structure confirmed that this interaction can only occur at the pointed end. We used this structure to build a model of Lmod at the pointed end and to propose a mechanism of controlled elongation of the thin filament. Key to this mechanism is the ability of the N-terminal ABS to switch from a “closed gate” conformation to an “open gate” conformation, allowing the addition of an actin monomer and initiating elongation. Using circular dichroism (CD) and NMR, we determined the secondary structure of the N-terminal ABS and mapped residues that participate in actin binding. This site binds to actin with a disordered “hinge”, and with an amphipathic α-helical “latch”, which supports the model. The dissociation of this site from actin is allowed because the linker connecting it to the LRR is sufficiently long. Using NMR, we showed that the linker serves as a Ca2+-binding site. Then, using pyrene actin polymerization assay and transmission electron microscopy (TEM), we showed that the nucleation activity of Lmod is attenuated when it is Ca2+ bound, indicating that Lmod binding to actin is regulated by Ca2+.
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Details
- Title
- Roles of the N-terminal region of leiomodin in thin filament formation
- Creators
- Garry Edward Smith Jr
- Contributors
- Alla S Kostyukova (Advisor)John R Cort (Committee Member)Wenji Dong (Committee Member)Bertrand Tanner (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- School of Chemical Engineering and Bioengineering
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Publisher
- Washington State University
- Number of pages
- 174
- Identifiers
- 99901019538101842
- Language
- English
- Resource Type
- Dissertation