Nucleotide Binding to ARL2 in the TBCD ∙ ARL2 ∙ β-Tubulin Complex Drives Conformational Changes in β-Tubulin
| Autor: | Bruce D. Pascal, Eric A. Ortlund, Emily R. Weikum, Scott J. Novick, Richard A. Kahn, Patrick R. Griffin, Joshua W. Francis, Devrishi Goswami |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Protein Folding GTP' Protein Conformation Trimer macromolecular substances Microtubules Article 03 medical and health sciences Protein structure GTP-Binding Proteins Tubulin Structural Biology Microtubule Humans Molecular Biology Tubulin complex 030102 biochemistry & molecular biology biology Chemistry Prefoldin HEK293 Cells 030104 developmental biology Biochemistry Guanosine 5'-O-(3-Thiotriphosphate) biology.protein Protein folding Microtubule-Associated Proteins Signal Transduction |
| Zdroj: | Journal of Molecular Biology. 429:3696-3716 |
| ISSN: | 0022-2836 |
| Popis: | Microtubules are highly dynamic tubulin polymers that are required for a variety of cellular functions. Despite the importance of a cellular population of tubulin dimers, we have incomplete information about the mechanisms involved in the biogenesis of αβ-tubulin heterodimers. In addition to prefoldin and the TCP-1 Ring Complex, five tubulin-specific chaperones, termed cofactors A-E (TBCA-E), and GTP are required for the folding of α- and β-tubulin subunits and assembly into heterodimers. We recently described the purification of a novel trimer, TBCD•ARL2•β-tubulin. Here, we employed hydrogen/deuterium exchange coupled with mass spectrometry to explore the dynamics of each of the proteins in the trimer. Addition of guanine nucleotides resulted in changes in the solvent accessibility of regions of each protein that led to predictions about each's role in tubulin folding. Initial testing of that model confirmed that it is ARL2, and not β-tubulin, that exchanges GTP in the trimer. Comparisons of the dynamics of ARL2 monomer to ARL2 in the trimer suggested that its protein interactions were comparable to those of a canonical GTPase with an effector. This was supported by the use of nucleotide-binding assays that revealed an increase in the affinity for GTP by ARL2 in the trimer. We conclude that the TBCD•ARL2•β-tubulin complex represents a functional intermediate in the β-tubulin folding pathway whose activity is regulated by the cycling of nucleotides on ARL2. The co-purification of guanine nucleotide on the β-tubulin in the trimer is also shown, with implications to modeling the pathway. |
| Databáze: | OpenAIRE |
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