Kinesin's light chains inhibit the head- and microtubule-binding activity of its tail - PubMed (original) (raw)

Kinesin's light chains inhibit the head- and microtubule-binding activity of its tail

Yao Liang Wong et al. Proc Natl Acad Sci U S A. 2010.

Abstract

Kinesin-1 is a microtubule-based motor comprising two heavy chains (KHCs) and two light chains (KLCs). Motor activity is precisely regulated to avoid futile ATP consumption and to ensure proper intracellular localization of kinesin-1 and its cargoes. The KHC tail inhibits ATPase activity by interacting with the enzymatic KHC heads, and the tail also binds microtubules. Here, we present a role for the KLCs in regulating both the head- and microtubule-binding activities of the kinesin-1 tail. We show that KLCs reduce the affinity of the head-tail interaction over tenfold and concomitantly repress the tail's regulatory activity. We also show that KLCs inhibit tail-microtubule binding by a separate mechanism. Inhibition of head-tail binding requires steric and electrostatic factors. Inhibition of tail-microtubule binding is largely electrostatic, pH dependent, and mediated partly by a highly negatively charged linker region between the KHC-interacting and cargo-binding domains of the KLCs. Our data support a model wherein KLCs promote activation of kinesin-1 for cargo transport by simultaneously suppressing tail-head and tail-microtubule interactions. KLC-mediated inhibition of tail-microtubule binding may also influence diffusional movement of kinesin-1 on microtubules, and kinesin-1's role in microtubule transport/sliding.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Constructs used in this study. (A) Tail (blue), KLC (red/light gray), and head (dark gray) constructs are shown beside a cartoon of kinesin-1. Coiled-coil regions in each construct are striped. Residues are numbered according to the Drosophila kinesin-1 sequence. Tail975 is a KHC tail N terminally fused to maltose-binding protein and is colored blue to indicate its basic nature. KLC-FL is the full-length KLC protein. KLCΔTPR is truncated immediately N-terminal to the cargo-binding TPR domains, whereas KLC-CC consists of only the coiled-coil region of the KLCs. Parts of the KLCs are colored red to indicate acidic regions. Head401 is a KHC head dimer with all surface-exposed Cys residues removed. (B) Crystal structure of the KHC head (PDB ID code 1mkj) (42) with S195 indicated in green space-fill representation. Acidic residues on the head’s tail-interacting face, particularly helix _α_3, are colored red. There are no basic residues on this face of the motor domain. (C) Figure legend for component depictions in subsequent figures.

Fig. 2.

KLCs inhibit tail binding to the heads. (A) Fluorescence anisotropy binding curves of Tail975 to 0.01 μM Head401 in the absence of KLCs (n = 13; squares), and in the presence of KLC-FL (n = 8; diamonds) or KLCΔTPR (n = 9; circles). Data points are mean ± SD. (Inset) A Scatchard plot of the data and corresponding fits, along with K d values calculated by nonlinear regression. (B) Head401 ATPase activity in the presence of 2 μM MTs is progressively inhibited by increasing Tail975 (squares), with a maximum extent of inhibition of 76%. In the presence of KLC-FL (diamonds) or KLCΔTPR (circles), the K d of the functional inhibition of heads by tails is increased. Data points are normalized mean ± SD (n = 5–15 for each point).

Fig. 3.

KLCs inhibit tail binding to MTs. (A) KLC-FL did not interact with MTs by itself, and it inhibited the tail-MT interaction in a concentration-dependent manner. Tail975 concentration was fixed at 2 μM. (B) Increasing the amount of KLC-FL resulted in a linear decrease in the percentage of pelleted Tail975 (n = 6–13). Maximum inhibition was observed at 2-μM KLC-FL. Beyond the 1∶1 ratio of KLC-FL:Tail975, the amount of pelleted tail did not decrease significantly (p > 0.05). At saturating KLC-FL levels, the K d for tail-MT binding was approximately 9 μM.

Fig. 4.

Inhibition of tail-MT binding does not require the KLC TPR domains and is pH dependent. Protein concentrations were set at 2-μM Tail975 ± 2.5-μM KLCΔTPR or KLC-CC. (A) KLCΔTPR and KLC-CC inhibit Tail975 binding to MTs. (B) The differences in amount of pelleted tail between Tail975 alone (n = 13), Tail975 + KLCΔTPR (n = 7), and Tail975 + KLC-CC (n = 5) were all highly significant (*, p < 0.001). Estimated tail-MT _K_ _d_ values were > 14 μM in the presence of KLCΔTPR and 4 μM in the presence of KLC-CC. (C) MT-binding assay performed at pH 6.6 and pH 7.4. (D) Tail975 alone binds equally well to MTs at pH 6.6 or pH 7.4. However, KLC-mediated inhibition of the tail-MT interaction is significantly reduced at pH 6.6, with the K d for tail-MT binding in the presence of KLCΔTPR decreasing to approximately 5 μM (*, p < 0.001, n = 5).

Fig. 5.

A model for KLC-mediated regulation of the kinesin-1 tail. Kinesin-1 is colored as in Fig. 1. Without KLCs (Left), kinesin-1 would be either regulated in solution or bound to MTs with the tail tethered. Due to the high affinity of tails for heads/MTs, the motor cannot access its cargo transport-competent state. In the presence of KLCs (Right), tail-head and tail-MT interactions are inhibited. Strong inhibition of tail-MT binding means that the regulated conformation of kinesin-1 becomes the predominant form, but tail-head affinity is also reduced such that the motor is in a poised state that can be easily activated for cargo transport.

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Kinesin's light chains inhibit the head- and microtubule-binding activity of its tail - PubMed (original) (raw)