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