Analysis of Crystal Structure and Solution Structure of the Motor Domain of Rice Kinesin K16

Abstract

The novel rice plant specific kinesin K16 has several unique enzymatic characteristics comparing with conventional kinesin. The most interesting property is that the ADP-free K16 motor domain is very stable, contrast to conventional kinesin that is very labile in ADP-free state. Recently, we have determined crystal structure of the novel rice kinesin K16 motor domain (K16MD) in complex with MgADP at 2.5A resolution. The overall structure of the K16MD is similar to that of conventional kinesin motor domains, as expected from the high similarity of amino acid sequence (43.2 %). However, the neck-linker of the ADP bound K16 motor domain showed an ordered conformation in a position quite different from that observed in conventional kinesin, which may reflect the unique enzymatic characteristics of rice kinesin K16. In the present study, we analyzed the inner structure of the K16 motor domain in detail and compared the structure with Eg5 and other related kinesins. It has been revealed that K16MD does not have interaction of amino acids side chains, which stabilizes the docking conformation of neck-linker. We have also analyzed the conformation of neck-linker in the solution using the K16 by FRET. Motor domain mutant G220C chimera protein fused with GFP at the neck-linker has been prepared and labeled IAE-DANS. The FRET efficiencies between the fluorescent probe DANS at C220 and GFP in the presence and absence of nucleotides were analyzed.