Abstract
GhKCH2 belongs to a group of plant-specific kinesins (KCHs) containing an actin-binding calponin homology (CH) domain in the N-terminus. Previous studies revealed that the GhKCH2 CH domain (GhKCH2-CH) had a higher affinity for F-actin (Kd = 0.42 ± 0.02 µM) than most other CH-domain-containing proteins. To understand the underlying mechanism, prokaryotically expressed GhKCH2-CH (amino acids 30-166) was purified and crystallized. Crystals were grown by the sitting-drop vapour-diffusion method using 0.1 M Tris-HCl pH 7.0, 20%(w/v) PEG 8000 as a precipitant. The crystals diffracted to a resolution of 2.5 Å and belonged to space group P21, with unit-cell parameters a = 41.57, b = 81.92, c = 83.00 Å, α = 90.00, β = 97.31, γ = 90.00°. Four molecules were found in the asymmetric unit with a Matthews coefficient of 2.22 Å(3) Da(-1), corresponding to a solvent content of 44.8%.
Highlights
Microfilaments and microtubules, two vital cytoskeleton systems in cells, together take part in a variety of cellular activities, such as cell division and proliferation, transportation of organelles and vesicles, and the organization and formation of plant preprophase bands, phragmoplasts and cell plates (Wasteneys & Galway, 2003; Petrasˇek & Schwarzerova, 2009)
The kinesins are a superfamily of microtubule-based motor proteins (Howard, 1996), some of which (KCHs) contain a single N-terminal calponin homology (CH) domain that is able to bind to both microfilaments and microtubules
GhKCH2 (GenBank accession No EF432568), a KCH previously cloned from cotton (Gossypium hirsutum) fibres in our laboratory, has a motor domain with microtubulestimulated ATPase activity and a CH domain that strongly interacts with microfilaments, suggesting it to be a candidate for a linker between microfilaments and microtubules (Xu et al, 2007, 2009)
Summary
Microfilaments and microtubules, two vital cytoskeleton systems in cells, together take part in a variety of cellular activities, such as cell division and proliferation, transportation of organelles and vesicles, and the organization and formation of plant preprophase bands, phragmoplasts and cell plates (Wasteneys & Galway, 2003; Petrasˇek & Schwarzerova , 2009). The kinesins are a superfamily of microtubule-based motor proteins (Howard, 1996), some of which (KCHs) contain a single N-terminal calponin homology (CH) domain that is able to bind to both microfilaments and microtubules. Our laboratory and others identified further KCHs (GhKCH2, O12/OsKCH1, AtKinG and NtKCH1), all of which were able to bind to both microfilaments and microtubules in vitro or in vivo (Frey et al, 2009; Xu et al, 2009; Buschmann et al, 2011; Umezu et al, 2011; Klotz & Nick, 2012). GhKCH2 (GenBank accession No EF432568), a KCH previously cloned from cotton (Gossypium hirsutum) fibres in our laboratory, has a motor domain with microtubulestimulated ATPase activity and a CH domain that strongly interacts with microfilaments, suggesting it to be a candidate for a linker between microfilaments and microtubules (Xu et al, 2007, 2009). The expression, purification, crystallization and preliminary X-ray diffraction studies of the CH domain of GhKCH2 are described
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
