Investigations on the nucleotide binding domain of KdpB by NMR spectroscopy and solution structure of an {α4β7 [alpha-4-beta-7] integrin antagonist [Elektronische Ressource] / Melina Haupt

Description

Institutfur¨ OrganischeChemieundBiochemiederTechnischenUniversitat¨ Munchen¨InvestigationsontheNucleotideBindingDomainofKdpBbyNMRSpectroscopyandSolutionStructureofanα4β7IntegrinAntagonistMelinaHauptVollstandiger¨ AbdruckdervonderFakultat¨ fur¨ ChemiederTechnischenUni versitat¨ Munchen¨ zurErlangungdesakademischenGradeseinesDoktorsderNaturwissenschaftengenehmigtenDissertation.Vorsitzender: Univ. Prof.Dr. SteffenJ.GlaserPrufer¨ derDissertation:1. Univ. Prof.Dr.HorstKessler2. Univ. Prof.Dr.SevilWeinkauf3. Univ. Prof.Dr.KarlheinzAltendorf,Universitat¨ Osnabruck¨DieDissertationwurdeam18.08.2004beiderTechnischenUniversitat¨ Munchen¨eingereichtunddurchdieFakultat¨ fur¨ Chemieam17.09.2004angenommen.meinenElternDie vorliegende Arbeit wurde am Institut fur¨ Organische Chemie undBiochemiederTechnischenUniversitat¨ Munchen¨ inderZeitvonNovember2000bisAugust2004unterderLeitungvonProf.Dr.H.Kesslerangefertigt.AcknowledgementsFirstandforemost,IwishtoexpressmysincereappreciationtoProf.Dr.HorstKessler.Ideeplyappreciatehiscontinuoussupportoverthewholeperiodwithallitsupsanddowns. Hisencouragementtokeepongoingandreinforcestrengthwhenthefirstdoublelabelledproteinsamplewentdownthedrain. Fortheexcellent working conditions with first rate spectrometers. For giving methe opportunity to present my work at various international conferencesandmeetings. Andfinallyforsomewonderfulhikingtourswiththegroup,ourAK Fahrten ,intheItalianAlps.

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2004
• Nombre de lectures: 154
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Institutf¨urOrganischeChemieundBiochemie derTechnischenUniversit ¨ atM ¨ unchen

Investigations on the Nucleotide Binding Domain of KdpB by NMR Spectroscopy

and

Solution Structure of anα4β7 Integrin Antagonist

Melina Haupt

Vollsta¨ ndiger Abdruck der von der Fakulta¨ t fu¨ r Chemie der Technischen Uni- versit ¨ atM ¨ nchenzurErlangungdesakademischenGradeseines u

Doktors der Naturwissenschaften

genehmigten Dissertation.

Vorsitzender: Pru¨ fer der Dissertation:

Univ.-Prof. Dr. Steffen J. Glaser

1. Univ.-Prof. Dr. Horst Kessler 2. Univ.-Prof. Dr. Sevil Weinkauf 3. Univ.-Prof. Dr. Karlheinz Altendorf, Universita¨ t Osnabru¨ ck

DieDissertationwurdeam18.08.2004beiderTechnischenUniversita¨tMu¨nchen eingereicht und durch die Fakulta¨  t  fu¨  r Chemie am 17.09.2004 angenommen.

meinen Eltern

DievorliegendeArbeitwurdeamInstitutf¨urOrganischeChemieund BiochemiederTechnischenUniversit ¨ atM ¨ uncheninderZeitvonNovember 2000 bis August 2004 unter der Leitung von Prof. Dr. H. Kessler angefertigt.

Acknowledgements

First and foremost, I wish to express my sincere appreciation to

Prof. Dr. Horst Kessler.

I deeply appreciate his continuous support over the whole period with all its ups and downs. His encouragement to keep on going and reinforce strength when the first double labelled protein sample went down the drain. For the excellent working conditions with first-rate spectrometers. For giving me the opportunity to present my work at various international conferences and meetings. And finally for some wonderful hiking tours with the group, ourAK-Fahrten, in the Italian Alps.

I would also like to express my gratitude toProf. Dr. Karlheinz Altendorffrom theUniversityofOsnabr¨uckforthepossibilitytogaininsightsintothefasci- nating Kdp system and for his support and vivid interest in our cooperation.

This is where I also want to thankDr. Marc BramkampandBrigitte Herkenhoff- Hesselmann-itewrohpnimginhsfrrukcofbr¨uOsnatyofersivinUehtmorf tein samples in ridiculously high concentrations and purities for the NMR investigations and for their introduction into biochemistry during my short stay at the Microbiology Department. And Marc in special for many exten- sive and informative discussions about the Kdp system.

I am very thankful toDr. Murray Colesfor the wonderful cooperation in the completion of the KdpBN project and for proof-reading parts of the manuscript.

I would like to acknowledge the excellent work of our administratorsDr. Rainer HaeßnerandAlex Frenzel, who always took a great effort to keep spec- trometers and computers running.

I would like to thank FrauE. Bruckmaier, FrauM. Machuleand FrauB. Diaw for their professional work in the office and prompt information when it

ii

was time to prolong the contract of employment.

Among my colleagues who all contributed to the special atmosphere of our group I would like to emphasise those from the 32 304 laboratory. Merci `aDr. Vincent Truffaultpour m’introduire dans les premier pas de l’RMN. Danke anMichael John,Markus HellerundJochen Klagesfu¨ r die Diskussionen im Labor und am Spektrometer, die Zusammenarbeit und das angenehme Arbeitsklima.

