Three-Dimensional Structure of CAP-Gly Domain of Mammalian Dynactin Determined by Magic Angle Spinning NMR Spectroscopy: Conformational Plasticity and Interactions with End-Binding Protein EB1

Abstract

Microtubules (MTs) and their associated proteins (MAPs) play important roles in vesicle and organelle transport, cell motility and cell division. Perturbation of these processes by mutation typically gives rise to severe pathological conditions. In our efforts to obtain atomic information on MAP/MT interactions with the goal to understand mechanisms that might potentially assist in the development of treatments for these diseases, we have determined the 3D structure of CAP-Gly domain of mammalian dynactin by MAS NMR spectroscopy. We observe two conformations in the β2 strand encompassing residues T43-V44-A45, residues that are adjacent to the disease associated mutation, G59S. Upon binding of CAP-Gly to microtubule plus-end tracking protein EB1, the CAP-Gly shifts to a single conformer. We find extensive chemical shift perturbations in several stretches of residues of CAP-Gly upon binding to EB1, from which we define accurately the CAP-Gly/EB1 binding interface. We also observe that the loop regions may exhibit unique flexibility, especially in the GKNDG motif, which participates in the microtubule binding. This study in conjunction with our previous reports suggests that conformational plasticity is an intrinsic property of CAP-Gly likely due to its unusually high loop content and may be required for its biological functions.

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

“…At the same time, as shown in Fig. 3D, loop regions in CAP-Gly underwent pronounced conformational changes upon the formation of a complex with MTs, compared with the free CAP-Gly and CAP-Gly/EB1 complex (15,23,26). These changes resulted in sidechains adopting different orientations in free and MTand EB1-bound states (Fig.…”

“…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).…”

“…The structure was determined with a total of 1,194 nonredundant 13 C-13 C/ 15 N-13 C distance restraints (after excluding low-probability restraints), of which 408 and 237 are long-and medium-range restraints, respectively. We note that this number of restraints is significantly higher than that used for MAS NMR structure determination of CAP-Gly (917 total, 197 long-range, and 97 medium-range restraints) (23), as well as in the MAS NMR structures of similar-size proteins determined recently (27)(28)(29). To further evaluate the distance restraint quality, we summarized the results in a 2D contact map displayed in Fig.…”

“…In A, the residues that belong to intermolecular interfaces of CAP-Gly with MT and EB1 are color coded as follows: with direct contacts in dREDOR-and HETCOR-based spectra (green); with large (> 1.0 ppm) and moderate (0.5-1.0 ppm) chemical shift perturbations (purple and orange, respectively). CAP-Gly residues at the interface with EB1 determined from chemical shift perturbations (23) are shown in orange. The primary sequence of CAP-Gly is depicted at the Bottom of A, and the residues are color coded as described above.…”

“…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…”