Microtubule-associated Protein-like Binding of the Kinesin-1 Tail to Microtubules

Mark A Seeger,Sarah E Rice

doi:10.1074/jbc.m109.068247

Mark A Seeger, Sarah E Rice

Open Access

https://doi.org/10.1074/jbc.m109.068247

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Abstract

The kinesin-1 molecular motor contains an ATP-dependent microtubule-binding site in its N-terminal head domain and an ATP-independent microtubule-binding site in its C-terminal tail domain. Here we demonstrate that a kinesin-1 tail fragment associates with microtubules with submicromolar affinity. Binding is largely electrostatic in nature, and is facilitated by a region of basic amino acids in the tail and the acidic E-hook at the C terminus of tubulin. The tail binds to a site on tubulin that is independent of the head domain-binding site but overlaps with the binding site of the microtubule-associated protein Tau. Surprisingly, the kinesin tail domain stimulates microtubule assembly and stability in a manner similar to Tau. The biological function of this strong kinesin tail-microtubule interaction remains to be seen, but it is likely to play an important role in kinesin regulation due to the close proximity of the microtubule-binding region to the conserved regulatory and cargo-binding domains of the tail.

Highlights

We found that co-sedimentation was the more accurate approach for determining low-affinity interactions, and fluorescence anisotropy was more accurate for evaluating high-affinity interactions
We have shown that the kinesin-1 tail binds to microtubules with a submicromolar affinity in an electrostatic-dependent manner
Kd values determined by co-sedimentation are more accurate for the lower affinity interactions (1 ␮M Ͻ Kd Ͻ 10 ␮M), because fluorescence anisotropy measurements are hampered by prohibitive amounts of light scattering at microtubule concentrations above 10 ␮M

Summary

EXPERIMENTAL PROCEDURES

Constructs and Protein Purification—All proteins were grown in standard Luria-Bertani medium plus appropriate antibiotics in BL21(DE3) RP cells (Stratagene, La Jolla, CA). K349 G234A, a monomeric human kinesin-1 head construct spanning residues 1–349 and containing a G234A mutation in its Switch I domain, which allows it to bind to microtubules in its low affinity ADP-bound state (24), was purified as previously described (10), and quantified by Coomassie Protein Assay (Thermo Fisher Scientific, Rockford, IL). To determine the ratio of ligand to tubulin necessary to induce microtubule assembly, aliquots of samples prepared as described above, except with varying concentrations of ligand, were centrifuged at 50,000 ϫ g at 20 °C for 15 min, and equal amounts of the recovered supernatants and. Electron Microscopy—5 ␮M tubulin was incubated in Binding Buffer ϩ 1 mM GTP with or without 5 ␮M Tail[944] or 5 ␮M Tail[944] A905C Mutant AϩB or 5 ␮M hTau-40 for 15 min at room temperature. Spectra were collected in triplicate from three separate samples and averaged after subtracting the spectrum of the buffer alone

RESULTS

Fluorescence anisotropy

DISCUSSION