I would also like to thankGustav(aka Dr. Gerd Gemmecker) for generously distributing measurement time at the spectrometers when time was press- ing and for some amusing interruptions of the daily laboratory business.

Furthermore, I would like to acknowledge those who contributed in the completion of the written work: Kai Tetzlafffor the competent aid in setting up the computer and for his per- suasiveness that L A TEX was the right program to choose. Dr. Guy Deutscherfor proof-reading large parts of the manuscript. And not to forget my friends who were not cross that I stayed absent quite often during the final stage of this work.

Finally, I want to thank my family to whom this work is dedicated and who have a considerable part in the completion of this work. I will always be grateful for their support during my studies, their faith and confidence that I would take the right decision and their help and encouragement when science was not going the way it ought to.

and KdpE . . . . . . . . . . . . . . . . . . . . .

The Proposed Reaction Cycle – The E1E2 Model . . .

Regulation of the kdp Operon - The Subunits

2.3.4

The Subunits KdpC and KdpF . . . . . . . . .

2.3.3

The KdpB Subunit . . . . . . . . . . . . . . . .

2.3.2

20

. . . . .

19

KdpD . . . . .

. . . . .

18

. . . . .

16

2.1.2 Ion Pumps . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2.1.1 Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

2.1 Types of Transport . . . . . . . . . . . . . . . . . . . . . . . . . 3

3

1

2.3.1

The KdpA Subunit . . . . . . . . . . . . . . . .

. . . . .

15

. . . . .

14

The Kdp System . . . . . . . . . . . . . . . . . . . . . .

P-type ATPases . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

3.2.1

Assignment Strategies . . . . . . . . . . . . . . . .

. . .

29

3.1

Prerequisites – The Magnetic Phenomenon . . . .

. . .

28

31

Biochemical Context

2

Scope of the Work

3.2.2

3.2.3

NOESY-spectra – Key to 3 Dimensional Structures

. . .

2.3

2.4

Nuclear Magnetic Resonance

2.2

. . . . .

23

2.5

Towards a New Understanding of the Reaction Cycle

. . .

Protein Structure Determination by NMR . . . . . . . . .

3.2

28

27

A Short History . . . . . . . . . . . . . . . . . . . . . . . . . . .

27

3

Contents

1

Abstract and

iii

iv

C ONTENTS

4 Structure Determination of KdpBN 33 4.1 Protein Expression and Labelling . . . . . . . . . . . . . . . . . 33 4.2 Assignment & Secondary Structure Prediction . . . . . . . . . 40 4.3 The Tertiary Structure of KdpBN . . . . . . . . . . . . . . . . . 47

5

6

7

8

Nucleotide Binding Studies on KdpBN 5.1 TNP-nucleotide Displacement Experiments . . . . . . . . . . . 5.2 NMR Titration – Location of the Binding Pocket . . . . . . . . 5.3 Resonance Assignment of AMP-PNP Bound KdpBN . . . . . . 5.4 Defining the Binding Pocket . . . . . . . . . . . . . . . . . . . . 5.4.1 The Isotope-Filtered 2D-NOESY Experiment . . . . . . 5.4.2 Saturation Transfer Difference Spectroscopy . . . . . . 5.5 The Model of Nucleotide Bound KdpBN . . . . . . . . . . . . .

Methods and Experiments 6.1 Protein Expression and Isotope Labelling . . . . . . . . . . . . 6.2 NMR spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Structure calculations . . . . . . . . . . . . . . . . . . . . . . . . 6.4 Extent of Assignment . . . . . . . . . . . . . . . . . . . . . . . . 6.5 Data Deposition . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6 Artwork . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Discussion 7.1 The 3D Structure of KdpBN and Nucleotide Binding . . . . . . 7.2 The Reaction Cycle of the KdpFABC Complex . . . . . . . . . 7.3 Evolutionary Aspects . . . . . . . . . . . . . . . . . . . . . . . .

55 56 58 66 67 67 69 71

73 73 74 76 77 77 77

79 79 84 92

NMR Structure of anα4β7 Integrin Antagonist 95 8.1 Integrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 8.2α4β 99. . . . . . . . . . . . . . . . . . . .  .7 Integrin Antagonists 8.3 Prerequisites for Drug Design . . . . . . . . . . . . . . . . . . . 100 8.4 Peptidomimetics . . . . . . . . . . . . . . . . . . . . . . . . . . 102

.

.

.

105

.

.

.

.

. . . . .

. . 106

v

C ONTENTS

8.5

8.5.1

Resonance Assignment . . . . . .

. . . . .

. .

. . . . .

8.6.1

NMR Spectroscopy . . . . . . . . . . . . . .

. . . . .

8.6.2

Structure Determination . . . . . . . . . . .

115

.

NMR Investigations on c(-Tic-L-D-T-D-p-)

115

. .

.

.

.

.

8.5.3

. . . . .

108

Temperature Coefficients . . . . . . . . . .

The Solution Structure of c(Tic-L-D-T-D-p)

8.5.4

. .

111

112

Materials and Methods . . . . . . . . . . . . . . . .

. . . . .

. .

115

8.6

. . . . .

. .

. . . . .

8.6.3 Molecular Dynamics . . . . . . . . . . . . .

116

. .

Bibliography

117

8.5.2

ROESY Spectra for Distance Restraints . .

. .

. . . . .

vi

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