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

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

Mapping protein–protein interactions by double-REDOR-filtered magic angle spinning NMR spectroscopy

“…G30-S31, G39 and V44–45) and experience allosteric effects but are not involved in the formation of the intermolecular interface. This result is consistent with our prior observations for CAP-Gly/MT complex (Yan et al 2015a). Thus, dREDOR is a more direct method than CSP for mapping out intermolecular interfaces, especially when the binding affinity is moderate or weak.…”

“…However, the presence of such water-protein contacts can be easily probed by the dREDOR-HETCOR experiments and indicated by signals that appear at 1 H chemical shift of water in the dREDOR-HETCOR spectra. The possibility of this kind of interaction was examined by us previously (Yan et al 2015a). In that study, the 2D dREDOR-HETCOR experiment was conducted in the U- 2 H, 13 C, 15 N-CAP-Gly/microtubule complex prepared in 99.8% D 2 O, and there was no evidence for water-protein interaction.…”

“…We have therefore explored an alternative approach for gaining atomic-resolution information into the interfaces formed by U- 13 C, 15 N-CAP-Gly and natural abundance microtubules, dubbed dREDOR. The experiment starts by the application of a simultaneous 1 H− 13 C and 1 H− 15 N REDOR (double REDOR, dREDOR) filters of various durations to the complexes of U- 13 C, 15 N labeled protein with its unlabeled binding partner, followed by 1 H- 13 C or 1 H- 15 N cross polarization transfer and, if desired, the subsequent 13 C- 13 C, 15 N- 13 C or 13 C- 1 H mixing periods (Yan et al 2015a). The dREDOR filter in the first step dephases the magnetization of the protons that are directly bonded to 13 C and 15 N labeled sites (i.e., all protons in the U- 13 C, 15 N labeled protein).…”

Structural Analysis of Human Cofilin 2/Filamentous Actin Assemblies: Atomic-Resolution Insights from Magic Angle Spinning NMR Spectroscopy

“…To discriminate between those possibilities and identify cofilin residues located at the interface with F-actin, we employed a dREDOR filtered approach33. In this method, simultaneous 1 H- 13 C/ 1 H- 15 N REDOR filter dephases all cofilin protons that are directly bonded to either 13 C or 15 N atoms (i.e., 1 H belonging to the U- 13 C, 15 N-cofilin).…”

“…The resulting spectrum contains specific information about the residues forming intermolecular interface. We note that dREDOR based experiment is the only approach to identify intermolecular interfaces when chemical shift perturbations are small, such as in cofilin/actin complex here or CAP-Gly/microtubule complex studied by us previously33. To perform residue assignments of cofilin residues at the interface, this experiment is implemented as a 2D dREDOR-CORD sequence (Fig.…”