Structure of the C-terminal domain of FliG, a component of the rotor in the bacterial flagellar motor.

Many motile species of bacteria are propelled by flagella, which are rigid helical filaments turned by rotary motors in the cell membrane. The motors are powered by the transmembrane gradient of protons or sodium ions. Although bacterial flagella contain many proteins, only three-MotA, MotB and FliG-participate closely in torque generation. MotA and MotB are ion-conducting membrane proteins that form the stator of the motor. FliG is a component of the rotor, present in about 25 copies per flagellum. It is composed of an amino-terminal domain that functions in flagellar assembly and a carboxy-terminal domain (FliG-C) that functions specifically in motor rotation. Here we report the crystal structure of FliG-C from the hyperthermophilic eubacterium Thermotoga maritima. Charged residues that are important for function, and which interact with the stator protein MotA, cluster along a prominent ridge on FliG-C. On the basis of the disposition of these residues, we present a hypothesis for the orientation of FliG-C domains in the flagellar motor, and propose a structural model for the part of the rotor that interacts with the stator.     

Direct observation of steps in rotation of the bacterial flagellar motor

The bacterial flagellar motor is a rotary molecular machine that rotates the helical filaments that propel many species of swimming bacteria. The rotor is a set of rings up to 45 nm in diameter in the cytoplasmic membrane; the stator contains about ten torque-generating units anchored to the cell wall at the perimeter of the rotor. The free-energy source for the motor is an inward-directed electrochemical gradient of ions across the cytoplasmic membrane, the protonmotive force or sodium-motive force for H+-driven and Na+-driven motors, respectively. Here we demonstrate a stepping motion of a Na+-driven chimaeric flagellar motor in Escherichia coli at low sodium-motive force and with controlled expression of a small number of torque-generating units. We observe 26 steps per revolution, which is consistent with the periodicity of the ring of FliG protein, the proposed site of torque generation on the rotor. Backwards steps despite the absence of the flagellar switching protein CheY indicate a small change in free energy per step, similar to that of a single ion transit.     

In situ structure of the complete Treponema primitia flagellar motor

The bacterial flagellar motor is an amazing nanomachine: built from approximately 25 different proteins, it uses an electrochemical ion gradient to drive rotation at speeds of up to 300 Hz (refs 1, 2). The flagellar motor consists of a fixed, membrane-embedded, torque-generating stator and a typically bidirectional, spinning rotor that changes direction in response to chemotactic signals. Most structural analyses so far have targeted the purified rotor, and hence little is known about the stator and its interactions. Here we show, using electron cryotomography of whole cells, the in situ structure of the complete flagellar motor from the spirochaete Treponema primitia at 7 nm resolution. Twenty individual motor particles were computationally extracted from the reconstructions, aligned and then averaged. The stator assembly, revealed for the first time, possessed 16-fold symmetry and was connected directly to the rotor, C ring and a novel P-ring-like structure. The unusually large size of the motor suggested mechanisms for increasing torque and supported models wherein critical interactions occur atop the C ring, where our data suggest that both the carboxy-terminal and middle domains of FliG are found.     

Torque-generating units of the flagellar motor of Escherichia coli have a high duty ratio

Rotation of the bacterial flagellar motor is driven by an ensemble of torque-generating units containing the proteins MotA and MotB. Here, by inducing expression of MotA in motA- cells under conditions of low viscous load, we show that the limiting speed of the motor is independent of the number of units: at vanishing load, one unit turns the motor as rapidly as many. This result indicates that each unit may remain attached to the rotor for most of its mechanochemical cycle, that is, that it has a high duty ratio. Thus, torque generators behave more like kinesin, the protein that moves vesicles along microtubules, than myosin, the protein that powers muscle. However, their translation rates, stepping frequencies and power outputs are much higher, being greater than 30 microm s(-1), 12 kHz and 1.5 x 10(5) pN nm s(-1), respectively.     

