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Biophysical dissection of ABC Transporter mechanism

Biophysical dissection of ABC Transporter mechanism. John Ramos Paul “The Man” Smith Nathan Karpowich Bo Chen Oksana Martsinkovich. Linda Millen Jonathan Moody Phillip Thomas UT Southwestern Physiology. Funding: NIH, MOD, CFF. ABC domains. ABC dimer?.

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Biophysical dissection of ABC Transporter mechanism

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  1. Biophysical dissection of ABC Transporter mechanism John Ramos Paul “The Man” Smith Nathan Karpowich Bo Chen Oksana Martsinkovich Linda Millen Jonathan Moody Phillip Thomas UT Southwestern Physiology Funding: NIH, MOD, CFF

  2. ABC domains ABC dimer? ATP-Binding Cassette (ABC) Transporters

  3. F1 F0 www.sanken.osaka-u.ac.jp ABC domains are “F1-like” ATPases. Walker A B

  4. The “A-Protein” paradigm for (F1-like) mechanical ATPases - - Mg++ + + + + + • 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. • Therefore, ATP binding (NOT HYDROLYSIS) drives the mechanochemical powerstroke. Walker A B • 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).

  5. ABC domains ABC dimer? ATP-Binding Cassette (ABC) Transporters

  6. No consistent pattern of oligomerization of ABC domains! But the ATPase active site was not located at an interdomain interface in 5 different ABC crystal structures… Completely solvent-exposed ATPase active site -- no where near an interdomain interface! ?

  7. E171 E171Q Concluded that the problem was the inability to observe the inherently transient complex with ATP (combined with the fact that AMP-PNP and ATP--S are lousy analogs)

  8. E171 E171Q MJ07962•ATP2: Rfree= 25.1% @ 1.9 Å Using enzymological subterfuge to block ATP hydrolysis yields hyper-stable ABC dimerization!

  9. 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.

  10. E171 E171Q MJ07962•ATP2: Rfree= 25.1% @ 1.9 Å ABC motor domain mechanism is fairly well understood … but how do the associated TM domains drive transport?

  11. Divergent transmembrane domain structures within the ABC Transporter superfamily

  12. B12 ABSORBANCE • - - - 488 Alexa Fluor Excitation • ___ 488 Alexa Fluor Emission • - - 546 Alexa Fluor Excitation • ___ 546 Alexa Fluor Emission Wavelength (nm) FRET … to vitamin B12! BtuCD-F transports vitamin B12 (structures from Locher, Rees, et al.)

  13. Purification of BtuCD & Alexa-Fluor546-labeled BtuF* A C-terminal cys engineered into BtuF BtuF+Cys E142QCD BtuCD Fluorescecne scan Coomasie stain 29kD

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

  15. 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) )

  16. Avoid topological problems by using bicelles

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

  18. FRET / anisotropy provide rich information concerning transport reaction mechanism

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

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

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

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

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

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

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

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

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

  28. Biophysical dissection of ABC Transporter mechanism John Ramos Paul “The Man” Smith Nathan Karpowich Bo Chen Oksana Martsinkovich Linda Millen Jonathan Moody Phillip Thomas UT Southwestern Physiology Funding: NIH, MOD, CFF

  29. 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. Observations (conclusions?): • 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. Funding: NIH R21!

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