Reversible and Irreversible Coiled Coils in the Stalk Domain of ncd Motor Protein

Abstract

Ncd is a microtubule minus end-directed motor protein from Drosophila, a member of the kinesin-14 family, and an essential protein in mitosis and meiosis. Full-length ncd exists as a dimer via the formation of an alpha-helical coiled coil in its central stalk domain (P192-R346), which is thought to be one of the key regions for its motility. In our previous studies, however, none of the various synthetic polypeptide fragments (up to 46 residues) from the stalk domain formed a coiled coil. Herein, we have investigated the structural properties of the full-length ncd stalk domain using recombinant polypeptides together with shorter segments. These new fragments did form coiled coils as verified by far-UV circular dichroism (CD) spectroscopy and analytical ultracentrifugation, suggesting that a certain length of polypeptide would be required for dimer formation. Moreover, deletion mapping revealed that the cooperativity among the neighboring subdomains in the stalk domain is required for formation of the coiled coil. Interestingly, the intact stalk domain segments showed three-state transition in thermal unfolding measurements with CD, indicating the presence of two regions: (i) a coiled-coil region (P227-R306) that exhibits reversible denaturation at a lower temperature (20-30 degrees C) and (ii) a more rigid coiled-coil region (T307-E334) that exhibits irreversible denaturation at a high temperature (ca. 60 degrees C). These results imply that the N-terminal region of the stalk domain might be able to adopt both a coiled-coil conformation and a dissociated one, which might be relevant to the functions of ncd.

Biochemical and Molecular Dynamic Simulation Analysis of a Weak Coiled Coil Association between Kinesin-II Stalks

“…Next, to verify the secondary structure of the purified polypeptides, we subjected them to Far-UV circular dichroism (CD) analysis, which is the appropriate secondary structure determination tool used for coiled-coil estimation [19], [38], [39]. The CD spectra of His-KLP68D/64D-S and His-KLP64D/68D-S heterodimers, collected in the P2 fractions, contained alpha-helical characteristic double minima at 208 and 222 nm (Figure 2C).…”

Molecular mechanisms of kinesin-14 motors in spindle assembly and chromosome segregation

“…Furthermore, in case of the X. laevis XCTK2, it has been demonstrated that, if the number of kinesins bound to microtubules is too high, gliding speed is significantly reduced, presumably owing to the non-synchronized state of multiple bound motors (Hentrich and Surrey, 2010). As mentioned previously, the presence of reversibly stable regions that can collapse in the coiledcoil domain (Hallen et al, 2008(Hallen et al, , 2011Ito et al, 2006;Makino et al, 2007) might be able to partially compensate for any braking that results from unsynchronized power force generation.…”

“…In Drosophila, the neck region at the proximal end of the coiled-coil stalk domain, adjacent to the motor, is essential for the regulation of lever-like rotations and step size (Hallen et al, 2011). Moreover, biochemical studies of Ncd stalk peptides (Ito et al, 2006) and additional studies (Makino et al, 2007) indicate the presence of sequence features that generate reversible and irreversible regions within the Ncd coiled-coil region. It has been proposed that a partial collapse of the Ncd stalk prevents braking that can occur when multiple Ncd motors are bound to a microtubule but without synchronized ATP hydrolysis and forcestroke generation (Makino et al, 2007).…”

“…Moreover, biochemical studies of Ncd stalk peptides (Ito et al, 2006) and additional studies (Makino et al, 2007) indicate the presence of sequence features that generate reversible and irreversible regions within the Ncd coiled-coil region. It has been proposed that a partial collapse of the Ncd stalk prevents braking that can occur when multiple Ncd motors are bound to a microtubule but without synchronized ATP hydrolysis and forcestroke generation (Makino et al, 2007). Several lines of evidence point to the tail domain as providing some of the most crucial functional cues to diversify kinesin-14 function.…”

Kinesin-14: the roots of reversal

“…[1] show that the two lever positions are each stabilized by multiple salt bridges and how the lever might be driven from one stable position to another is unclear. Furthermore, the lever (the coiled-coil tail of the motor) is potentially forbiddingly long: perhaps as long as 150 nm in the wild-type molecule, albeit with some regions less stable than others [7]. …”