Structural study of the hook-filament junction
     HAP3 (FlgL), together with HAP1 (FlgK) forms the junction between the hook and the helical filament of the bacterial flagellum. The hook is relatively flexible and functions as a universal joint, while the filament has a more rigid structure to work as a helical propeller for swimming of bacteria. Thus, the junction connects the two helical polymers of rather different mechanical properties. The junction is therefore presumed to be a helical assembly of HAP1 and HAP3, and from the length of the junction it appears to contain only a couple of turns of 1-start helix for each protein, which corresponds to about 11 subunits. It is interesting to see how the helical structures can be regulated to have such short segments.
     In order to understand the assembly regulation mechanisms of HAP1 and HAP3, we overexpressed the two proteins and successfully purified them in large amounts. We have been studying their assembly behaviors in solution. HAP3 forms two types of assemblies: fibrils and a ring of well-defined diameter. X-ray diffraction patterns from the fibril revealed that it contains an extended beta-sheet structure just like beta-amyloid fibers. Since HAP3 has disordered terminal regions in its monomer form in solution, just as flagellin and hook, this fibril formation appear to be a result of misaggregation of the disordered terminal parts into beta-sheet structures, although the terminal regions are predicted their amino-acid sequence to form alpha-helices.
     Since the ring may be a short helical structure corresponding to the junction complex, with alpha-helices formed in the terminal regions, the search for conditions under which only the ring formation is observed is now underway. Once the conditions are found, we will carry on the structural study of the ring by X-ray crystallography and electron cryomicroscopy.