Analysis of Conformational Change of Novel Rice Kinesin K16 Using Small Angle X-ray Solution Scattering and EPR

Abstract

We have previously revealed that rice kinesin K16 has several unique enzymatic characteristics comparing with conventional kinesin. The most interesting property is that the ADP-free K16 motor domain(MD) is very stable, contrast to conventional kinesin that is very labile in ADP-free state. Recently, we have successfully dissolved the crystal structure of ADP bound K16 motor domain. The overall structure of the K16MD is similar to that of conventional kinesin MD, as expected from the high similarity of amino acid sequence. However, neck-linker region showed an ordered conformation in a position quite different from conventional kinesin. In this study, we designed the K16MD chimera protein fused with GFP at the neck-linker in order to monitor the conformational change of the neck-linker during ATP hydrolysis by small angle X-ray solution scattering and EPR. We determined the Radius gyration (Rg) values of K16-GFP in the presence or absence of nucleotides by X-ray solution scattering. The Rg of nucleotide-free K16-GFP was about 42A. In the presence of ADP and ATP, the Rg values were 38A and 39A, respectively. These results may suggest that the neck-linker of nucleotide free K16 is in the docked conformation, on the other hand, the neck-linker of nucleotide bound state is in the novel conformation observed in crystal structure. We also analyzed conformational change of K16 in the solution by EPR. We constructed K16 mutants which have single cysteine at 331, 335 or 340 and labeled with 4-maleimido-2,2,6,6-tetramethyl-1-piperidinyloxy. But we could not observe notable change of mobility during ATP hydrolysis in the absence of microtubule for the three mutants. Currently, we are analyzing the distance between kinesin core reagion at 47 and neck linker at 328 using the dipolar EPR method.