Structural basis of Irgb6 inactivation by Toxoplasma gondii through the phosphorylation of switch I.

Irgb6 is a priming immune-related GTPase (IRG) that counteracts Toxoplasma gondii. It is known to be recruited to the low virulent type II T. gondii parasitophorous vacuole (PV), initiating cell-autonomous immunity. However, the molecular mechanism by which immunity-related GTPases become inactivated after the parasite infection remains obscure. Here, we found that Thr95 of Irgb6 is prominently phosphorylated in response to low virulent type II T. gondii infection. We observed that a phosphomimetic T95D mutation in Irgb6 impaired its localization to the PV and exhibited reduced GTPase activity in vitro. Structural analysis unveiled an atypical conformation of nucleotide-free Irgb6-T95D, resulting from a conformational change in the G-domain that allosterically modified the PV membrane-binding interface. In silico docking corroborated the disruption of the physiological membrane binding site. These findings provide novel insights into a T. gondii-induced allosteric inactivation mechanism of Irgb6.

Structural model of microtubule dynamics inhibition by kinesin-4 from the crystal structure of KLP-12 -tubulin complex.

Kinesin superfamily proteins are microtubule-based molecular motors driven by the energy of ATP hydrolysis. Among them, the kinesin-4 family is a unique motor that inhibits microtubule dynamics. Although mutations of kinesin-4 cause several diseases, its molecular mechanism is unclear because of the difficulty of visualizing the high-resolution structure of kinesin-4 working at the microtubule plus-end. Here, we report that KLP-12, a C. elegans kinesin-4 ortholog of KIF21A and KIF21B, is essential for proper length control of C. elegans axons, and its motor domain represses microtubule polymerization in vitro. The crystal structure of the KLP-12 motor domain complexed with tubulin, which represents the high-resolution structural snapshot of the inhibition state of microtubule-end dynamics, revealed the bending effect of KLP-12 for tubulin. Comparison with the KIF5B-tubulin and KIF2C-tubulin complexes, which represent the elongation and shrinking forms of microtubule ends, respectively, showed the curvature of tubulin introduced by KLP-12 is in between them. Taken together, KLP-12 controls the proper length of axons by modulating the curvature of the microtubule ends to inhibit the microtubule dynamics.

From meter-long structures that allow nerve cells to stretch across a body to miniscule 'hairs' required for lung cells to clear mucus, many life processes rely on cells sporting projections which have the right size for their role. Networks of hollow filaments known as microtubules shape these structures and ensure that they have the appropriate dimensions. Controlling the length of microtubules is therefore essential for organisms, yet how this process takes place is still not fully elucidated. Previous research has shown that microtubules continue to grow when their end is straight but stop when it is curved. A family of molecular motors known as kinesin-4 participate in this process, but the exact mechanisms at play remain unclear. To investigate, Tuguchi, Nakano, Imasaki et al. focused on the KLP-12 protein, a kinesin-4 equivalent which helps to controls the length of microtubules in the tiny worm Caenorhabditis elegans. They performed genetic manipulations and imaged the interactions between KLP-12 and the growing end of a microtubule using X-ray crystallography. This revealed that KLP-12 controls the length of neurons by inhibiting microtubule growth. It does so by modulating the curvature of the growing end of the filament to suppress its extension. A 'snapshot' of KLP-12 binding to a microtubule at the resolution of the atom revealed exactly how the protein helps to bend the end of the filament to prevent it from growing further. These results will help to understand how nerve cells are shaped. This may also provide insights into the molecular mechanisms for various neurodegenerative disorders caused by problems with the human equivalents of KLP-12, potentially leading to new therapies.

CAMSAP2 organizes a γ-tubulin-independent microtubule nucleation centre through phase separation.

Microtubules are dynamic polymers consisting of αß-tubulin heterodimers. The initial polymerization process, called microtubule nucleation, occurs spontaneously via αß-tubulin. Since a large energy barrier prevents microtubule nucleation in cells, the γ-tubulin ring complex is recruited to the centrosome to overcome the nucleation barrier. However, a considerable number of microtubules can polymerize independently of the centrosome in various cell types. Here, we present evidence that the minus-end-binding calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) serves as a strong nucleator for microtubule formation by significantly reducing the nucleation barrier. CAMSAP2 co-condensates with αß-tubulin via a phase separation process, producing plenty of nucleation intermediates. Microtubules then radiate from the co-condensates, resulting in aster-like structure formation. CAMSAP2 localizes at the co-condensates and decorates the radiating microtubule lattices to some extent. Taken together, these in vitro findings suggest that CAMSAP2 supports microtubule nucleation and growth by organizing a nucleation centre as well as by stabilizing microtubule intermediates and growing microtubules.

