The catalytic cycle of the nucleotide-binding domain of the ... (Dr. phil. nat.) im Fach 14 nachdem...

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  • The Catalytic Cycle of the Nucleotide-Binding

    Domain of the ABC-Transporter HlyB

    Dissertation zur Erlangung des Doktorgrades

    der Naturwissenschaften

    vorgelegt beim Fachbereich 14 der Johann Wolfgang Goethe - Universität

    in Frankfurt am Main

    von

    Jelena Zaitseva aus Moskau

    Frankfurt (2005) (D 30)

  • Vom Fachbereich………………………………………………………….der

    Johann Wolfgang Goethe - Universität als Dissertation angenommen.

    Dekan : ...

    Gutachter : ...

    Datum der Disputation: ...

  • Fachbereich 14 der

    Johann Wolfgang Goethe - Universität

    Der Bewerber ...

    hat heute das Promotionsverfahren im Fach

    ...

    mit der Gesamtnote ...

    abgeschlossen.

    Die einzelnen Prüfungsleistungen wurden wie folgt bewertet :

    Dissertation: .……………………………….

    Disputation: ………………………………...

    Das Recht zur Führung des Doktortitels wird nicht durch diese

    Bescheinigung, sondern erst durch die Aushändigung der Urkunde erworben.

    Frankfurt am Main, den ...

    (Siegel)

    Der Dekan

    (Unterschrift)

  • (Siegel der Universität)

    Der Fachbereich ...

    der

    Johann Wolfgang Goethe - Universität

    verleiht

    Jelena Zaitseva

    aus Moscau

    den Grad eines Doktors der Naturwissenschaften

    (Dr. phil. nat.)

    im Fach 14

    nachdem er in ordnungsgemäßem Promotionsverfahren

    durch die Arbeit

    The catalytic cycle of the nucleotide-binding domain of

    the ABC-transporter HlyB

    und eine öffentliche Disputation

    seine wissenschaftliche Befähigung erwiesen hat.

    Die Promotionsleistung wurde mit

    (Gesamtnote)

    beurteilt.

    Frankfurt am Main, den (Datum der Disputation)

    (Siegel)

    Der Dekan

    (Unterschrift)

  • The Catalytic Cycle of the Nucleotide-Binding

    Domain of the ABC-Transporter HlyB

    by

    Jelena Zaitseva

    Submitted to the Institute of Biochemistry, the Johann Wolfgang Goethe-

    University of Frankfurt in partial fulfillment of the requirements for the degree

    of Doctor of Philosophy

    Date

  • VI

    Summary

    Nucleotide-binding domains (NBDs), roughly 27 kDa in size, are conservative

    components of the large family of ABC (ATP-binding cassette) transporters, which includes

    importers, exporters, and receptors. NBDs or ABC-ATPases supply energy for the

    translocation of a vast variety of substrates across biological membranes. Despite their

    hydrophilic sequence, many NBDs tend to aggregate and precipitate in solution upon isolation

    from the complete transporter. The conditions stabilizing an extremely labile NBD component

    of the E.coli HlyA transporter, HlyB-NBD, were developed. As a result, the pure highly

    concentrated enzyme was protected from precipitation for months that allowed screening of

    the unlimited crystallization conditions in the presence of different substrates and performance

    of the reproducible functional assays. HlyB-NBD was characterized in regard to its uncoupled

    ATPase activity, oligomeric state, and stability in solution. Comparative analysis of protein

    stability and ATPase activity in various buffers suggested an inverse relationship between the

    two. Kinetic analysis of ATPase activity revealed ATP-induced protein dimerization. Gel-

    filtration experiments with the wild type protein and H662A-mutant of HlyB-NBD provided

    further evidence of protein dimerization in the presence of ATP. The crystal structures in post-

    and pre-hydrolysis nucleotide-bound states of HlyB-NBD were determined at 1.6Å and 2.5Å

    resolution, respectively. While the hydrolytically deficient H662A mutant of HlyB-NBD was

    crystallized as a stable dimer in the presence of ATP or ATP-Mg2+, with two nucleotide

    molecules sandwiched between the two monomers, the same protein was shown to be a

    monomer in the ADP-loaded state. The wild type protein failed to develop crystals with bound

    ATP, yet formed ADP-bound crystals identical to those of the H662A-mutant. The X-ray

    structures of HlyB-NBD in various states of the hydrolytic cycle and the functional studies of

    the enzyme have provided an opportunity to characterize enzyme-substrate complexes and

    protein-protein interactions between the NBD subunits in great detail. Comparison of the

    nucleotide-free, the ADP-, and the ATP-loaded states revealed oligomeric and conformational

    changes of the protein upon substrate binding and resulted in a molecular picture of the

    catalytic cycle. The correlated results of the structural and functional investigations of HlyB-

    NBD are discussed with relation to the mechanism of action of ABC transporters.