Adaptation at the output of the chemotaxis signalling pathway

In the bacterial chemotaxis network, receptor clusters process input, and flagellar motors generate output. Receptor and motor complexes are coupled by the diffusible protein CheY-P. Receptor output (the steady-state concentration of CheY-P) varies from cell to cell. However, the motor is ultrasensitive, with a narrow operating range of CheY-P concentrations. How the match between receptor output and motor input might be optimized is unclear. Here we show that the motor can shift its operating range by changing its composition. The number of FliM subunits in the C-ring increases in response to a decrement in the concentration of CheY-P, increasing motor sensitivity. This shift in sensitivity explains the slow partial adaptation observed in mutants that lack the receptor methyltransferase and methylesterase and why motors show signal-dependent FliM turnover. Adaptive remodelling is likely to be a common feature in the operation of many molecular machines.     

Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization

The structural change that generates force and motion in actomyosin motility has been proposed to be tilting of the myosin light chain domain, which serves as a lever arm. Several experimental approaches have provided support for the lever arm hypothesis; however, the extent and timing of tilting motions are not well defined in the motor protein complex of functioning actomyosin. Here we report three-dimensional measurements of the structural dynamics of the light chain domain of brain myosin V using a single-molecule fluorescence polarization technique that determines the orientation of individual protein domains with 20-40-ms time resolution. Single fluorescent calmodulin light chains tilted back and forth between two well-defined angles as the myosin molecule processively translocated along actin. The results provide evidence for lever arm rotation of the calmodulin-binding domain in myosin V, and support a 'hand-over-hand' mechanism for the translocation of double-headed myosin V molecules along actin filaments. The technique is applicable to the study of real-time structural changes in other biological systems.     

Structure of the bacterial flagellar hook and implication for the molecular universal joint mechanism

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.     

Cell surface clustering of Cadherin adhesion complex induced by antibody coated beads

Cadherin receptors mediate cell-cell adhesion, signal transduction and assembly of cytoskeletons. How a single transmembrane molecule Cadherin can be involved in multiple functions through modulating its binding activities with many membrane adhesion molecules and cytoskeletal components is an unanswered question which can be elucidated by clues from bead experiments. Human lung cells expressing N-Cadherin were examined. After co-incubation with anti-N-Cadherin monoclonal antibody coated beads, cell surface clustering of N-Cadherin was induced. Immunofluorescent detection demonstrated that in addition to Cadherin, β-Catenin, α-Catenin, α-Actinin and Actin fluorescence also aggregated respectively at the membrane site of bead attachment. Myosin heavy chain (MHC), another major component of Actin cytoskeleton, did not aggregate at the membrane site of bead attachment. Adhesion unrelated protein Con A and polylysine conjugated beads did not induce the clustering of adhesion molecules. It is indicated that the Cadherin/Catenins/α-Actinin/Actin complex is formed at Cadherin mediated cell adherens junction; occupancy and cell surface clustering of Cadherin is crucial for the formation of Cadherin adhesion protein complexes.     

Polar and lateral flagellar motors of marine Vibrio are driven by different ion-motive forces.

Various species of marine Vibrio produce two distinct types of flagella, each adapted for a different type of motility. A single, sheathed polar flagellum is suited for swimming in liquid medium, and numerous unsheathed lateral flagella, which are produced only under viscous conditions, are suited for swarming over viscous surfaces. Both types of flagella are driven by reversible motors embedded in the cytoplasmic membrane. Here we report that the energy source for the polar flagellar motor of Vibrio parahaemolyticus is the sodium-motive force, whereas the lateral flagellar motors are driven by the proton-motive force. This is evidence that two distinct types of flagella powered by different energy sources are functionally active in one cell.     

Distinct roles of the FliI ATPase and proton motive force in bacterial flagellar protein export

Translocation of many soluble proteins across cell membranes occurs in an ATPase-driven manner. For construction of the bacterial flagellum responsible for motility, most of the components are exported by the flagellar protein export apparatus. The FliI ATPase is required for this export, and its ATPase activity is regulated by FliH; however, it is unclear how the chemical energy derived from ATP hydrolysis is used for the export process. Here we report that flagellar proteins of Salmonella enterica serovar Typhimurium are exported even in the absence of FliI. A fliH fliI double null mutant was weakly motile. Certain mutations in FlhA or FlhB, which form the core of the export gate, substantially improved protein export and motility of the double null mutant. Furthermore, proton motive force was essential for the export process. These results suggest that the FliH-FliI complex facilitates only the initial entry of export substrates into the gate, with the energy of ATP hydrolysis being used to disassemble and release the FliH-FliI complex from the protein about to be exported. The rest of the successive unfolding/translocation process of the substrates is driven by proton motive force.     

