biophysical dissection of abc transporter mechanism

Membrane Protein Roadmap 03/26/09 Slide #1 of 27

Biophysical dissection of ABC Transporter mechanism

John RamosPaul “The Man” SmithNathan KarpowichBo ChenOksana Martsinkovich

Funding: NIH, MOD, CFF

Linda Millen

Jonathan Moody

Phillip ThomasUT Southwestern Physiology


ATP-Binding Cassette (ABC) Transporters

ABC domains ABC dimer?


F1

F0

www.sanken.osaka-u.ac.jp

ABC domains are “F1-like” ATPases.

Walker A B


The “A-Protein” paradigmfor (F1-like) mechanical ATPases

• ATP binds at a domain-domain interface.

• The flanking domains act as an ATP-dependent mechanical clamp.

• ATP encapsulation in the interfacial active site drives the mechanochemical “power-stroke” which is closure of the clamp formed by the flanking domains.

• Non-hydrolyzable analogues typically have much lower binding affinity than ATP … and therefore often fail to drive the powerstroke of ATPase motors.

• Same principles apply to at least some non-F1-like mechanical ATPases (definitely GroEL, DnaK/Hsp70, perhaps even myosin).

Mg++

++

+++

--

• Therefore, ATP binding (NOT HYDROLYSIS) drives the mechanochemical powerstroke.Walker A B


ATP-Binding Cassette (ABC) Transporters

ABC domains ABC dimer?


But the ATPase active site was not located at an interdomain interface in 5 different ABC crystal structures…

No consistent pattern of oligomerization of ABC domains!

?Completely solvent-exposed ATPase active site --

no where near an interdomain interface!


Concluded that the problem was the inability to observe the inherently transient complex with ATP

E171

E171Q

(combined with the fact that AMP-PNP and ATP--S are lousy analogs)


Using enzymological subterfuge to block ATP hydrolysis yields hyper-stable ABC dimerization!

MJ07962•ATP2: Rfree= 25.1% @ 1.9 Å

E171

E171Q


The -helical subdomain rotates away from the core in the absence of the -phosphate of ATP

• Up to 20˚ rotation of -helical subdomain observed in some non-ATP-form ABC domain structures.


ABC motor domain mechanism is fairly well understood … but how do the associated TM domains drive transport?

MJ07962•ATP2: Rfree= 25.1% @ 1.9 Å

E171

E171Q


Divergent transmembrane domain structures within the ABC Transporter superfamily


FRET … to vitamin B12!

B12 ABSORBANCE

- - - 488 Alexa Fluor Excitation___ 488 Alexa Fluor Emission

- - - 546 Alexa Fluor Excitation___ 546 Alexa Fluor Emission

Wavelength (nm)

BtuCD-Ftransports

vitamin B12(structures

from Locher, Rees, et al.)


Purification of BtuCD & Alexa-Fluor546-labeled BtuF*

BtuF+CysBtuCD

E142QCD

29kD

Fluorescecnescan

Coomasiestain

A C-terminal cys engineered into BtuF


FRET provides a ruler measuring B12 movement relative to a fixed point in the transporter


Anisotropy can monitor molecular associationalthough it also (weakly) influenced by quenching/lifetime effects

Perrin Equation:

1/r = (1/r0)(1 + (/rot) = (1/r) (1 + (RT•/V) )


Avoid topological problems by using bicelles

QuickTime™ and a decompressor

are needed to see this picture.

Monitoring Bicelle formation usingLight Scattering (Ex.:297-Em.:315nm)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 200

5.0×10 4

1.0×10 5

1.5×10 5bicellerange~1.0-2.5

SolublizedRegion

Long to Short Chain Lipid Mass Ratio(diC6PC/3Polar-1PC)

(0Intensity

o)Detector


Model for the conformational reaction cycle of BtuCD(partially, but not fully, validated)


FRET / anisotropy provide rich information concerning transport reaction mechanism


The E-to-Q active-site mutation (E142Q) in the active traps ATP in BtuD (just like in the isolated MJ0796 ABC domain)


BtuF binds to BtuCD with higher affinity whenATP is locked in the active site


The E142Q mutation in BtuD traps B12 in BtuCD just like the wild-type transporter -- but does not release it


A non-BtuCD-interacting mutant variant of BtuF (E50R/E180R) can monitor free B12 concentration


Correspondence of crystal to functional states is obscure -- except for E-to-Q MalEFG-K


Biophysical dissection of ABC Transporter mechanism

John RamosPaul “The Man” SmithNathan KarpowichBo ChenOksana Martsinkovich

Funding: NIH, MOD, CFF

Linda Millen

Jonathan Moody

Phillip ThomasUT Southwestern Physiology


Update on E. coli IMP overexpression physiology project

• Expressed 5 IMP’s (4 ABC Transporters, 1 MFS) in MG1655 via pQE60/pRep4 plasmids; compare 2 soluble proteins (enolase & the cytoplasmic domain of one of the ABC Transporters).

• Characterize cell growth rates, morphology +/- membrane stains, transcriptome (via microarray), expression of selected reporter genes (for factors), and protein expression level / physical state.

Funding: NIH R21!

• Expressing cells suffer from “gigantism” -- when expressing either soluble or membrane proteins.

• IMP expressing cells are growth-inhibited -- & toxicity level correlates with amount of detergent solulizable IMP produced.

• Evidence of intracellular lipid-rich inclusions in overexpressing cells, whether or IMP is recoverable.

• No activation of s, E, or Cpx systems (or s32).• ~200 genes reduced in expression -- most shared by soluble proteins &

IMPs, many annotated to be related to acid stress response.• ~50 genes increased in expression -- most specific to IMPs, 45 part of

flagellar biosynthesis / chemotaxis regulon.• ~5 overexpressed genes may be specific for IMP overexpression.

Observations (conclusions?):

biophysical dissection of abc transporter mechanism

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