The inability to obtain atomic structures on actin cytoskeleton has severely hindered our understanding of this most abundant eukaryotic protein and its dynamic turnovers in performing a myriad of cellular functions such as cell motility, cytokinesis and morphogenesis through interactions with hundreds of actin-binding proteins. This application describes an innovative double-mutant strategy to overcome this longstanding barrier in the actin cytoskeleton field. Indeed, this novel double-mutant strategy has enabled the PIs' successful determination of the very first atomic crystal structure of actin-nucleator complex (actin-Cobl complex). In addition, a similar approach has been used to solve the structure of actin with a bacterial effector, VopL. The observed non-filament-like conformation in actin-Cobl structure and filament-like conformation in actin-VopL structure together suggest that both types of conformation are fully accessible to an actin complex obtained via the double-mutant strategy, thus its true conformation is most likely preserved. The experiments proposed in this application will apply the double-mutant strategy to three most divergent members of the latest class actin nucleators characterized by the presence of tandem actin-binding sites. The goal is to decipher their molecular mechanisms of actin nucleation, the roles of ATP hydrolysis in their functional cycle, and how they collaborate with specific cellular components to fulfill their functions. This goal will be achieved in three Specific Aims using combined structural and functional approaches:
Aim 1 : Mechanisms of Cobl-mediated actin nucleation;
Aim 2 : Mechanistic study of actin nucleation by Lmod;
and Aim 3 : Mechanistic study of APC-mediated actin nucleation. The extensive preliminary studies presented in this application suggest a high promise of success for the proposed research. Results from this study will be significant not only in elucidating the molecular mechanisms of their respective roles in neurogenesis, muscle development and tumor initiation, but also in unraveling some long-sought-after general underpinnings for de novo actin nucleation, a process underlying every stage of mammalian development as well as many types of pathogenic infection. By providing a detailed atomic gallery of how de novo actin nucleation is accomplished and regulated, this study will stimulate deeper mechanistic investigations on these nucleators as well as discovery and characterization of new actin nucleators. More importantly, this application will validate the double-mutant strategy on proteins/protein fragments with and without actin nucleation activities, thus providing sufficient proof-of-principle for extending this approach to many other actin-involved biological processes beyond actin nucleation. Ultimately, the research along this line is expected to directly benefit the treatment of many forms of human diseases due to actin cytoskeleton malfunctions including neurodegenerative disorders, muscular dystrophy, tumorigenesis and metastasis.

Public Health Relevance

Actin cytoskeleton performs a myriad of fundamental cellular functions and causes fatal diseases when malfunctions. In this application we describe a double-mutant strategy that has allowed the determination of the very first atomic structure of an actin-nucleator. The proposed study will provide sufficient proof-of-principle for applying this approach to many other actin-involved biological processes beyond actin nucleation. A mechanistic understanding of actin cytoskeleton and dynamics will directly benefit the treatment of many forms of human diseases including neurodegenerative disorders, muscular dystrophy, tumorigenesis and metastasis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM116280-01
Application #
8979897
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Gindhart, Joseph G
Project Start
2016-01-01
Project End
2019-12-31
Budget Start
2016-01-01
Budget End
2016-12-31
Support Year
1
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Baylor College of Medicine
Department
Biochemistry
Type
Schools of Medicine
DUNS #
051113330
City
Houston
State
TX
Country
United States
Zip Code
77030
Du, Junqing; Kirk, Brian; Zeng, Jia et al. (2018) Three classes of response elements for human PRC2 and MLL1/2-Trithorax complexes. Nucleic Acids Res 46:8848-8864
Xu, Gang; Ma, Tianqi; Zang, Tianwu et al. (2018) OPUS-CSF: A C-atom-based scoring function for ranking protein structural models. Protein Sci 27:286-292
Ni, Fengyun; Kondrashkina, Elena; Wang, Qinghua (2018) Determinant of receptor-preference switch in influenza hemagglutinin. Virology 513:98-107
Lin, Xingcheng; Noel, Jeffrey K; Wang, Qinghua et al. (2018) Atomistic simulations indicate the functional loop-to-coiled-coil transition in influenza hemagglutinin is not downhill. Proc Natl Acad Sci U S A 115:E7905-E7913
Yu, Linglin; Lu, Mingyang; Jia, Dongya et al. (2017) Modeling the Genetic Regulation of Cancer Metabolism: Interplay between Glycolysis and Oxidative Phosphorylation. Cancer Res 77:1564-1574
Xu, Gang; Ma, Tianqi; Zang, Tianwu et al. (2017) OPUS-DOSP: A Distance- and Orientation-Dependent All-Atom Potential Derived from Side-Chain Packing. J Mol Biol 429:3113-3120
Zhang, Cheng; Drake, Justin A; Ma, Jianpeng et al. (2017) Optimal updating magnitude in adaptive flat-distribution sampling. J Chem Phys 147:174105
Lin, Xingcheng; Noel, Jeffrey K; Wang, Qinghua et al. (2016) Lowered pH Leads to Fusion Peptide Release and a Highly Dynamic Intermediate of Influenza Hemagglutinin. J Phys Chem B 120:9654-60
Chen, Xiaorui; Ni, Fengyun; Kondrashkina, Elena et al. (2015) Mechanisms of leiomodin 2-mediated regulation of actin filament in muscle cells. Proc Natl Acad Sci U S A 112:12687-92