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. 2015 Nov 24;112(47):14611-6.
doi: 10.1073/pnas.1509852112. Epub 2015 Nov 10.

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

Si Yan  1 , Changmiao Guo  1 , Guangjin Hou  1 , Huilan Zhang  1 , Xingyu Lu  1 , John Charles Williams  2 , Tatyana Polenova  3
Affiliations

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

Si Yan et al. Proc Natl Acad Sci U S A. .

Abstract

Microtubules and their associated proteins perform a broad array of essential physiological functions, including mitosis, polarization and differentiation, cell migration, and vesicle and organelle transport. As such, they have been extensively studied at multiple levels of resolution (e.g., from structural biology to cell biology). Despite these efforts, there remain significant gaps in our knowledge concerning how microtubule-binding proteins bind to microtubules, how dynamics connect different conformational states, and how these interactions and dynamics affect cellular processes. Structures of microtubule-associated proteins assembled on polymeric microtubules are not known at atomic resolution. Here, we report a structure of the cytoskeleton-associated protein glycine-rich (CAP-Gly) domain of dynactin motor on polymeric microtubules, solved by magic angle spinning NMR spectroscopy. We present the intermolecular interface of CAP-Gly with microtubules, derived by recording direct dipolar contacts between CAP-Gly and tubulin using double rotational echo double resonance (dREDOR)-filtered experiments. Our results indicate that the structure adopted by CAP-Gly varies, particularly around its loop regions, permitting its interaction with multiple binding partners and with the microtubules. To our knowledge, this study reports the first atomic-resolution structure of a microtubule-associated protein on polymeric microtubules. Our approach lays the foundation for atomic-resolution structural analysis of other microtubule-associated motors.

Keywords: dynactin's CAP-Gly domain; intermolecular interface determination; magic angle spinning NMR; microtubules; structure determination.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
(A) Overview of the structure of the dynactin complex. (Top Left) Overall structural organization of dynactin based on the recent cryo-EM structure (21). (Middle and Bottom Left) Domain structure of p150Glued microtubule-binding subunit of dynactin. The CAP-Gly domain (colored purple) is part of the p150Glued. (B) SDS/PAGE cosedimentation gel of CAP-Gly with microtubules. The lanes are labeled in order as follows: M, molecular mass markers; F, free CAP-Gly standard; P, pellet obtained by ultracentrifugation of CAP-Gly/microtubule solution; S, supernatant after ultracentrifugation of the pellet. (C) Transmission electron microscopy (TEM) images of microtubules (Top Left); U-13C,15N,2H-CAP-Gly/MT NMR sample (Top Right). The Bottom images were recorded before and after 2 wk of continuous multidimensional MAS NMR experiments.
Fig. 2.
Fig. 2.
Expansions around the aliphatic regions of 2D CORD (A and B) and 2D PAIN-CP (C) spectra of sparsely labeled CAP-Gly bound to polymerized microtubules, demonstrating examples of long-range, medium-range, and sequential distance restraints used in the structure calculation. CORD spectrum of [1,6-13C-glucose,U-15N]-CAP-Gly/MT is shown in A; CORD spectrum of [U-13C,15N]-CAP-Gly/MT is shown in B; PAIN-CP spectra of [2-13C-glucose,U-15N]-CAP-Gly/MT is shown in C. In all datasets, the cross-peaks corresponding to the different distance restraint ranges are color coded as follows: long-range, green; medium-range, orange; sequential, black. Both ambiguous and unambiguous restraints were used in the structure calculation. All spectra were recorded at 20.0 T; the CORD mixing time was 500 ms.
Fig. 3.
Fig. 3.
(A) The 3D structure of CAP-Gly bound to polymerized microtubules, calculated from MAS NMR restraints (PDB ID code 2MPX); the structure is an average from the ensemble of 10 lowest energy structures. The CAP-Gly residues are color coded according to the number (noted as “n”) of long-range distance restraints per residue detected in the various MAS NMR spectra. (B) A matrix representation of all intra- and interresidue contacts generated from the MAS NMR distance restraints. (C) The number of long-range distance restraints plotted vs. the residue number. The CAP-Gly secondary structure is shown on the top. (D) The structure of CAP-Gly bound to polymerized MTs (purple) shown in A superimposed with the average structure of free CAP-Gly (23) (orange; PDB ID code 2M02) and CAP-Gly in complex with EB1 (15) (light green; PDB ID code 2HKQ). (E) Loop conformations of CAP-Gly in free, MT-bound, and EB1-bound states corresponding to those in D: β3-β4 loop (Top) and β2-β3 loop (Bottom).
Fig. 4.
Fig. 4.
(A) Intermolecular interfaces of CAP-Gly with microtubules and end-binding protein EB1, derived from dREDOR (two Left panels) and chemical shift perturbations (two Right panels) in MAS NMR experiments, mapped onto the structure of CAP-Gly bound with MTs and in complex with EB1, respectively. The intermolecular interface of CAP-Gly with MT was obtained from dREDOR-based CPMAS and CORD experiments on U-13C,15N-CAP-Gly/MT (B, Top and Middle spectra, gray) and from CORD and 1H-13C-REDOR-HETCOR (35) experiments on U-13C,15N,2H-CAP-Gly/MT sample (B, Middle and Bottom spectra, red). Resonances obtained from both dREDOR-CORD and REDOR-HETCOR are labeled in green in B. (C) dREDOR-based CPMAS and CORD spectra of U-13C,15N-CAP-Gly/EB1. 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.
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