Cells are able to hold their shape thanks to tube-like structures called microtubules that are made of hundreds of tubulin proteins. Microtubules are responsible for maintaining the uneven distribution of molecules throughout the cell, a phenomenon known as polarity that allows cells to differentiate into different types with various roles. A protein complex called the γ-tubulin ring complex (γ-TuRC) is necessary for microtubules to form. This protein helps bind the tubulin proteins together and stabilises microtubules. However, recent research has found that in highly polarized cells such as neurons, which have highly specialised regions, microtubules can form without γ-TuRC. Searching for the proteins that could be filling in for γ-TuRC in these cells some evidence has suggested that a group known as CAMSAPs may be involved, but it is not known how. To characterize the role of CAMSAPs, Imasaki, Kikkawa et al. studied how one of these proteins, CAMSAP2, interacts with tubulins. To do this, they reconstituted both CAMSAP2 and tubulins using recombinant biotechnology and mixed them in solution. These experiments showed that CAMSAP2 can help form microtubules by bringing together their constituent proteins so that they can bind to each other more easily. Once microtubules start to form, CAMSAP2 continues to bind to them, stabilizing them and enabling them to grow to full size. These results shed light on how polarity is established in cells such as neurons, muscle cells, and epithelial cells. Additionally, the ability to observe intermediate structures during microtubule formation can provide insights into the processes that these structures are involved in.

Structural basis of membrane recognition of Toxoplasma gondii vacuole by Irgb6.

The p47 immunity-related GTPase (IRG) Irgb6 plays a pioneering role in host defense against Toxoplasma gondii infection. Irgb6 is recruited to the parasitophorous vacuole membrane (PVM) formed by T. gondii and disrupts it. Despite the importance of this process, the molecular mechanisms accounting for PVM recognition by Irgb6 remain elusive because of lack of structural information on Irgb6. Here we report the crystal structures of mouse Irgb6 in the GTP-bound and nucleotide-free forms. Irgb6 exhibits a similar overall architecture to other IRGs in which GTP binding induces conformational changes in both the dimerization interface and the membrane-binding interface. The membrane-binding interface of Irgb6 assumes a unique conformation, composed of N- and C-terminal helical regions forming a phospholipid binding site. In silico docking of phospholipids further revealed membrane-binding residues that were validated through mutagenesis and cell-based assays. Collectively, these data demonstrate a novel structural basis for Irgb6 to recognize T. gondii PVM in a manner distinct from other IRGs.

Architecture of the Bacterial Flagellar Distal Rod and Hook of Salmonella.

The bacterial flagellum is a large molecular complex composed of thousands of protein subunits for motility. The filamentous part of the flagellum, which is called the axial structure, consists of the filament, the hook, and the rods, with other minor components-the cap protein and the hook associated proteins. They share a common basic architecture of subunit arrangement, but each part shows quite distinct mechanical properties to achieve its specific function. The distal rod and the hook are helical assemblies of a single protein, FlgG and FlgE, respectively. They show a significant sequence similarity but have distinct mechanical characteristics. The rod is a rigid, straight cylinder, whereas the hook is a curved tube with high bending flexibility. Here, we report a structural model of the rod constructed by using the crystal structure of a core fragment of FlgG with a density map obtained previously by electron cryomicroscopy. Our structural model suggests that a segment called L-stretch plays a key role in achieving the distinct mechanical properties of the rod using a structurally similar component protein to that of the hook.

Identification and functional analysis of 2-hydroxyflavanone C-glucosyltransferase in soybean (Glycine max).

C-Glucosyltransferase is an enzyme that mediates carbon-carbon bond formation to generate C-glucoside metabolites. Although it has been identified in several plant species, the catalytic amino acid residues required for C-glucosylation activity remain obscure. Here, we identified a 2-hydroxyflavanone C-glucosyltransferase (UGT708D1) in soybean. We found that three residues, His20, Asp85, and Arg292, of UGT708D1 were located at the predicted active site and evolutionarily conserved. The substitution of Asp85 or Arg292 with alanine destroyed C-glucosyltransferase activity, whereas the substitution of His20 with alanine abolished C-glucosyltransferase activity but enabled O-glucosyltransferase activity. The catalytic mechanism is discussed on the basis of the findings.

