Plant Kinesin-Like Calmodulin Binding Protein Employs Its Regulatory Domain for Dimerization

Sarah E Rice,Robert J Fletterick,Joshua S Waitzman,Maia V Vinogradova,Galina G Malanina

doi:10.1371/journal.pone.0066669

Sarah E Rice, Robert J Fletterick + Show 3 more

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https://doi.org/10.1371/journal.pone.0066669

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Abstract

Kinesin-like calmodulin binding protein (KCBP), a Kinesin-14 family motor protein, is involved in the structural organization of microtubules during mitosis and trichome morphogenesis in plants. The molecular mechanism of microtubule bundling by KCBP remains unknown. KCBP binding to microtubules is regulated by Ca2+-binding proteins that recognize its C-terminal regulatory domain. In this work, we have discovered a new function of the regulatory domain. We present a crystal structure of an Arabidopsis KCBP fragment showing that the C-terminal regulatory domain forms a dimerization interface for KCBP. This dimerization site is distinct from the dimerization interface within the N-terminal domain. Side chains of hydrophobic residues of the calmodulin binding helix of the regulatory domain form the C-terminal dimerization interface. Biochemical experiments show that another segment of the regulatory domain located beyond the dimerization interface, its negatively charged coil, is unexpectedly and absolutely required to stabilize the dimers. The strong microtubule bundling properties of KCBP are unaffected by deletion of the C-terminal regulatory domain. The slow minus-end directed motility of KCBP is also unchanged in vitro. Although the C-terminal domain is not essential for microtubule bundling, we suggest that KCBP may use its two independent dimerization interfaces to support different types of bundled microtubule structures in cells. Two distinct dimerization sites may provide a mechanism for microtubule rearrangement in response to Ca2+ signaling since Ca2+- binding proteins can disengage KCBP dimers dependent on its C-terminal dimerization interface.

Highlights

Kinesin-like calmodulin binding protein (KCBP) is a molecular motor found in plants [1]
Our model indicates that the domain swap does not play a role in positioning of the negative coil over the microtubule-binding surface of KCBP in the Arabidopsis KCBP crystals
We have found that the negative coil of KCBP, a part of its regulatory domain, stabilizes KCBP dimers, which are formed by hydrophobic interactions between the residues of the calmodulin binding helices, another part of its regulatory domain

Summary

Introduction

Kinesin-like calmodulin binding protein (KCBP) is a molecular motor found in plants [1]. Its activation and silencing is crucial mainly for normal trichome morphogenesis [4] Both mitosis and trichome morphogenesis, though discrete processes, rely on correct cytoskeleton structure, which is based on microtubules and actin filaments. KCBP belongs to the kinesin family of molecular motors. KCBP has a typical kinesin motor domain often referred to as a head. This domain attaches to microtubules and contains a functional nucleotide-binding site. KCBP has an unusual N-terminal tail domain that relates KCBP to another family of molecular motors, myosins, which move along actin filaments. Just like the tails of myosins VIIa and X, the tail of KCBP contains talin-like FERM domains and MyTH4 homology regions with additional affinity to microtubules [7] (Fig. 1)

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