The haunted delimitation of subjectivity in the Work of Nicolas Abraham

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 Ca(2+)-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 Ca(2+) signaling since Ca(2+)- binding proteins can disengage KCBP dimers dependent on its C-terminal dimerization interface

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

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 Ca(2+)-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 Ca(2+) signaling since Ca(2+)- binding proteins can disengage KCBP dimers dependent on its C-terminal dimerization interface

Differential interference contrast microscopy of microtubules in the presence of different constructs of KCBP.

<p><i>A-</i> microtubules alone; <i>B-</i> microtubules plus KCBP (884–1253); <i>C-</i> microtubules plus KCBP (884–1225); <i>D-</i> microtubules plus Ca²⁺-KIC; <i>E-</i> microtubules plus KCBP (884–1253) and Ca²⁺-KIC; <i>F</i>- microtubules plus KCBP (884–1225) and Ca²⁺-KIC.</p

Schematic presentation of the domain organization in KCBP.

<p>Schematic presentation of the domain organization in KCBP.</p

Structural similarity between KCBP and kinesin-1.

<p><i>A-</i> Structure of Arabidopsis KCBP dimer. Color coding: grey – motor core, red – regulatory domain, blue – ADP. <i>B-</i> Structure of conventional human kinesin-1 (PDB ID 3KIN). Color coding: dark grey – motor core, green – neck, blue – ADP. <i>C-</i> Structural alignment of KCBP and kinesin-1 monomers. <i>D</i> – Structural alignment of KCBP and kinesin-1 dimers.</p

Data collection and model refinement statistics.

*<p>Numbers in parentheses are given for reflections in the highest resolution shell.</p

Dimer organization of KCBP.

<p>A- Crystal structure of <i>Arabidopsis</i> KCBP solved at resolution of 2.4 Å. Two molecules of KCBP (a.a. 876–1261) found in the crystal asymmetric unit are shown. Structural elements are presented as cartoon. ADP and Mg ions are shown as space-filling model in blue. Motor domain is in grey. The regulatory domain is highlighted in red. The unstructured fragment of the regulatory domain in one of the molecules is shown in dotted line. The structural elements of the regulatory domain and N- and C-termini are indicated. Helices α4 and α6 are indicated for the viewer orientation. B- Close-up presentation of the dimer interface made of the regulatory helices. The interacting residues are shown as sticks and indicated. Color-coding is by element: carbonyls in green, nitrogens in blue, sulphur in yellow.</p

Gel-filtration profiles of the isolated KCBP constructs on Superdex200Profiles corresponding to the different KCBP samples are color coded and indicated.

<p>Profiles of the protein standards are shown as dashed lines in black.</p