Crystallization and preliminary X-ray analysis of the periplasmic domain of FliP, an integral membrane component of the bacterial flagellar type III protein-export apparatus.

The bacterial flagellar proteins are transported via a specific export apparatus to the distal end of the growing structure for their self-assembly. FliP is an essential membrane component of the export apparatus. FliP has an N-terminal signal peptide and is predicted to have four transmembrane (TM) helices and a periplasmic domain (FliPP) between TM-2 and TM-3. In this study, FliPP from Thermotoga maritima (TmFliPP) and its selenomethionine derivative (SeMet-TmFliPP) were purified and crystallized. TmFliPP formed a homotetramer in solution. Crystals of TmFliPP and SeMet-TmFliPP were obtained by the hanging-drop vapour-diffusion technique with 2-methyl-2,4-pentanediol as a precipitant. These two crystals grew in the hexagonal space group P6222 or P6422, with unit-cell parameters a = b = 114.9, c = 193.8 Å. X-ray diffraction data were collected from crystals of TmFliPP and SeMet-TmFliPP to 2.4 and 2.8 Šresolution, respectively.

Conformational analysis of isolated domains of Helicobacter pylori CagA.

The CagA protein of Helicobacter pylori is associated with increased virulence and gastric cancer risk. CagA is translocated into the host cell by a H. pylori type IV secretion system via mechanisms that are poorly understood. Translocated CagA interacts with numerous host factors, altering a variety of host signalling pathways. The recently determined crystal structure of C-terminally-truncated CagA indicated the presence of two domains: the smaller, flexible N-terminal domain and the larger, middle domain. In this study, we have investigated the conformation, oligomeric state and stability of the N-terminal, middle and glutamate-proline-isoleucine-tyrosine-alanine (EPIYA)-repeats domains. All three domains are monomeric, suggesting that the multimerisation of CagA observed in infected cells is likely to be mediated not by CagA itself but by its interacting partners. The middle and the C-terminal domains, but not the N-terminal domain, are capable of refolding spontaneously upon heat denaturation, lending support to the hypothesis that unfolded CagA is threaded C-terminus first through the type IV secretion channel with its N-terminal domain, which likely requires interactions with other domains to refold, being threaded last. Our findings also revealed that the C-terminal EPIYA-repeats domain of CagA exists in an intrinsically disordered premolten globule state with regions in PPII conformation--a feature that is shared by many scaffold proteins that bind multiple protein components of signalling pathways. Taken together, these results provide a deeper understanding of the physicochemical properties of CagA that underpin its complex cellular and oncogenic functions.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of a core fragment of FlgG, a bacterial flagellar rod protein.

FlgG is a bacterial flagellar rod protein and constructs the distal rod connecting to the hook. FlgG of Salmonella enterica serovar Typhimurium is a 260-amino-acid protein composed of a folded core region and N- and C-terminal regions that are unfolded in solution. A core fragment of FlgG (FlgG47-227) was expressed, purified and crystallized. Crystals of native and SeMet-labelled FlgG47-227 were obtained by the sitting-drop vapour-diffusion technique with PEG MME 2000 as precipitant. The native crystal belonged to the primitive orthorhombic space group P212121, with unit-cell parameters a = 47.78, b = 68.94, c = 110.57 Å. The SeMet crystal also belonged to space group P212121, with unit-cell parameters a = 47.53, b = 67.04, c = 110.27 Å.

Isolation of Salmonella mutants resistant to the inhibitory effect of Salicylidene acylhydrazides on flagella-mediated motility.

