Molecular Mechanisms of self-assembly and motion of the bacterial - - PowerPoint PPT Presentation

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Molecular Mechanisms of self-assembly and motion of the bacterial flagellum Mexico-Japan Workshop on Pharmacobiology and Nanobiology Universidad Nacional Autonoma de Mxico (UNAM) Mexico City, Mexico 2009.02.25-26 Keiichi Namba Graduate

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Molecular Mechanisms of self-assembly and motion of the bacterial flagellum

Mexico-Japan Workshop on Pharmacobiology and Nanobiology Universidad Nacional Autonoma de México (UNAM) Mexico City, Mexico 2009.02.25-26

Keiichi Namba Graduate School of Frontier Biosciences Osaka University

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Protonic NanoMachine

Bacteria Swimming

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Electron micrograph of Salmonella

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Flagellar bundle rotation

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Bacterial flagellum (Basal body-Hook-Filament)

By Nao Moriya

basal body Hook-filament junction hook filament

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Flagellar motor driven by the proton motive force

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Filament L ring P ring Hook HAP2 S ring C ring HAP3 HAP1 M ring FliG FliM FliN Inner membrane Outer membrane Peptidoglycan layer FliF MotB MotA Rod Export apparatus

Structures and functions

  • f

the bacterial flagellum

Cytoplasmic chaperones (FlgN,FliJ,FliS,FliT) (FlhA,FlhB,FliH,FliI,FliO,FliP,FliQ,FliR)

Protein export system (ATPase driven) Rotary motor (proton driven) Universal joint Helical propeller Assembly Promoter

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Protonic NanoMachine Lab

SPring-8 at Harima, Hyogo, Japan

Super Photon ring 8 GeV (giga electron volt) Highly brilliant X-ray with variable wavelength by synchrotron radiation

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Crystallography of flagllar proteins (update December 2007)

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Electron CryoMicroscope JEM3000SFF

(300 kV FEG & specimen holder cooled at 4 K)

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Electron cryomicroscopy of flagellar subcomplexes

Miyata & Kato, unpublished

Hook basal body Polyrod basal body

Fujii & Kato, unpublished

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Filament L ring P ring Hook HAP2 S ring C ring HAP3 HAP1 M ring FliG FliM FliN Inner membrane Outer membrane Peptidoglycan layer FliF MotB MotA Rod Export apparatus

Structures and functions

  • f

the bacterial flagellum

Cytoplasmic chaperones (FlgN,FliJ,FliS,FliT) (FlhA,FlhB,FliH,FliI,FliO,FliP,FliQ,FliR)

Protein export system (ATPase driven) Rotary motor (proton driven) Universal joint Helical propeller Assembly Promoter

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Structure of the filament as a helical propeller with switchable helical hand

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Flagellar bundle rotation

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Run and Tumble

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Switching from left-handed to right-handed supercoil by quick reversal of the motor rotation

Macnab & Ornston (1977) J. Mol. Biol.

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A model of polymorphic supercoiling

Based on the proposal by Asakura (1970) and Calladine (1975)

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Image recorded by H. Hotani

Polymorphic transition - Normal to Curly 1 & 2

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Repeat distance along the protofilament

L-type R-type

Yamashita et al. (1998) Nature Struct. Biol.

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Models of 10 supercoils with 2 straight filaments

#: the number of the R-type protofilaments

Left handed Right handed

2 3 4 5 1 6 9 10 11 7 8

# L R

Run Tumble

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Comparison of the two structures at 10 Å resolution

100 Å

Mimori-Kiyosue et al. (1996) P.N.A.S. Subtle changes in the conformation still not visible at this resolution

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Disassembly into subunits for crystallization

D3 D3 D2 D2 D1 D1 D0 D0

D3 D2 D1 D0 D2 D3 D1 D0

○ ○ ×

(1) (494)

SJW1103

F40 F27

(66) (178) (422) (450)

D1 D2 D1 D0 D3 D0

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Crystal of the F41 fragment of flagellin

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Cα backbone trace of F41

Samatey et al. (2001) Nature

D1 D3 D2

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Crystal packing of F41 (a-c plane)

51.9 Å

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A protofilament on EM map and the filament model

Samatey, Imada et al. (2001) Nature

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Crystal packing of F41 (a-c plane)

51.9 Å 52.7 Å

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Simulated extension of the protofilament

51.9 Å 52.7 Å

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Distribution and packing of hydrophobic side chains

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Image of a single filament and its Fourier transform

Mimori et al. (1995) J. Mol. Biol.

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High-resolution cryoEM map of the filament

(Resolution: meridional, 4 Å; equatorial, 5 Å)

Average of 100 filament images (~ 41,000 molecules) with unbending and solvent flattening refinement

Yonekura & Maki-Yonekura et al. (2003) Nature

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Complete atomic model of the flagellar filament

Yonekura & Maki- Yonekura et al. (2003) Nature

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Structure of the hook for universal joint mechanism

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The flagellar hook

Filament L ring P ring Hook HAP2 S ring C ring HAP3 HAP1 M ring FliG FliM FliN Inner membran e Outer membrane Peptidoglycan layer FliF MotB MotA Rod E xport apparatus (FliE,FlgB,FlgC,FlgF,FlgG)

hook Functions as a universal joint, transmitting the motor torque to the helical propeller

  • riented in different directions.

Length: 55 nm (±6 nm) Number of subunits: ~130

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Flagellar hook working as a universal joint

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Run and Tumble

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Samatey, Matsunami, Imada et al. (2004) Nature

Cα Backbone of FlgE-32

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Bacterial flagellum (Basal body-Hook-Filament)

By Nao Moriya

basal body Hook-filament junction hook filament

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Universal joint motion of the flagellar hook

Basal body

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Universal joint motion of the flagellar hook

Basal body

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Simulated extension of a hook protofilament model

Molecular dynamic simulation of hook protofilament extension by Akio Kitao Samatey, Matsunami, Imada et al. (2004) Nature

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Universal joint motion of the flagellar hook

Basal body

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Self-assembly of the flagellum

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The assembly process of the bacterial flagellum

(update 2002)

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Nature of lateral intersubnit interactions in the filament and inner surface of the central channel

* D1-D1: hydrophilic * D0-D0: hydrophobic * Surface of the central channel: hydrophilic (Inner diameter of the channel: 20 Å)

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Three caps for flagellar assembly

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Filament L ring P ring Hook HAP2 S ring C ring HAP3 HAP1 M ring FliG FliM FliN Inner membrane Outer memb rane Peptidoglycan layer FliF MotB MotA Rod Export apparatus

Flagellar protein export at the base and self-assembly at the distal end

Cytoplasmic chaperones (FlgN,FliJ,FliS,FliT) (FlhA,FlhB,FliH,FliI,FliO,FliP,FliQ,FliR)

Assembly Promoter

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3D structure of the HAP2 pentamer cap

2D average of side-view images Pentamer cap as one half of the HAP2 decamer

Maki-Yonekura & Yonekura et al. (2003) P.N.A.S.

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Rotary mechanism of the flagellar cap promoting the self-assembly of flagellin

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Single particle cryoEM image analysis of the basal body

Miyata & Kato unpublished

100 Å

C ring M ring S ring P ring L ring

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Structural information of important part still largely missing

Where? Where? Where?

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Lab members

April 5, 2007 at EXPO Park

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Protonic NanoMachine Project, ERATO, JST (1997-2002) Dynamic NanoMachine Project, ICORP, JST (2002-2007)