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ADPr-actin trimer is a short-F-actin oligomer that binds selectively to F-actin binding proteins

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Title: ADPr-actin trimer is a short-F-actin oligomer that binds selectively to F-actin binding proteins
Author: Sidhu, Navneet
Department: Department of Molecular and Cellular Biology
Program: Molecular and Cellular Biology
Advisor: Dawson, John F.
Abstract: Actin is one of the most abundant and conserved proteins involved in a myriad of biological functions critical for maintaining eukaryotic life. The functions of actin rely on dynamic reorganization of actin filaments, which is modulated by several Actin Binding Proteins (ABPs). Despite the significance of these interactions in regulating actin function, the field lacked atomic details of interactions of F-actin with ABPs until the first decade of the 2000s. With advances in cryo-Electron Microscopy (cryo-EM) since 2015, structural models of F-actin alone and bound to ABPs have been proposed. The resolution of earlier EM structures lacked important atomic details; consequently, traditional X-ray crystallography remains the mainstay technique to obtain high resolution structures. However, the ability of actin to self-assemble into filaments of varying lengths limits the application of X-ray crystallography. To overcome this challenge posed by actin, a short, non-polymerizable F-actin oligomer called ADPr-trimer was generated. The aim of my project was to generate heterocomplexes of ADPr-trimer with F-ABPs for structural work. A candidate-based approach was applied to determine interactions of ADPr-trimer with purified ABPs (myosin subfragment-1, cofilin and gelsolin). Cofilin bound to ADPr-trimer and overcome polymerization inhibition properties of ADPr-trimer and forming filaments, and myosin did not bind ADPr-trimer. Gelsolin bound to ADPr-trimer; however, the complex did not yield crystals. Therefore, a negative staining EM approach was used to determine the suitability of the gelsolin:trimer complex for cryo-EM structure determination. The EM-micrographs revealed the presence of heterogeneous species in the gelsolin:trimer complex sample. EM work led to identifying a workflow for future EM work in the lab. In addition, an unbiased approach was applied to identify binding proteins involving pull-down of ABPs from cell lysates on ADPr-trimer affinity columns coupled with mass spectrometry (MS). An identified potential interactor, myosin light chain (MLC6B), was produced for biochemical validation of the interaction. Apo-MLC6B and MLC6B associated with myosin bound to ADPr-trimer in pull-down assays, supporting the MS results. Understanding the interactions of actin with ABPs through the application of proteomics and cryo-EM structural determination will advance our knowledge of the critical role of actin and its binding proteins in maintaining eukaryotic life.
URI: http://hdl.handle.net/10214/17714
Date: 2020-01-03
Rights: Attribution 4.0 International
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Attribution 4.0 International Except where otherwise noted, this item's license is described as Attribution 4.0 International