  • VII

    Acknowledgements

    The completion of this dissertation would not have been possible without help and

    support of numerous people. First of all, I would like to thank my thesis adviser Robert

    Tampé. His generous invitation to visit Germany almost 5 years ago gave me a unique

    opportunity to see all the people in the lab and have informal conversations with them, to

    present my previous work and have a genuine scientific discussion with my future colleagues.

    I am also thankful to Robert Tampé for his support, his advice, and his guidance throughout

    the course of my stay at Frankfurt University.

    I owe an enormous debt of gratitude to my co-adviser Lutz Schmitt, whose integral

    view on research, overly enthusiasm, stimulating suggestions, and encouragement helped me

    all the time during experimental work and writing of this thesis. I would also like to

    acknowledge Lutz Schmitt for his immense willingness to share his knowledge, skills, time,

    and his expertise.

    I would like to express my sincere gratitude to Barry Holland for his seminal work on

    HlyB, who provided me with invaluable guidance and vital feedback on the project.

    My special thanks go to Lutz Schmitt, Nils Hanekop, Alexander Wiedenmann, Robert

    Ernst, Robin Klemm, and Carsten Horn for their tremendous help with my adjustment to a

    new place, a new life style, and a new working environment. I am very grateful to Lutz and

    Nils, who always had time to help me with all the problems I encountered in my everyday life

    and science. I would also like to extend my gratitude to Renate Guntrum, Alexander

    Wiedenmann, Robert Ernst, Robin Klemm, and Stefan Jenewein for truly enjoyable

    atmosphere they fostered both in and out of the lab and for being my very good friends and

    the best colleagues I would ask for. I simply cannot put into words my appreciation of all of

    you, who made me feel truly home in a foreign country.

    I would like to thank Christine Le Gal, Susanne Heintke, Martynas Gavutis, and

    Rupert Abele for their indispensable assistance with the thesis submission. I am also grateful

    to all other members of the group: Hans Bäumert, Gerhard Spatz-Kümbel, Eckhard Linker,

    Chris van der Does, Eva Janas, Christoph Kyritsis, Ramunas Valiokas, Stanislav Gorbulev,

    Silke Hutschenreiter, Min Chen, Chiara Presenti, Christian Kolbe, Karin Busch, Dirk

    Schaible, Claudia Detje, Joachim Koch, Jennifer Strunk, Matthias Hofacker, Meike Herget,

    Simone Gompf, Stephanie Becker, Peter Lamken, Suman Lata, Eva Jaks, Annett Reichel, Pia

    Müller, Gudrun Illig, Silke Beismann-Driemeyer. I want to thank you for many valuable and

  • VIII

    stimulating discussions, for your support, and for being my colleagues I have had the pleasure

    of interacting with during my time at Frankfurt University.

    I would like to acknowledge the staff of the BW6 beamline (DESY, Hamburg) and

    especially Gleb Bourenkov for the support during data acquisition and the crucial suggestions.

    I am also grateful to Uli Ermler and Harmut Michel for the in-house facilities.

    I am enormously grateful to my best friends for their support during all these years.

    Larisa Avramova, thanks for your amazing patience to listen and give the never-failing

    answers to all my questions, thanks for being you, who always made me feel good just being

    around you.

    Dmitri Nizovtsev, not only you motivated me to achieve my best, you inspired me by

    your encouragement, insight, enthusiasm, and sharing you inner philosophy. Thanks for your

    tremendous help with writing the thesis, advices, great conversations, and the laughs. I am

    also deeply thankful for your continuous support throughout the years.

    Irina Kolesnikova, thank you for your readiness to help any minute in any situation,

    for being the most caring person in the world, for keeping in touch, and for all the good times

    throughout my life.

    Elisabeth Gerle, I am grateful to you for being my best friend for almost as long as I

    remember myself and for being you, openhearted and trusting.

    Last but certainly not least I would like to say big “THANK YOU” to my family for

    their love and generosity. I am thankful to my sons, Georg and Daniel, for their smiles, for

    their trust, and for