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Structure-Function Analysis of the Catalytic Domain of the Histidine Kinase Chea

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Structure-Function Analysis of the Catalytic Domain of the Histidine Kinase Chea Loyola University Chicago Loyola eCommons Dissertations Theses and Dissertations 1997 Structure-Function Analysis of the Catalytic Domain of the Histidine Kinase Chea Dolph David Ellefson Loyola University Chicago Follow this and additional works at: https://ecommons.luc.edu/luc_diss Part of the Microbiology Commons Recommended Citation Ellefson, Dolph David, "Structure-Function Analysis of the Catalytic Domain of the Histidine Kinase Chea" (1997). Dissertations. 3425. https://ecommons.luc.edu/luc_diss/3425 This Dissertation is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Dissertations by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. Copyright © 1997 Dolph David Ellefson LOYOLA UNIVERSITY MEDICAL CENTER LIBRARY LOYOLA UNIVERSITY OF CHICAGO STRUCTURE-FUNCTION ANALYSIS OF THE CATALYTIC DOMAIN OF THE HISTIDINE KINASE CHEA A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL IN CANDIDACY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MICROBIOLOGY AND IMMUNOLOGY BY DOLPH DAVID ELLEFSON CHICAGO, ILLINOIS MAY, 1997 Copyright by Dolph David Ellefson, 1997 All Rights Reserved ii ACKNOWLEDGEMENTS I would like to thank my director, Dr. Alan J. Wolfe, for his support, advice, and encouragment during the many years in his laboratory. In his laboratory, I was given a rare opportunity to explore a new arena of science and interact with a field of gifted researchers who I would not known otherwise. I would also like to thank the members of my committee, Ors. Charles Lange, Hans-Martin Jack, Tom Gallagher, and Rick Stewart for their valuable advice and criticism. I would also like to thank Dr. Katharine Knight for her support and her example of excellence in science. I would like to thank Dr. Herb Mathews for his valuable advice and friendship while at Loyola. Finally, I would like to thank my family. My sisters JoAnn, Laurie, and Kara. My brothers Greg, Tim, Jim, and Sean. My father Woody, and my mother Rose. I did not walk alone through all of this. Without their love and constant support, this would not have been possible. All I have done and all I have yet to do will be as much theirs as mine. Thank you all so much for letting me do this selfish thing. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS .iii LIST OF ILLUSTRATIONS • • • . • . • . • • • • • • • • • • . • • . • • . • • . • • • • • • ix LIST OF TABLES ........................................... X Chapter 1. INTRODUCTION........................................ 1 2. LITERATURE REVIEW • • • • • • • • • • • • • • • • • • • • • . • • • • • • • • • • • • . 4 Sense and Response . 4 Chemotaxis and the Flagellum in Eschericia coli . 5 The Flagellar Complex. • • • • • • • • • • • . • • • • • • • • • • • • • • • • • • • 8 The basal body . 1 0 MotA and MotB . 11 The hook . 11 The filament . 12 The Chemotaxis Pathway. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 1 2 The Excitatory Response. • • • • • . • • • • • • • • • • • • • • • • • . • . 1 3 iv The chemoreceptors . 1 3 CheW . 15 Che Y . 15 CheZ . 16 The Adaptation Response . • . • . • . • . 1 6 CheR . 18 CheB . 1 9 Functional Organization of CheA • . • • . • . • . 20 Histidine Kinases . 24 3. MATERIALS AND METHODS .......................... 28 Chemicals and Reagents • • • . • • . • • • . • . • . • . 28 Bacterial Strains and Alleles • • . • • • • • • • . • . • . • . • • • 29 Media and Growth Conditions........................ 33 Swarm Assays . 33 Plasmid Construction . • • • . • • . • . • • • • • • • . • • . • . • . • 34 Competence Procedure. • . • • • • . • • . • . • . • . 37 Site-Directed Mutagenesis . • . • . • • . • . • . 37 Homologous Recombination • . • . • . 40 Purification of His-tagged proteins • . • . • . • . 41 V In Vitro Phosphorylation of Proteins, SOS-PAGE, Autoradiography and lmmunoblot Analysis . • • • • • . • • 43 Sequencing . 43 Primer End-Labeling . 44 Genomic DNA Preparation • • • • • • • • • • • • • • • • • • • • • • • • • • • 45 Western Analysis . 46 Generation of CheAL-Specific and CheA antisera • • • • • • 4 7 ATP Binding Assay . 49 4. RESULTS ........................................... 51 In Vivo Analysis of CheA Putative catalytic domain Mutants . 51 Rationale for design of catalytic mutants • • • • • • • • • • • 51 Mutant Alleles Defective in the Putative Catalytic Domain do not Support Chemotaxisln Vivo • . 53 The Inability to Perform Chemotaxis is not due to a Metabolic Defect . 55 CheAs, but not CheAL48HQ Restores of Chemotactic Ability to Kinase-Deficient CheAL Mutants • • • • • • • • • • • • 58 Dominance of Various CheA Catalytic Mutants • • • . 