Cycling of cell-surface MHC glycoproteins through primaquine-sensitive intracellular compartments

Class II major histocompatibility complex (MHC) glycoproteins associate with peptides derived from material endocytosed by antigen-presenting cells and processed along the endocytotic pathway. No consensus exists as to what extent class II molecules themselves are endocytosed and it is not known whether endocytosed MHC class II molecules can be recycled again to the cell surface--an itinerary which might allow a single cell-surface MHC molecule to associate with different peptides during its lifetime. We now show by using new cleavable labelling reagents that class II and class I MHC on B lymphoblastoid cells are continually endocytosed and recycled to the cell surface. The intracellular pool size is normally kept small by efficient recycling, but in the presence of primaquine the rate of recycling is slowed, thereby increasing the size of this pool substantially. On removal of the amine, the intracellular population recycles rapidly to restore the original distribution. These results reveal a cycle that might explain the rapid binding and turnover of some peptide/class II MHC complexes and the exchange of pre-existing for new peptides observed in living cells.     

Abrupt changes in flagellar rotation observed by laser dark-field microscopy

Bacteria such as Escherichia coli and Salmonella typhimurium swim by rotating their flagella, each of which consists of an external helical filament and a rotary motor embedded in the cell surface. The function of the flagellar motor has been examined mainly by tethering the flagellar filament to a glass slide and observing the resultant rotation of the cell body. But under these conditions the motor operates at a very low speed (about 10 r.p.s.) owing to the unnaturally high load conditions inherent in this technique. Lowe et al. analysed the frequency of light scattered from swimming cells to estimate the average rotation speed of flagellar bundles of E. coli as about 270 r.p.s. To analyse motor function in more detail, however, measurement of high-speed rotation of a single flagellum (at low load) with a temporal resolution better than 1 ms is needed. We have now developed a new method--laser dark-field microscopy--which fulfils these requirements. We find that although the average rotation speed of S. typhimurium flagella is rather stable, there are occasional abrupt slowdowns, pauses and reversals (accomplished within 1 ms). These changes were frequently observed in mutants defective in one of the motor components (called the switch complex), suggesting that this component is important not only in switching rotational direction but also in torque generation or regulation.     

Mechanism of colour discrimination by a bacterial sensory rhodopsin

A photosensitive protein resembling the visual pigments of invertebrates enables phototactic archaebacteria to distinguish colour. This protein exists in two spectrally-distinct forms, one of which is a transient photoproduct of the other and each of which undergoes photochemical reactions controlling the cell's swimming behaviour. Activation of a single pigment molecule in the cell is sufficient to signal the flagellar motor. This signal-transduction mechanism makes evident a colour-sensing capability inherent in the retinal/protein chromophore.     

Jeffcott转子-环流耦合系统动力学建模及分析

基于湍流整体流动理论和摄动分析,建立了Jeffcott转子-环流耦合系统的动力学模型,并以大型屏蔽电机泵转子系统为例,采用复模态分析方法分析了转速、转子偏心和定、转子壁面摩擦效应对转子-环流耦合系统动力学特性的影响.结果表明：环流将导致转子的固有频率大幅下降,并产生模态分叉和跳变;转子偏心将导致转子失稳并使其失稳转速下降;定、转子壁面的摩擦效应能够显著提高转子的失稳转速.
     

