Interactions between Bacterial Flagellar Axial Proteins in Their Monomeric State in Solution Yukio Furukawa 1,2 , Katsumi Imada 1 , Ferenc Vonderviszt 3 Hideyuki Matsunami 1 , Ken-ichi Sano 4 , Kazuhiro Kutsukake 5 and Keiichi Namba 1,6 * 1 Protonic NanoMachine Project ERATO, JST, 3-4 Hikaridai Seika, Kyoto 619-0237, Japan 2 Division of Biological Science Graduate School of Science Nagoya University, Chikusa Nagoya 464-8602, Japan 3 Department of Physics University of Veszpre ´m Egyetem u.10., H-8200 Hungary 4 RIKEN Harima Institute 1-1-1 Kouto, Mikazuki Hyogo 679-5198, Japan 5 Division of Biology Faculty of Science Okayama University 3-1-1 Tsushima-naka Okayama 700-8530, Japan 6 Advanced Technology Research Laboratories, Matsushita Electric Industrial Co., Ltd 3-4 Hikaridai, Seika, Kyoto 619-0237, Japan The axial structure of the bacterial flagellum is composed of many differ- ent proteins, such as hook protein and flagellin, and each protein forms a short or long axial segment one after another in a well-defined order along the axis. Under physiological conditions, most of these proteins are stable in the monomeric state in solution, and spontaneous polymeri- zation appears to be suppressed, as demonstrated clearly for flagellin, probably to avoid undesirable self-assembly in the cytoplasmic space. However, no systematic studies of the possible associations between monomeric axial proteins in solution have been carried out. We therefore studied self and cross-association between hook protein, flagellin and three hook-associated proteins, HAP1, HAP2 and HAP3, in all possible pairs, by gel-filtration and analytical centrifugation, and found inter- actions in the following two cases only. Flagellin facilitated HAP3 aggre- gation into b-amyloid-like filaments, but without stable binding between the two. Addition of HAP3 to HAP2 resulted in disassembly of preformed HAP2 decamers and formation of stable HAP2–HAP3 heterodimers. HAP2 missing either of its disordered terminal regions did not form the heterodimer, whereas HAP3 missing either of its disordered terminal regions showed stable heterodimer formation. This polarity in the hetero- dimer interactions suggests that the interactions between HAP2 and HAP3 in solution are basically the same as those in the flagellar axial structure. We discuss these results in relation to the assembly mechanism of the flagellum. q 2002 Elsevier Science Ltd. All rights reserved Keywords: bacterial flagellum; flagellar axial proteins; analytical ultracentrifugation; self-assembly; b-amyloid fiber *Corresponding author Introduction Most bacteria swim by means of flagella. The flagellum can be divided into three major portions, the basal body, the hook and the filament (Figure 1). The basal body is embedded in the cell mem- brane and functions as a rotary motor to drive the rotation of a long helical filament that propels the cell movements. The filament is a helical propeller and the hook is a universal joint that transmits the torque to the helical filament that can be oriented in various directions. There are three other minor components in the extracellular portion of the flagellum, and they are called hook-associated pro- teins, HAP1 (FlgK), HAP2 (FliD) and HAP3 (FlgL). 1,2 HAP1 and HAP3 are located between the hook and the filament in this order and form the junction between these two filamentous structures with distinct mechanical characteristics. 3,4 HAP2 forms the cap structure at the distal end of the 0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved E-mail address of the corresponding author: keiichi@crl.mei.co.jp Abbreviations used: MALDI-TOF MS, matrix- associated laser desorption/ionization time-of-flight mass spectroscopy; API, aminopeptidase I; CPY, carboxypeptidase Y; CP, clostripain; cv, column volume. doi: 10.1016/S0022-2836(02)00139-0 available online at http://www.idealibrary.com on B w J. Mol. Biol. (2002) 318, 889–900