Reversible and irreversible coiled coils in the stalk domain of ncd motor protein.
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.
Makino
Tsukasa
T
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Morii
Hisayuki
H
Shimizu
Takashi
T
Arisaka
Fumio
F
Kato
Yusuke
Y
Nagata
Koji
K
Tanokura
Masaru
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000235:genetics; D002942:Circular Dichroism; D019281:Dimerization; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics; D016547:Kinesins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D011392:Proline / Q000737:chemistry / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017384:Sequence Deletion; D013696:Temperature; D014461:Ultracentrifugation
D029721:Drosophila Proteins; D011994:Recombinant Proteins; C066696:ncd protein, Drosophila; D001120:Arginine; D011392:Proline; D016547:Kinesins