Internal Dynamics of Dynactin CAP-Gly Is Regulated by Microtubules and Plus End Tracking Protein EB1

Abstract

Background:The role of microtubules on conformational dynamics of microtubule-associated proteins remains poorly understood. Results: Magic angle spinning NMR studies indicate significant differences in the dynamics of CAP-Gly going from the unbound to the MT-bound or EB1-bound state. Conclusion: Environment-dependent motions occurring on multiple time scales are functionally (biologically) relevant. Significance: Conformational dynamics of a MT-associated protein bound to microtubules at atomic resolution is established for the first time.

Atomic-resolution structure of the CAP-Gly domain of dynactin on polymeric microtubules determined by magic angle spinning NMR spectroscopy

“…Furthermore, owing to its flexibility the CAP-Gly domain and the entire dynactin's shoulder projection are not visible in the high-resolution EM maps of the dynactin complex (21), precluding their structural analysis. Indeed, as we reported recently (24), CAP-Gly(19-107) bound to polymeric MTs is dynamic on the timescales spanning nano-to milliseconds, and the lack of electron density in the cryo-EM studies is consistent with our results. In contrast to the EM-based studies, MAS NMR experiments permit detection of dynamic complexes, as shown in our current work.…”

“…occurring over six decades of motional timescales (nano-to milliseconds), in its free form, assembled on MTs, and bound to EB1 (24). We discovered that loop regions of CAP-Gly are dynamic when both free and bound to MTs whereas their mobility is attenuated in complex with EB1.…”

“…Such reorientations may be important for fine-tuning the CAP-Gly's binding to EB1 and MTs. As discussed in Structural Plasticity of CAP-Gly, the loop regions are structurally well-defined, and the conformational rearrangements are related to CAP-Gly's conformational plasticity reported by us (23,24).…”

“…CAP-Gly possesses conformational plasticity that is important for dynactin's interactions with multiple binding partners (23). This conformational plasticity is intimately connected to CAP-Gly's inherent mobility that spans many decades of motional timescales, from nano-to milliseconds (24). CAP-Gly's motional profile is modulated by its environment, and, remarkably, both free and MT-bound proteins are dynamic in loops whereas CAP-Gly in complex with EB1 is considerably more rigid (24).…”

“…More importantly, diffraction and EM methods do not report on dynamics, perhaps the most fundamental missing link needed to understand how MTs and MT motor protein produce force, how "information" is transmitted (allostery), and how mutations related to disease manifest themselves in terms of function. Magic angle spinning (MAS) NMR spectroscopy is uniquely poised to bridge this gap and provide atomicresolution insights on microtubule-associated proteins bound to polymeric MTs (22)(23)(24). Recently, using a hybrid MAS NMR/molecular dynamics (MD) approach, we have gained a comprehensive view of the residue-specific conformational dynamics of CAP-Gly…”