Schriemer, David C.Rafiei, Atefeh2021-01-292021-01-292021-01-21Rafieh, A. (2021). Mass Spectrometry-based Integrative Structural Modeling of the Doublecortin-Microtubule Interaction (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.http://hdl.handle.net/1880/113038A comprehensive understanding of protein function requires the structural elucidation of physical contacts with other proteins, known as protein-protein interactions (PPIs). Many PPIs evade classical structural determination techniques for many reasons. One class of PPIs that is challenging to canonical techniques involves large multi-subunit protein complexes comprised of proteins with intrinsically disordered regions. The interaction between doublecortin (DCX) and microtubules (MTs) is a particularly intriguing example of such PPIs. Doublecortin is a critically important protein involved in neuronal development, which appears to function by engaging MTs in a complex interaction pattern involving both ordered and disordered domains. In this work, I developed and used a mass spectrometry-based integrative structural modeling (ISM) approach to generate a unifying structural model of DCX-MT interaction. A crosslinking-mass spectrometry (XL-MS) workflow was developed for the analysis of interactions involving MTs in general. Different crosslinker reagents targeting different amino acid classes were tested and the integrity of the MTs in response to the crosslinking reactions were monitored. Then, a DCX-MT construct was reconstituted in-vitro and XL-MS performed using different reagents. Doublecortin self-association was investigated using isotopically-labeled DCX, to differentiate between inter- and intra- DCX crosslink peptides. It was found that DCX self-associates only in the presence of MTs. The majority of the inter-DCX crosslinks were observed in the C-terminus regions of the DCX sequence. Finally, the structural elucidation of DCX-MT interaction was carried out in a step-wise approach. The residue-based distance restraints from XL-MS along with the cryo-EM map of DCX-MT and the X-ray structures of protein subunits were combined in the Integrative Modeling Platform to identify the MT-binding domain. It was found that the N-terminus doublecortin-like domain is the primary MT binding domain, while the C-terminus doublecortin-like domain and C-tail are MT-dependent oligomerization domains. My modeling results support a DCX-MT interaction model in which DCX can self-associate between all immediate neighbors. Finally, it is shown that fast crosslinking chemistries (i.e. diazirine based photo-crosslinkers) were required to generate a converging model, as the more conventional long-lived crosslinkers are prone to conformational “kinetic trapping”. This has implications for ISM of similar systems, and modeling in general.engUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.Mass Spectrometry, Chemical crosslinking, integrative modeling, microtubule, Doublecortin, Structural biologyEducation--SciencesHealth SciencesBiochemistrydoctoral thesis10.11575/PRISM/38605