The bacterial flagellar filament is a helical propeller constructed from 11 protofilaments of a single protein, flagellin. The filament switches between left- and right-handed supercoiled forms when bacteria switch their swimming mode between running and tumbling. Supercoiling is produced by two different packing interactions of flagellin called L and R. In switching from L to R, the intersubunit distance ( approximately 52 A) along the protofilament decreases by 0.8 A. Changes in the number of L and R protofilaments govern supercoiling of the filament. Here we report the 2.0 A resolution crystal structure of a Salmonella flagellin fragment of relative molecular mass 41,300. The crystal contains pairs of antiparallel straight protofilaments with the R-type repeat. By simulated extension of the protofilament model, we have identified possible switch regions responsible for the bi-stable mechanical switch that generates the 0.8 A difference in repeat distance.

The bacterial flagellum is a motile organelle, and the flagellar hook is a short, highly curved tubular structure that connects the flagellar motor to the long filament acting as a helical propeller. The hook is made of about 120 copies of a single protein, FlgE, and its function as a nano-sized universal joint is essential for dynamic and efficient bacterial motility and taxis. It transmits the motor torque to the helical propeller over a wide range of its orientation for swimming and tumbling. Here we report a partial atomic model of the hook obtained by X-ray crystallography of FlgE31, a major proteolytic fragment of FlgE lacking unfolded terminal regions, and by electron cryomicroscopy and three-dimensional helical image reconstruction of the hook. The model reveals the intricate molecular interactions and a plausible switching mechanism for the hook to be flexible in bending but rigid against twisting for its universal joint function.

No abstract

The apocytochrome b gene, exclusively encoded by the mitochondrial genome, was engineered so that it could be expressed in the yeast cytoplasm. Different combinations of the apocytochrome b transmembrane domains were produced in the form of hybrid proteins fused to both the N-terminal mitochondrial targeting sequence of the ATPase subunit 9 from Neurospora crassa and to a cytoplasmic vers,ion of the b14 RNA maturase, localised on the N-terminal and C-terminal sides, respectively, of the hydrophobic stretches. The b14 RNA maturase, which can complement mitochondrial mutations, was used as an in vivo reporter to assess the mitochondrial import of the different groups of transmembrane helices. This new, reliable and sensitive reporter activity allowed us to experimentally determine the limitations to the mitochondrial import of hydrophobic proteins. All eight transmembrane helices of apocytochrome b could be imported into mitochondria, either alone or in combination, but no more than three to four transmembrane helices could be imported together at one time. This limit is close to that observed in the population of nuclear-encoded mitochondrial proteins. The hydrophobic characteristics of engineered and natural proteins targeted to the mitochondrial inner membrane revealed two factors important in the import process. These were (a) the local hydrophobicity of a transmembrane segment, and (b) the average regional hydrophobicity of the protein over an extended length of 60-80 residues. Such features may have played a major role in the evolution of mitochondria1 genomes.

No abstract

The bacterial flagellar hook is a tubular helical structure made by the polymerization of multiple copies of a protein, FlgE. Here we report the structure of the hook from Campylobacter jejuni by cryo-electron microscopy at a resolution of 3.5 Å. On the basis of this structure, we show that the hook is stabilized by intricate inter-molecular interactions between FlgE molecules. Extra domains in FlgE, found only in Campylobacter and in related bacteria, bring more stability and robustness to the hook. Functional experiments suggest that Campylobacter requires an unusually strong hook to swim without its flagella being torn off. This structure reveals details of the quaternary organization of the hook that consists of 11 protofilaments. Previous study of the flagellar filament of Campylobacter by electron microscopy showed its quaternary structure made of seven protofilaments. Therefore, this study puts in evidence the difference between the quaternary structures of a bacterial filament and its hook.

The axial proteins of the bacterial flagellum function as a drive shaft, universal joint, and propeller driven by the flagellar rotary motor; they also form the putative protein export channel. The Nand C-terminal sequences of the eight axial proteins were predicted to form interlocking ␣-domains generating an axial tube. We report on an Ϸ1-nm resolution map of the hook from Salmonella typhimurium, which reveals such a tube made from interdigitated, 1-nm rod-like densities similar to those seen in maps of the filament. Atomic models for the two outer domains of the hook subunit were docked into the corresponding outermost features of the map. The N and C termini of the hook subunit fragment are positioned next to each other and face toward the axis of the hook. The placement of these termini would permit the residues missing in the fragment to form the rod-like features that form the core domain of the hook. We also fit the hook atomic model to an Ϸ2-nm resolution map of the hook from Caulobacter crescentus. The hook protein sequence from C. crescentus is largely homologous to that of S. typhimurium except for a large insertion (20 kDa). According to difference maps and our fitting, this insertion is found on the outer surface of the hook, consistent with our modeling of the hook.bacterial chemotaxis ͉ bacterial motility ͉ electron cryomicroscopy T he bacterial flagellum is the organ of motility for many species of bacteria. About 40 genes are needed to assemble the structure; Ϸ22 of the genes contribute structural proteins found in the completed flagellum. Of these 22 proteins, six appear to be key components of the rotary motor. An additional protein is likely to function as an adaptor connecting the motor to the axial component (1). Nine more proteins make up the axial component consisting of a rod (drive shaft), hook (universal joint), junction, filament (propeller), and cap. Two of the remaining proteins make up rings, which serve as a bushing that allows passage of the drive shaft through the cell wall and outer membrane. The rest of the proteins are associated with the flagellar-specific protein export.The rotary motor, powered by the proton-motive gradient across the cell membrane, turns the filament, which converts torque into thrust. The helical hook of the bacterial flagellum acts as a universal joint, allowing the motor to drive the filament off-axis. The hook connects the rod to the hook-filament junction, which in turn is connected to the filament. The hook, which is assembled before the more distal segments of the axial component, plays a role in the assembly of the filament. The flagellar filament elongates by subunit addition at its distal tip (2, 3). Subunits exported by the cell are thought to diffuse along a channel in the hook (and also the rod within the basal body and partially assembled filament). Three-dimensional reconstructions reveal a 3-nm channel running along the axis of the rod (D.R.T., D. G. Morgan, and D.J.D., unpublished data), hook (4), and filament (5), although higher-resolu...

Bacterial flagellar filament is a macromolecular assembly consisting of a single protein, flagellin. Bacterial swimming is controlled by the conformational transitions of this filament between leftand right-handed supercoils induced by the flagellar motor torque. We present a massive molecular dynamics simulation that was successful in constructing the atomic-level supercoil structures consistent with various experimental data and further in elucidating the detailed underlying molecular mechanisms of the polymorphic supercoiling. We have found that the following three types of interactions are keys to understanding the supercoiling mechanism. ''Permanent'' interactions are always maintained between subunits in the various supercoil structures. ''Sliding'' interactions are formed between variable hydrophilic or hydrophobic residue pairs, allowing intersubunit shear without large change in energy. The formation and breakage of ''switch'' interactions stabilize inter-and intrasubunit interactions, respectively. We conclude that polymorphic supercoiling is due to the energy frustration between them. The transition between supercoils is achieved by a ''transform and relax'' mechanism: the filament structure is geometrically transformed rapidly and then slowly relaxes to energetically metastable states by rearranging interactions.bacterial swimming ͉ molecular dynamics ͉ supercoiling ͉ flagellin ͉ transform and relax mechanism