Structural and biochemical investigation of the regulation of Rab11a by the guanine nucleotide exchange factors SH3BP5 and TRAPPII

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dc.contributor.author Jenkins, Meredith L.
dc.date.accessioned 2019-11-29T18:30:05Z
dc.date.copyright 2019 en_US
dc.date.issued 2019-11-29
dc.identifier.uri http://hdl.handle.net/1828/11343
dc.description.abstract Rab11 is a critical GTPase involved in the regulation of membrane trafficking in the endocytic pathway, and it’s misregulation is involved in a variety of human diseases including Huntington’s disease and Alzheimer’s disease. Additionally, de novo mutations (DNMs) of Rab11 have been identified in patients with developmental disorders, and interestingly several parasites, viruses, and bacteria can subvert membrane trafficking through Rab11 positive vesicles to allow for replication and evasion from the immune system. Although Rab11 is one of the best characterized Rab GTPases, hindering the capability to completely understand Rab11 regulation and its role in human disease is the lack of detail describing how Rab11 proteins are activated by their cognate guanine nucleotide exchange factors (GEFs). This thesis is therefore focused on revealing the molecular mechanisms of the GEFs responsible for the activation of Rab11: SH3BP5 and TRAPPII. To investigate the recently discovered GEF SH3BP5, we solved the 3.1Å structure of Rab11 bound to SH3BP5 and revealed a coiled coil architecture of SH3BP5 that mediates exchange through a unique Rab-GEF interaction. The structure revealed a unique rearrangement of the switch-I region of Rab11 compared to other solved Rab-GEF structures, with a constrained conformation when bound to SH3BP5. Mutational analysis of switch-I revealed the molecular determinants that allow for Rab11 selectivity over evolutionarily similar Rab GTPases, and GEF deficient mutants of SH3BP5 show greatly decreased Rab11 activation in cellular assays of active Rab11. To interrogate the highly controversial GEF TRAPPII, we recombinantly expressed and purified the 9 subunit, 427 kDa complex in Spodoptera frugiperda 9(Sf9) cells. We found that the TRAPPII complex is a GEF for both Rab1 and Rab11, and we discovered novel activity for another Rab GTPase. To interrogate the role of these GEFs in human disease, we used HDX-MS and nucleotide exchange assays to show that some DNMs destabilize Rab11 either through a complete or partial disruption of nucleotide binding. Importantly, we discovered that one of these DNMs, K13N, completely prevented SH3BP5 and TRAPPII mediated nucleotide exchange, revealing a putative mechanism of disease. Overall the work completed in this thesis leads to a greater understanding of the molecular mechanisms underlying the activation of Rab11 by its cognate GEFs. en_US
dc.language English eng
dc.language.iso en en_US
dc.rights Available to the World Wide Web en_US
dc.subject Structural Biology en_US
dc.subject Rab GTPase en_US
dc.subject Guanine Nucleotide Exchange Factor en_US
dc.title Structural and biochemical investigation of the regulation of Rab11a by the guanine nucleotide exchange factors SH3BP5 and TRAPPII en_US
dc.type Thesis en_US
dc.contributor.supervisor Burke, John E
dc.degree.department Department of Biochemistry and Microbiology en_US
dc.degree.level Master of Science M.Sc. en_US
dc.identifier.bibliographicCitation Jenkins ML, Margaria JP, Stariha JTB, Hoffmann RM, McPhail JA, Hamelin DJ, Boulanger MJ, Hirsch E, Burke JE. 2018. Structural determinants of Rab11 activation by the guanine nucleotide exchange factor SH3BP5. Nat Commun 9:3772. en_US
dc.description.scholarlevel Graduate en_US
dc.description.embargo 2020-11-25

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