© 2005 The Japanese Biochemical Society
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Removal of Tightly Bound ADP Induces Distinct Structural Changes of the Two Tryptophan-Containing Regions of the ncd Motor Domain
1 Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraqi 305-8566; 2 Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902; 3 Center for Radioisotope Facilities, Okazaki Research Facilities, Okazaki 444-8787; and 4 Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657
* To whom correspondence should be addressed. Tel: +81-29-861-6191, Fax: +81-29-861-9494, E-mail: tak-shimizu{at}aist.go.jp
ncd is a molecular motor belonging to the kinesin superfamily. In solution, it is a homo-dimer of a 700 amino acid polypeptide. The C-terminus of each polypeptide forms a globular domain of about 40 kDa, the motor domain with ATPase activity. The ATPase site of the motor domain of kinesin family members, including ncd, binds ADP tightly, the release of which is facilitated by microtubules during the mechanochemical ATPase cycle. Previously, we studied the spectroscopic characteristics of the ncd motor domain, focusing on interactions of the transition-moment-dipoles between ADP and aromatic amino acid side chains using circular dichroism (CD) spectroscopy. In the present study, we generated several ncd motor domain mutants. In each, a tryptophanyl or specific tyrosyl residue was mutated. We found that Trp370 and Tyr442, the latter of which stacks directly with the adenine moiety of bound ADP, caused the bound ADP to exhibit peculiar CD signals. In addition, fluorescence measurements revealed that Trp370, but not Trp473, was responsible for the emission intensity change depending on the presence or absence of bound ADP. This fluorescence result implies that the structural change induced at the ADP-binding site (on the release of the ADP) is transmitted to the region that includes Trp370, which is relatively close to the ADP-binding site but not in direct contact with the ADP-binding region. In contrast, Trp473 in the region that is in contact with the 
-helical coiled coil stalk did not experience the structural changes caused on removal of ADP. The distinct behavior of these two tryptophanyl residues suggests that the ncd motor domain has a bifacial architecture made up of a relatively deformable side including the nucleotide binding site and a more rigid one.
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