Abstract
Thirteen tubulin protofilaments, made of αβ-tubulin heterodimers, interact laterally to produce cytoskeletal microtubules. Microtubules exhibit the striking property of dynamic instability, manifested in their intermittent growth and shrinkage at both ends. This behavior is key to many cellular processes, such as cell division, migration, maintenance of cell shape, etc. Although assembly and disassembly of microtubules is known to be linked to hydrolysis of a guanosine triphosphate molecule in the pocket of β-tubulin, detailed mechanistic understanding of corresponding conformational changes is still lacking. Here we take advantage of the recent generation of in-microtubule structures of tubulin to examine the properties of protofilaments, which serve as important microtubule assembly and disassembly intermediates. We find that initially straight tubulin protofilaments, relax to similar non-radially curved and slightly twisted conformations. Our analysis further suggests that guanosine triphosphate hydrolysis primarily affects the flexibility and conformation of the inter-dimer interface, without a strong impact on the shape or flexibility of αβ-heterodimer. Inter-dimer interfaces are significantly more flexible compared to intra-dimer interfaces. We argue that such a difference in flexibility could be key for distinct stability of the plus and minus microtubule ends. The higher flexibility of the inter-dimer interface may have implications for development of pulling force by curving tubulin protofilaments during microtubule disassembly, a process of major importance for chromosome motions in mitosis.
Highlights
The ability to self-assemble from tubulin dimers in presence of guanosine triphosphate (GTP) and spontaneously disassemble, when GTP molecules in tubulin pockets are hydrolyzed, is a dramatic and essential feature of microtubules
We find that the nucleotide primarily affects the interface between two tubulin dimers, making it more flexible in the GTP state
This makes the GTP-bound tubulin protofilament easier to incorporate into microtubule lattice, providing a simple mechanism for microtubule dynamic instability
Summary
Molecular models of the straight GDP-bound tubulin and Mg-GTP-bound tubulin structures (dimers, tetramers and hexamers) were based on 3J6F and 3J6E PDB structures [22]. The latter contained the slowly hydrolysable GTP analog (GMPCPP) in the exchangeable nucleotide-binding site of β-tubulin. Propka software [48] was used to calculate the unknown degree of protonation of ionizable amino acid residues and Dowser program [49] was used to identify and solvate cavities inside the protein. The parameters of the GTP and GDP molecules were taken from the CHARMM27 force field, and parameters of their phosphate groups were set in accordance with [53], similar to the phosphate groups of ATP and ADP
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