Salicylidene acylhydrazides identified as inhibitors of virulence-mediating type III secretion systems (T3SSs) potentially target their inner membrane export apparatus. They also lead to inhibition of flagellar T3SS-mediated swimming motility in Salmonella enterica serovar. Typhimurium. We show that INP0404 and INP0405 act by reducing the number of flagella/cell. These molecules still inhibit motility of a Salmonella ΔfliH-fliI-fliJ/flhB((P28T)) strain, which lacks three soluble components of the flagellar T3S apparatus, suggesting that they are not the target of this drug family. We implemented a genetic screen to search for the inhibitors' molecular target(s) using motility assays in the ΔfliH-fliI/flhB((P28T)) background. Both mutants identified were more motile than the background strain in the absence of the drugs, although HM18 was considerably more so. HM18 was more motile than its parent strain in the presence of both drugs while DI15 was only insensitive to INP0405. HM18 was hypermotile due to hyperflagellation, whereas DI15 was not hyperflagellated. HM18 was also resistant to a growth defect induced by high concentrations of the drugs. Whole-genome resequencing of HM18 indicated two alterations within protein coding regions, including one within atpB, which encodes the inner membrane a-subunit of the F(O)F(1)-ATP synthase. Reverse genetics indicated that the alteration in atpB was responsible for all of HM18's phenotypes. Genome sequencing of DI15 uncovered a single A562P mutation within a gene encoding the flagellar inner membrane protein FlhA, the direct role of which in mediating drug insensitivity could not be confirmed. We discuss the implications of these findings in terms of T3SS export apparatus function and drug target identification.

Functional defect and restoration of temperature-sensitive mutants of FlhA, a subunit of the flagellar protein export apparatus.

The flagellar axial component proteins are exported to the distal end of the growing flagellum for self-assembly by the flagellar type III export apparatus. FlhA is a key membrane protein of the export apparatus, and its C-terminal cytoplasmic domain (FlhA(C)) is a part of an assembly platform for the three soluble export components, FliH, FliI, and FliJ, as well as export substrates and chaperone-substrate complexes. FlhA(C) is composed of a flexible linker region and four compact domains (A(C)D1-A(C)D4). At 42 °C, a temperature-sensitive (TS) G368C mutation in FlhA(C) blocks the export process after the FliH-FliI-FliJ-substrate complex binds to the assembly platform, but it remains unknown how it does so. In this study, we analyzed a TS mutant variant, FlhA(C)(G368C), and its pseudorevertant variants FlhA(C)(G368C/L359F), FlhA(C)(G368C/G364R), FlhA(C)(G368C/R370S), and FlhA(C)(G368C/P550S) using far-ultraviolet circular dichroism. Whereas the denaturation of the wild-type FlhA(C) occurs in a single step, FlhA(C)(G368C) and its pseudorevertant variants showed thermal transitions, at least, in two steps. The first transition of FlhA(C)(G368C) can further be divided into reversible and following irreversible transitions, which correspond to the denaturation of A(C)D2 and A(C)D1, respectively. We show the relation between the reversible transition and the TS defect in the exporting function of FlhA(C)(G368C) and that the loss of function is caused by denaturation of A(C)D2. We suggest that A(C)D2 is directly involved in the translocation of export substrates.

Structural analysis of the essential resuscitation promoting factor YeaZ suggests a mechanism of nucleotide regulation through dimer reorganization.

BACKGROUND: The yeaZ gene product forms part of the conserved network YjeE/YeaZ/YgjD essential for the survival of many gram-negative eubacteria. Among other as yet unidentified roles, YeaZ functions as a resuscitation promoting factor required for survival and resuscitation of cells in a viable but non-culturable (VBNC) state. METHODOLOGY/PRINCIPAL FINDINGS: In order to investigate in detail the structure/function relationship of this family of proteins we have performed X-ray crystallographic studies of Vibrio parahaemolyticus YeaZ. The YeaZ structure showed that it has a classic actin-like nucleotide-binding fold. Comparisons of this crystal structure to that of available homologues from E. coli, T. maritima and S. typhimurium revealed two distinctly different modes of dimer formation. In one form, prevalent in the absence of nucleotide, the putative nucleotide-binding site is incomplete, lacking a binding pocket for a nucleotide base. In the second form, residues from the second subunit complete the nucleotide-binding site. This suggests that the two dimer architectures observed in the crystal structures correspond to a free and a nucleotide-bound form of YeaZ. A multiple sequence alignment of YeaZ proteins from different bacteria allowed us to identify a large conserved hydrophobic patch on the protein surface that becomes exposed upon nucleotide-driven dimer re-arrangement. We hypothesize that the transition between two dimer architectures represents the transition between the 'on' and 'off' states of YeaZ. The effect of this transition is to alternately expose and bury a docking site for the partner protein YgjD. CONCLUSIONS/SIGNIFICANCE: This paper provides the first structural insight into the putative mechanism of nucleotide regulation of YeaZ through dimer reorganization. Our analysis suggests that nucleotide binding to YeaZ may act as a regulator or switch that changes YeaZ shape, allowing it to switch partners between YjeE and YgjD.

