Mechanical properties of tubulin intra- and inter-dimer interfaces and their implications for microtubule dynamic instability

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.

Atomistic basis of microtubule dynamic instability assessed via multiscale modeling

“…This finding is consistent with previous in vitro (50) and in vivo (51) experiments that reported highly variable tapered tips for microtubules by high resolution imaging and microtubule tip tracking. It is also in line with previous work that suggested there is a flexibility difference between the two nucleotide-states, with GTP being softer at its intraand inter-dimer interface (29,35,41), or a bending preference (24,33) with GTPprotofilaments growing less curved or nearly straight compared to the extensive outward peeling observed for GDP-protofilaments.…”

“…Our simulations confirmed the two major bending modes for the oligomers, tangential and radial outward bending, regardless of the nucleotide or lattice constraints ( Fig S1A, Supplemental Movies S1 and S2), as previously shown for single tubulin dimers (29,40). To compare our data to previously published tubulin MD simulations (29,35), the last 300ns of our trajectories were analyzed further in terms of root mean squared fluctuations (RMSF) and bending angle dynamics ( Fig S1-S3, Table S1A, S1B, S2A, S2B), which confirmed behavior consistent with these previous studies. We avoid drawing strong quantitative conclusions about nucleotide dependence of bending angles based solely on these results since the angle data is highly variable and is not likely to be converged due to longer relaxation times.…”

“…Next, as argued in previous studies (29,35), we assessed the possibility of a flexibility difference between different nucleotide states as the main cause of dynamic instability (Fig 7D, 7E). Interestingly, assuming that GTP-dimer is more flexible than GDP-dimer (~ 2 fold based on Table S1A) and both dimers prefer a radially kinked or straight conformation (θx=22°, 0°) did not result in normal microtubule dynamics, i.e.…”

“…More generally, as stated by the authors (31), analysis of residue contact map and CG bond strength and length is not a measure of the actual binding strength of tubulin dimers; rather, an energy landscape of the lateral and longitudinal interaction of the dimers needs to be calculated. In addition, while a number of other MD studies have contributed toward an atomistic understanding of intradimer bending mechanics (29,30), to our knowledge, previous studies have not examined the free energy of interdimer bending as a function of its nucleotide besides equilibrium trajectory analysis (35,36,41). Finally, previous studies have not estimated the nucleotide dependence of the strength of the longitudinal bond, generally regarded as stronger than the lateral bond.…”

“…Previous studies of microtubule dynamic instability posited that the energetic difference between the two nucleotide states of tubulin in models of microtubule assembly (24,25,(34)(35)(36)(37)(26)(27)(28)(29)(30)(31)(32)(33) is due to differences in the tubulin-tubulin lateral bond strength (25,27,28,31,32,34), tubulin-tubulin longitudinal bond strength (31,32,37), the bending preference or flexibility (24,26,29,30,33,35,36), or indirectly dependent on the lateral bond as a result of nucleotide-dependent bending strain energy (38). At the level of atomic structure, a recent cryo-electron microscopy (cryo-EM) study showed that GDP, GDP-Pi and GTPγS all have compacted lattices (~82 Å) compared to the GMPCPP extended lattice (~ 83.7 Å) (32), consistent with conclusions from earlier cryo-EM studies (37,39).…”