Mechanism of Self-association and Filament Capping by Flagellar HAP2 Ferenc Vonderviszt 1,5 *, Katsumi Imada 1 , Yukio Furukawa 1,2 Hatsuho Uedaira 3 , Hisaaki Taniguchi 4 and Keiichi Namba 1 1 International Institute for Advanced Research, Matsushita Electric Industrial Co., Ltd 3-4 Hikaridai, Seika 619-0237, Japan 2 Graduate School of Biological Science, Nara Advanced Institute of Science and Technology, 8916-5 Takayamacho, Ikoma 630-01 Japan 3 National Institute of Bioscience and Human-Technology Higashi, Tsukuba 305-8566, Japan 4 Institute for Comprehensive Medical Science, Fujita Health University, Kutsukake-cho Toyoake 470-1192, Japan 5 Department of Physics University of Veszpre Âm H-8200 Veszpre Âm, Egyetem u.10, Hungary HAP2 forms a capping structure, which binds very tightly to the distal end of ¯agellar ®laments and still allows insertion of ¯agellin subunits below the cap by an unknown mechanism. Terminal regions of HAP2 from Salmonella typhimurium were found to be quickly degraded by var- ious proteases, indicating that HAP2 also possesses disordered terminal regions like other axial proteins of bacterial ¯agellum. Removal of these portions by trypsin results in a fragment of 40 kDa (HP40), which lacks 42 NH 2 -terminal and 51 COOH-terminal residues. HAP2 in solution readily associates into a decameric structure without any signi®cant population of intermediate oligomeric forms. The HP40 fragments, how- ever, do not form decamers, while they can assemble into pentamers, as revealed by chemical cross-linking and analytical ultracentrifugation. Decameric HAP2 also dissociates into pentamers and smaller oligomers upon a heat induced conformational transition around 36  C. While the highly mobile terminal regions are immobilized in decameric HAP2 com- plexes, they are still largely disordered in the pentameric state. These results demonstrate that the intersubunit interactions within the penta- mers are mainly through the HP40 portions, whereas the terminal regions are responsible for association of pentamers into decameric complexes. Several observations indicate that HAP2 performs its capping function as a pentamer. We suggest that binding of the pentameric HAP2 cap to the ®lament is mediated by the highly ¯exible terminal regions. Indeed, HP40 fragments are unable to cap the end of ®laments, while removal of about 30 residues from both terminal regions of HAP2 results in a highly reduced capping ability. A model is presented to explain the molecular mechanism of capping, in which conformational entropy in the disor- dered terminal regions moderates the otherwise too tight HAP2-®lament interactions to allow insertion of ¯agellin subunits below the cap. # 1998 Academic Press Keywords: bacterial ¯agellum; ¯agellar growth; cap structure; HAP2 assembly; FliD *Corresponding author Introduction Bacteria swim by means of ¯agella. The major part of the ¯agellum is the helical ®lament, which works as a propeller rotated by a membrane- embedded molecular motor at its base part (Macnab, 1995; DeRosier, 1995). The long helical ®lament is connected to the basal structure via a short, highly curved segment called the hook. Both the hook and the helical ®lament are self-assem- bling macromolecular structures composed of the hook protein and ¯agellin, respectively (Namba & Vonderviszt, 1997, and references therein). There are also three minor components, called hook associated proteins (HAP1, HAP2, HAP3; Homma et al., 1984a; Homma & Iino, 1985a). Estimates of HAP stoichiometries indicated that the ¯agellum roughly contains 10-15 copies of HAP1, 10-30 copies of HAP3 and 6-12 copies of HAP2 (Ikeda et al., 1987). E-mail address of the corresponding author feri@elod.vein.hu Abbreviations used: EDC, 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride; sulfo-NHS, N-hydroxysulfosuccinimide; HP42 and HP40, proteolytic fragments of HAP2 with relative molecular masses of 42,000 and 40,000 Da. Article No. mb982274 J. Mol. Biol. (1998) 284, 1399±1416 0022 ± 2836/98/501399±18 $30.00/0 # 1998 Academic Press