Structure of the cytoplasmic domain of FlhA and implication for flagellar type III protein export.

FlhA is the largest integral membrane component of the flagellar type III protein export apparatus of Salmonella and is composed of an N-terminal transmembrane domain (FlhA(TM)) and a C-terminal cytoplasmic domain (FlhA(C)). FlhA(C) is thought to form a platform of the export gate for the soluble components to bind to for efficient delivery of export substrates to the gate. Here, we report a structure of FlhA(C) at 2.8 A resolution. FlhA(C) consists of four subdomains (A(C)D1, A(C)D2, A(C)D3 and A(C)D4) and a linker connecting FlhA(C) to FlhA(TM). The sites of temperature-sensitive (ts) mutations that impair protein export are distributed to all four domains, with half of them at subdomain interfaces. Analyses of the ts mutations and four suppressor mutations to the G368C ts mutation suggested that FlhA(C) changes its conformation for its function. Molecular dynamics simulation demonstrated an open-close motion with a 5-10 ns oscillation in the distance between A(C)D2 and A(C)D4. These results along with further mutation analyses suggest that a dynamic domain motion of FlhA(C) is essential for protein export.

Role of the C-terminal cytoplasmic domain of FlhA in bacterial flagellar type III protein export.

For construction of the bacterial flagellum, many of the flagellar proteins are exported into the central channel of the flagellar structure by the flagellar type III protein export apparatus. FlhA and FlhB, which are integral membrane proteins of the export apparatus, form a docking platform for the soluble components of the export apparatus, FliH, FliI, and FliJ. The C-terminal cytoplasmic domain of FlhA (FlhA(C)) is required for protein export, but it is not clear how it works. Here, we analyzed a temperature-sensitive Salmonella enterica mutant, the flhA(G368C) mutant, which has a mutation in the sequence encoding FlhA(C). The G368C mutation did not eliminate the interactions with FliH, FliI, FliJ, and the C-terminal cytoplasmic domain of FlhB, suggesting that the mutation blocks the export process after the FliH-FliI-FliJ-export substrate complex binds to the FlhA-FlhB platform. Limited proteolysis showed that FlhA(C) consists of at least three subdomains, a flexible linker, FlhA(CN), and FlhA(CC), and that FlhA(CN) becomes sensitive to proteolysis by the G368C mutation. Intragenic suppressor mutations were identified in these subdomains and restored flagellar protein export to a considerable degree. However, none of these suppressor mutations suppressed the protease sensitivity. We suggest that FlhA(C) not only forms part of the docking platform for the FliH-FliI-FliJ-export substrate complex but also is directly involved in the translocation of the export substrate into the central channel of the growing flagellar structure.

Crystallization and preliminary X-ray analysis of the C-terminal cytoplasmic domain of FlhA, a membrane-protein subunit of the bacterial flagellar type III protein-export apparatus.

The axial components of the bacterial flagellum and the scaffolding proteins for its assembly are exported through the flagellar-specific type III protein-export apparatus, which is believed to be located on the cytoplasmic surface of the basal body. FlhA is an essential component of the type III export apparatus of Salmonella and consists of two major portions: an N-terminal transmembrane domain and a C-terminal cytoplasmic domain (FlhAC). FlhAC and a 38 kDa fragment of FlhAC (FlhAC38K) were purified and crystallized. The crystals were obtained by the sitting-drop vapour-diffusion technique with PEG 8000 as a precipitant. FlhAC crystals grew in the tetragonal space group I4(1)/I4(3), with unit-cell parameters a = b = 216.6, c = 65.0 A. FlhAC38K was crystallized in an orthorhombic form, with unit-cell parameters a = 53.0, b = 93.1, c = 186.5 A. X-ray diffraction data from crystals of FlhAC and the SeMet derivative of FlhAC were collected to 2.9 and 3.2 A, respectively.

Structural and functional analysis of the C-terminal cytoplasmic domain of FlhA, an integral membrane component of the type III flagellar protein export apparatus in Salmonella.

