Solid-state and solution NMR studies of the CAP-Gly domain of mammalian dynactin and its interaction with microtubules.
Microtubules (MTs) and microtubule binding proteins (MTBPs) play fundamental physiological roles including vesicle and organelle transport, cell motility, and cell division. Despite the importance of the MT/MTBP assemblies, there remains virtually no structural or dynamic information about their interaction at the atomic level due to the inherent insolubility and lack of long-range order of MTs. In this study, we present a combined magic angle spinning solid-state and solution NMR study of the MTBP CAP-Gly domain of mammalian dynactin and its interaction with paclitaxel-stabilized microtubules. We report resonance assignments and secondary structure analysis of the free CAP-Gly in solution and in the solid state by a combination of two- and three-dimensional homo- and heteronuclear correlation spectra. In solution, binding of CAP-Gly to microtubules is accompanied by the broadening of the majority of the peaks in HSQC spectra except for the residues at the termini, precluding further structural analysis of the CAP-Gly/microtubule complexes. In the solid state, DARR spectra of free CAP-Gly and its complex with microtubules display well-resolved lines, permitting residue-specific resonance assignments. Interestingly, a number of chemical shifts in the solid-state DARR spectra of the CAP-Gly/microtubule complex are perturbed compared to those of the free CAP-Gly, suggesting that conformational changes occur in the protein upon binding to the microtubules. These results indicate that CAP-Gly/microtubule assemblies are amenable to detailed structural characterization by magic angle spinning NMR spectroscopy and that solid-state NMR is a viable technique to study MT/protein interactions in general.
Sun
Shangjin
S
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
Siglin
Amanda
A
Williams
John C
JC
Polenova
Tatyana
T
D000072159:Dynactin Complex; D008869:Microtubule-Associated Proteins / Q000737:chemistry*; D008870:Microtubules / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015203:Reproducibility of Results
D000072159:Dynactin Complex; D008869:Microtubule-Associated Proteins