Microtubules (MTs) are key components of the cytoskeleton and play a central role in cell division and development. MT assembly is known to be associated with a structural change in αβ-tubulin dimers from kinked to straight conformations. How GTP binding renders individual dimers polymerization-competent, however, is still unclear. Here, we have characterized the conformational dynamics and energetics of unassembled tubulin using atomistic molecular dynamics and free energy calculations. Contradictory to existing allosteric and lattice models, we find that GTP-tubulin favors a broad range of almost isoenergetic curvatures, whereas GDP-tubulin has a much lower bending flexibility. Moreover, irrespective of the bound nucleotide and curvature, two conformational states exist differing in location of the anchor point connecting the monomers that affects tubulin bending, with one state being strongly favored in solution. Our findings suggest a new combined model in which MTs incorporate and stabilize flexible GTP-dimers with a specific anchor point state.
A step-by-step guide for reproducing the simulations and all custom-made scripts used in this study have been uploaded to a publicly available GitHub repository (https://github.com/moozzz/simulation-protocols/tree/master/free-tubulin-simulation).
- Maxim Igaev
- Helmut Grubmüller
- Maxim Igaev
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Luke M Rice, UT Southwestern Medical Cetner, United States
© 2018, Igaev & Grubmüller
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Clamp loaders place circular sliding clamp proteins onto DNA so that clamp-binding partner proteins can synthesize, scan, and repair the genome. DNA with nicks or small single-stranded gaps are common clamp-loading targets in DNA repair, yet these substrates would be sterically blocked given the known mechanism for binding of primer-template DNA. Here, we report the discovery of a second DNA binding site in the yeast clamp loader Replication Factor C (RFC) that aids in binding to nicked or gapped DNA. This DNA binding site is on the external surface and is only accessible in the open conformation of RFC. Initial DNA binding at this site thus provides access to the primary DNA binding site in the central chamber. Furthermore, we identify that this site can partially unwind DNA to create an extended single-stranded gap for DNA binding in RFC's central chamber and subsequent ATPase activation. Finally, we show that deletion of the BRCT domain, a major component of the external DNA binding site, results in defective yeast growth in the presence of DNA damage where nicked or gapped DNA intermediates occur. We propose that RFC’s external DNA binding site acts to enhance DNA binding and clamp loading, particularly at DNA architectures typically found in DNA repair.
Protein folding homeostasis in the endoplasmic reticulum (ER) is regulated by a signaling network, termed the unfolded protein response (UPR). Inositol-requiring enzyme 1 (IRE1) is an ER membrane-resident kinase/RNase that mediates signal transmission in the most evolutionarily conserved branch of the UPR. Dimerization and/or higher-order oligomerization of IRE1 are thought to be important for its activation mechanism, yet the actual oligomeric states of inactive, active, and attenuated mammalian IRE1 complexes remain unknown. We developed an automated two-color single-molecule tracking approach to dissect the oligomerization of tagged endogenous human IRE1 in live cells. In contrast to previous models, our data indicate that IRE1 exists as a constitutive homodimer at baseline and assembles into small oligomers upon ER stress. We demonstrate that the formation of inactive dimers and stress-dependent oligomers is fully governed by IRE1’s lumenal domain. Phosphorylation of IRE1’s kinase domain occurs more slowly than oligomerization and is retained after oligomers disassemble back into dimers. Our findings suggest that assembly of IRE1 dimers into larger oligomers specifically enables trans-autophosphorylation, which in turn drives IRE1’s RNase activity.