FlhA is an integral membrane component of the Salmonella type III flagellar protein export apparatus. It consists of 692 amino acid residues and has two domains: the N-terminal transmembrane domain consisting of the first 327 amino acid residues, and the C-terminal cytoplasmic domain (FlhAC) comprising the remainder. Here, we have investigated the structure and function of FlhAC. DNA sequence analysis revealed that temperature-sensitive flhA mutations, which abolish flagellar protein export at the restrictive temperature, lie in FlhAC, indicating that FlhAC plays an important role in the protein export process. Limited proteolysis of purified His-FlhAC by trypsin and V8 showed that only a small part of FlhAC near its N terminus (residues 328-351) is sensitive to proteolysis. FlhAC38K, the smallest fragment produced by V8 proteolysis, is monomeric and has a spherical shape as judged by analytical gel filtration chromatography and analytical ultracentrifugation. The far-UV CD spectrum of FlhAC38K showed that it contains considerable amounts of secondary structure. FlhA(Delta328-351) missing residues 328-351 failed to complement the flhA mutant, indicating that the proteolytically sensitive region of FlhA is important for its function. FlhA(Delta328-351) was inserted into the cytoplasmic membrane, and exerted a strong dominant negative effect on wild-type cells, suggesting that it retains the ability to interact with other export components within the cytoplasmic membrane. Overproduced FlhAC38K inhibited both motility and flagellar protein export of wild-type cells to some degree, suggesting that FlhAC38K is directly involved in the translocation reaction. Amino acid residues 328-351 of FlhA appear to be a relatively flexible linker between the transmembrane domain and FlhAC38K.

Domain organization and function of Salmonella FliK, a flagellar hook-length control protein.

Salmonella hook-length control protein FliK, which consists of 405 amino acid residues, switches substrate specificity of the type III flagellar protein export apparatus from rod/ hook-type to filament-type by causing a conformational change in the cytoplasmic domain of FlhB (FlhB(C)) upon completion of the hook assembly. An N-terminal region of FliK contains an export signal, and a highly conserved C-terminal region consisting of amino acid residues 265-405 (FliK((265-405))) is directly involved in the switching of FlhB(C). Here, we have investigated the structural properties of FliK. Gel filtration chromatography, multi-angle light scattering and analytical ultracentrifugation showed that FliK is monomeric in solution and has an elongated shape. Limited proteolysis showed that FliK consists of two domains, the N-terminal (FliK(N)) and C-terminal domains (FliK(C)), and that the first 203 and the last 35 amino acid residues are partially unfolded and subjected to proteolysis. Both FliK(N) and FliK(C) are more globular than full-length FliK, suggesting that these domains are connected in tandem. Overproduced His-FliK((199-405)) failed to switch export specificity of the export apparatus. Affinity blotting revealed that FlhB(C) binds to FliK and FliK((1-147)), but not to FliK((265-405)). Based on these results, we propose that FliK(N) within the central channel of the hook-basal body during the export of FliK is the sensor and transmitter of hook completion information and that the binding interaction of FliK(C) to FlhB(C) is structurally regulated by FliK(N) so as to occur only when the hook has reached a preset length. The conformational flexibility of FliK(C) may play a role in interfering with switching at an inappropriate point of flagellar assembly.

In vitro characterization of FlgB, FlgC, FlgF, FlgG, and FliE, flagellar basal body proteins of Salmonella.

The bacterial flagellar basal body is a rotary motor. It spans the cytoplasmic and outer membranes and drives rapid rotation of a long helical filament in the cell exterior. The flagellar rod at its central axis is a drive shaft that transmits torque through the hook to the filament to propel the bacterial locomotion. To study the structure of the rod in detail, we have established purification procedures for Salmonella rod proteins, FlgB, FlgC, FlgF, FlgG, and also for FliE, a rod adapter protein, from an Escherichia coli expression system. While FlgF was highly soluble, FlgB, FlgC, FlgG and FliE tended to self or cross-aggregate into fibrils in solutions at neutral pH or below, at high ionic strength, or at high protein concentration. These aggregates were characterized to be beta-amyloid fibrils, unrelated to the rod structure formed in vivo. Under non-aggregative conditions, no protein-protein interactions were detected between any pairs of these five proteins, suggesting that their spontaneous, template-free polymerization is strongly suppressed. Limited proteolyses showed that FlgF and FlgG have natively unfolded N and C-terminal regions of about 100 residues in total just as flagellin does, whereas FlgB, FlgC and FliE, which are little over 100 residues long, are unfolded in their entire peptide chains. These results together with other data indicate that all of the ten flagellar axial proteins share structural characteristics and folding dynamics in relation to the mechanism of their self-assembly into the flagellar axial structure.