61 In Vitro Autophosphorylation Analysis of CheA Variants Defective in G 1, F or G2. • • • • . • • • • • • • • • • • • • • • • • • • • . 64 vi ATP Binding . 68 The Non-Phosphorylatable Variants CheAs or CheAL48HQ can Mediate Transphosphorylation of Kinase-Deficient CheAL Mutants In Vitro............ 69 Structure Averaging Algorithms • • • • • . • . • • • • • • • . • • • • • • • • 73 CheA237-519 . 77 5. DISCUSSION AND SUMMARY •••••••••••••••••••••••••• 79 Mutagenesis strategy: The CheA catalytic domain • • • • • • • 79 Chemotaxis Requires Intact G1, F, and G2 Subdomains • • 81 Autophosphorylation Requires Intact G1, F, and G2 Subdomains . 82 G1 Residue G422 Plays an Intimate Role in Binding of ATP: G2 Residues G470, G472, and G474 do not • • • • • • •• 83 G2 Residues G470, G472, and/or G474 Play a Role in either the Docking of P1 and/or the Subsequent Phosphotransfer; G 1 Residue G422 and F Residues F455 and F459 do not • • • • • • • • • • • • • • • • • • • • • • • • . • • • • • • • 85 Clustering of Ternary Complexes Requires an Intact G2 Subdomain, but not a G1 Subdomain • • • • • • • • • • • • • • • • • • 86 G 1 and G2 Function in cis . • • . • . 88 Secondary Structure Prediction and Model for the Structure of the Catalytic Domain of the Histidine Kinase CheA..................... •• • . • 90 vii Significance of other Histidine Kinases that Lack Some of the Conserved Regions • • • • • • • • • • . • • • • . • • • • 92 WORKS CITED . 95 VITA ........................................................ 113 viii LIST OF ILLUSTRATIONS Figure Page 1. Th e " run " an d ''t um bl e " re s p o n se ••.•...•.......•........•...••. 7 2. The flagellar complex . ...................................... 9 3. Circuitry of the chemotactic signal transduction pathway .••••...• 14 4. Transfer of phosphate (P*) from the sensor CheA to the response regulator Che Y. 17 5. The domain organization of the two forms of CheA. • • • • • • • • • • . 22 6. Amino acid alignment of the N, G1, F, and G2 region of CheA homo logs. 25 7. Comparison of the N, G1, F, and G2 consensus sequences between CheA and orthodox TR domains. • • . • . • • . • . • . 26 8. Functional organization of CheA and schematic of cheA alleles used in this study. 32 9. Photographs of representative swarms. • • • . • . • • . • • . • . • . • • • 54 10. Photographs of representative swarm patterns. • • • • • • • • • • • • • • • . 56 11. Growthcurv-es ............................................. 57 12. Photographas of representative swarm patterns demonstrating that CheAs restores chemotaxis to cells that express the kinase- deficient mutants CheAL G 1 or CheAL G2 but not CheAL G 1/G2. • • 60 ix 13. Photograph of swarm plates demonstrating that allele cheAG2 confers a dominant-negative phenotype in a wild-type background ....................•..................•........ 62 14. Displacement of the outermost serine band plotted as a function of time demonstrating the effect of overexpressing plasmid-borne CheA variants transformed into wild-type cells •••••••••••••••••• 63 15. Purified variant CheA proteins •••••••••••••••••••••••••••••••• 66 16. In vitro autophosphorylation of wild-type and mutant CheA proteins . .................................................. 67 17. In vitro assays demonstrating that the non-phosphorylatable proteins CheAs and CheAL 48HQ can mediate transphosphorylation of kinase deficient mutants of CheAL •••••• 71 18. Phosphorylation of CheAL variants ••••••••••••••••••••••••••• 74 19. lmmunoblot analysis of using antisera specific for CheAL and CheA8 or CheAL alone . .................................... 75 20. Computer algorithm analysis of secondary structure of the transmitter domain of CheA ••••••••••••••••••••••••••••••••• 76 21. Model for the structure of the G1-G2 region of the transmitter domain of CheA . .......................................... 93 X LIST OF TABLES Table Page 1. Ligands of the four known methyl-accepting chemotaxis proteins. 16 2. Bacterial strains used in this study. • • . • • • • . • • • . • • • • • • • • 30 3. Mutagenesis and sequencing primers........................ 31 4. Plasmids used in this study. • • • • • • . • • • . • • . • • . • • . • • • 36 5. TNP-ADP binding affinities of CheAL proteins.................. 70 xi INTRODUCTION This dissertation project sought to determine the relationship between function and structure of the transmitter domain of the chemotaxis-associated histidine autokinase CheA of Eschericia coli. For some time, the transmitter domain of CheA has been proposed as the site of ATP binding and hydrolysis on the basis of homology to eukaryotic serine/threonine kinases. Here, I have tested this hypothesis, establishing that the transmitter region of CheA does act as the catalytic site. First,
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