Energy source of flagellar type III secretion

Bacterial flagella contain a specialized secretion apparatus that functions to deliver the protein subunits that form the filament and other structures to outside the membrane. This apparatus is related to the injectisome used by many gram-negative pathogens and symbionts to transfer effector proteins into host cells; in both systems this export mechanism is termed 'type III' secretion. The flagellar secretion apparatus comprises a membrane-embedded complex of about five proteins, and soluble factors, which include export-dedicated chaperones and an ATPase, FliI, that was thought to provide the energy for export. Here we show that flagellar secretion in Salmonella enterica requires the proton motive force (PMF) and does not require ATP hydrolysis by FliI. The export of several flagellar export substrates was prevented by treatment with the protonophore CCCP, with no accompanying decrease in cellular ATP levels. Weak swarming motility and rare flagella were observed in a mutant deleted for FliI and for the non-flagellar type-III secretion ATPases InvJ and SsaN. These findings show that the flagellar secretion apparatus functions as a proton-driven protein exporter and that ATP hydrolysis is not essential for type III secretion.     

Structural analysis of the essential self-cleaving type III secretion proteins EscU and SpaS

During infection by Gram-negative pathogenic bacteria, the type III secretion system (T3SS) is assembled to allow for the direct transmission of bacterial virulence effectors into the host cell. The T3SS system is characterized by a series of prominent multi-component rings in the inner and outer bacterial membranes, as well as a translocation pore in the host cell membrane. These are all connected by a series of polymerized tubes that act as the direct conduit for the T3SS proteins to pass through to the host cell. During assembly of the T3SS, as well as the evolutionarily related flagellar apparatus, a post-translational cleavage event within the inner membrane proteins EscU/FlhB is required to promote a secretion-competent state. These proteins have long been proposed to act as a part of a molecular switch, which would regulate the appropriate chronological secretion of the various T3SS apparatus components during assembly and subsequently the transported virulence effectors. Here we show that a surface type II beta-turn in the Escherichia coli protein EscU undergoes auto-cleavage by a mechanism involving cyclization of a strictly conserved asparagine residue. Structural and in vivo analysis of point and deletion mutations illustrates the subtle conformational effects of auto-cleavage in modulating the molecular features of a highly conserved surface region of EscU, a potential point of interaction with other T3SS components at the inner membrane. In addition, this work provides new structural insight into the distinct conformational requirements for a large class of self-cleaving reactions involving asparagine cyclization.     

Structural Changes of Water Molecules Upon the Reduction of Quinones in The Reaction Center from Rhodobactery Sphaeroides

1 Results The photosynthetic bacterial reaction center (RC) is a membrane protein complex.The RC is composed of three protein subunits and redox components such as bacteriochlorophylls, bacteriopheophytins,and quinones.The RC performs the photochemical electron transfer from the bacteriochlorophyll dimer through a series of electron donor and acceptor molecules to a secondary quinone,QB.QB accepts electrons from a primary quinone,QA,in two sequential electron transfer reactions.The second electron trans...     

Crystal structure of the CusBA heavy-metal efflux complex of Escherichia coli

Gram-negative bacteria, such as Escherichia coli, expel toxic chemicals through tripartite efflux pumps that span both the inner and outer membrane. The three parts are an inner membrane, substrate-binding transporter; a membrane fusion protein; and an outer-membrane-anchored channel. The fusion protein connects the transporter to the channel within the periplasmic space. A crystallographic model of this tripartite efflux complex has been unavailable because co-crystallization of the various components of the system has proven to be extremely difficult. We previously described the crystal structures of both the inner membrane transporter CusA and the membrane fusion protein CusB of the CusCBA efflux system of E. coli. Here we report the co-crystal structure of the CusBA efflux complex, showing that the transporter (or pump) CusA, which is present as a trimer, interacts with six CusB protomers and that the periplasmic domain of CusA is involved in these interactions. The six CusB molecules seem to form a continuous channel. The affinity of the CusA and CusB interaction was found to be in the micromolar range. Finally, we have predicted a three-dimensional structure for the trimeric CusC outer membrane channel and developed a model of the tripartite efflux assemblage. This CusC(3)-CusB(6)-CusA(3) model shows a 750-kilodalton efflux complex that spans the entire bacterial cell envelope and exports Cu I and Ag I ions.