Assembly Characteristics of Flagellar Cap Protein HAP2 of Salmonella: Decamer and Pentamer in the pH-sensitive Equilibrium Katsumi Imada 1 , Ferenc Vonderviszt 1,2 , Yukio Furukawa 1,3 Kenji Oosawa 4 and Keiichi Namba 1 * 1 International Institute for Advanced Research, Matsushita Electric Industrial Co., Ltd. 3-4 Hikaridai, Seika 619-02 Japan 2 Department of Physics University of Veszpre Âm Veszpre Âm, Egyetem u.10 H-8201, Hungary 3 Graduate School of Biological Science, Nara Advanced Institute of Science and Technology, 8916-5 Takayamacho, Ikoma 630-01 Japan 4 Graduate School of Polymathematics, Nagoya University, Chikusa-ku Nagoya 464-01 Japan The cap of the bacterial ¯agellum is an oligomeric assembly of HAP2 protein (also called FliD), tightly attached to the tip of the ¯agellar ®la- ment. Flagellar growth does not occur in ¯iD-de®cient mutants because ¯agellin monomers transported through the central channel of the ¯agel- lum leak out without polymerizing at the distal end. The structure of the cap complex is not known yet. An in vitro assembly of HAP2 proteins was found to have a pentagonal shape, while its molecular mass corre- sponded roughly to that of a dodecamer. To characterize the structure and assembly behavior of the complex formed in vitro in more detail, the stoichiometry of the complex and the association equilibrium have been studied. Crosslinking experiments now clearly show that the HAP2 com- plex is decameric. The assembly equilibrium is mainly between the monomer and decamer with a minor population of intermediate oligo- mers involved, and is highly dependent on the solution pH as well as the salt concentration: the fraction of the decamer sharply rises as the pH decreases from 8.5 to 8.0; the physiological concentration of salt partially suppresses the decamer formation. A preferential crosslinking within a pentameric unit together with a bipolar feature of the complex particle observed by electron microscopy suggests that the decamer is a bipolar pair of pentamers. Because of the polar nature of the ®lament cap struc- ture, the pentamer is suggested to be the cap complex with its decamer forming surface involved in interactions with the ®lament. # 1998 Academic Press Limited Keywords: bacterial ¯agellum; ¯agellar growth; cap structure; HAP2 assembly; FliD *Corresponding author Introduction Bacteria swim by rotating their ¯agellar ®la- ments (Berg & Anderson, 1973; Silverman & Simon, 1974; Larsen et al., 1974), each of which has a helical shape and works as a propeller. This unique motile apparatus, the bacterial ¯agellum, consists of three distinct major substructures: the basal-body, which contains a motor; the hook, working as a universal joint; and the ®lament, the helical propeller. The hook and the ®lament occupy a major portion of the extracellular part of the ¯agellum, which also contains three minor components called hook-associated protein 1, 2 and 3, or HAP1, HAP2 and HAP3, respectively (Homma et al., 1984a). There is a very short seg- ment between the hook and the ®lament, showing a thinner appearance on electron micrographs, where HAP1 and HAP3 are located in this order to form the junction (Ikeda et al., 1987). HAP2 is attached to the distal end of the ®lament, forming a cap structure (Ikeda et al., 1985, 1987). The hook and the ®lament are helical assemblies of single kinds of proteins, hook protein and ¯agellin, respectively. It is likely that HAP1 and HAP3 are also assembled in a helical manner similar to the hook and the ®lament. A little is known about the structural characteristics of the HAP2 cap, except for a thin and ¯at appearance at the distal end of the ®lament (Ikeda et al., 1985). Abbreviations used: BS 3 , Bis (sulfosuccinimidyl) suberate; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; sulfo-NHS, N-hydroxysulfosuccinimide; DTSSP, 3,3 0 - dithiobis(sulfosuccinimidyl propionate). J. Mol. Biol. (1998) 277, 883±891 0022±2836/98/140883±09 $25.00/0/mb981662 # 1998 Academic Press Limited