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Structural and Biochemical Studies of Σ54 Transcriptional Activation in Aquifex Aeolicus

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Structural and biochemical studies of σ54 transcriptional activation in Aquifex aeolicus by Natasha Keith Vidangos A dissertation in partial satisfaction of the degree of Doctor of Philosophy in Chemistry in the Graduate Division of the University of California, Berkeley Committee in charge: Professor David Wemmer, Chair Professor Jennifer Doudna Professor Sydney Kustu Fall 2010 Abstract Structural and biochemical studies of σ54 transcriptional activation in Aquifex aeolicus by Natasha Keith Vidangos Doctor of Philosophy in Chemistry University of California, Berkeley Professor David E. Wemmer, Chair This thesis addresses a diversity of questions regarding the structural details of σ54 transcriptional activation, and the function of σ54 activation in the hyperthermophile Aquifex aeolicus. In order to place each topic in its appropriate context, a general introduction is provided in the first chapter, and supplemented with additional, more detailed introductions in each subsequent chapter. The second chapter reflects the central project of this thesis, the determination of the structure of the DNA-binding domain of an NtrC-like σ54 transcriptional activator protein in Aquifex aeolicus, in complex with its high-affinity DNA binding site. Although this project was attempted by NMR, its structure was ultimately solved by X-ray crystallography. This structure, which shows slight DNA-bending, is compared to the recent structure of the homologous Fis protein in complex with DNA. In the third chapter, I describe a cross-comparison of DNA-binding domains from Aquifex aeolicus, including new structures of the DNA-binding domains of NtrC1 and NtrC2, and comparisons to NtrC4, ZraR and NtrC from Salmonella enterica serovar typhimurium, and Fis from Escherichia coli. These structures of DNA-binding domains from a single family enable a detailed comparison of structural changes that tune protein function. A trend is noted, in which larger dimerization interfaces in the DNA-binding domains appear to correlate with smaller dimerization interfaces in the other domains of the protein. The additional structure of the full-length NtrC1 protein is presented, but the DNA-binding domains were missing from the density, providing evidence for the flexibility of the linker between the central and DNA-binding domains. Together with structural information about the CD linker regions at the N-termini of the DNA-binding domains, I conclude that the CD linker regions are generally unstructured in the inactive state, functioning as tethers to bring the RC domains of NtrC into appropriate local concentrations for activity. In the fourth chapter, I depart from σ54 transcriptional activators, and discuss NMR studies on the σ54 factor. The Darst lab at Rockefeller University has produced a crystal structure of the full-length intact σ54 factor in complex with DNA. However, poor data quality prevents regions of the molecule from being traced unambiguously through the density. By applying new methods in NMR including TROSY spectroscopy and specific isotopic labeling, I attempted to resolve small regions of structure in this ambiguous region. However, these studies were complicated by protein aggregation. The fifth and final chapter takes a step back from structural perspective and compiles and discusses our current understanding of σ54 regulatory networks in Aquifex aeolicus. This analysis of genes with bioinformatics and biochemical techniques led me 1 to discover that NtrC3, an unstudied σ54 transcriptional activator, binds upstream of the dhsU gene, which is responsible for sulfur metabolism. NtrC3 also appears to be associated with a heme-binding, soluble histidine kinase, HksP4. In order to explore this two-component regulatory system further, the HksP4 protein was prepared and its gas- binding properties were studied in collaboration with Dr. Brian Smith of the Marletta lab. We find that the heme binds O2, NO, and CO gases in the reduced ferrous iron state, but did not observe any autophosphorylation activity in any state. I conclude that an additional element, such as an additional activator, or more physiological conditions, would be necessary for activity. Approved: Chair: ____________________________________________ Date: ____________ 2 To my grandfather, Serge Zaroodny, the Russian physicist I never met, but for whom a doctorate degree meant everything in the world. i Acknowledgements As with any thesis, there are so many people to thank. I thank my brothers for sharing in the stories, and my parents for their love and encouragement in the sciences for all these years, and their support and insistence that I should “hang on and finish”. I thank my husband, Ivan, for supporting me with good heart and humor through the six long years of a cross-costal long distance relationship, and with his insistence that I could “quit at any moment” and everything would still be all right. I thank my advisor David Wemmer, whose hands-off style, infinite patience, scientific rigor, and generous flexibility were deeply influential factors in my learning and enjoyment of my degree. Perhaps it is informative to say that in appreciation for Dave’s skill as a teacher and mentor, our lab nominated Dave for a graduate student mentoring award not once, but twice. I want to thank my old labmates for making the experience so fulfilling and the working environment harmonious. I thank Aaron Philips for his good humor and spirit that brought the Wemmer lab together with laughter, music, and West Virginian slang. I thank Jeff Pelton for hours of help with every aspect NMR. I thank Joe Batchelor for years of advice and shortcuts, and for the myriad of lessons I learned from him about letting go of old mistakes, learning to dream up new scientific ideas, and having a healthy supply of chocolate on hand for any emergencies. Then there was the series of strong, inspiring women who stepped in and became unasked-for, unlooked-for mentors when I needed them the most. In my first years in graduate school, it was Michaeleen Callahan-Doucleff, my “big-sister” graduate student who taught me everything I know and knew when to send me off into the woods, and when to sit me down with a plan. I took great comfort from my many professional soul- searching conversations and milk teas with my labmate Judy Hwang. And then there was Dr. Sonja Lorenz, who shared her enthusiasm with me in Schumann and Mendelssohn but also gave me unlimited scientific and personal wisdom on a daily basis, as well as proofreading a large part of this thesis. I have an especially deep gratitude to Professor Sydney Kustu, one of the most inspiring people I have known, who discovered me at the pit of a research slump, and taught me how to “stand up” and remember to be passionate about my work. I’m also grateful to Terry Lang for her random acts of kindness, Monica Smith for years of selfless support, and Terry Yen for endless friendship, laughs, and teaching me the mantra, “don’t stress – organize!” Scientifically, many people had a hand in the work presented in this thesis. Thanks goes to Dr. Brian Smith of the Marletta lab for hours of advising on the behavior of heme proteins. Thanks also to Dr. Artem Lyubimov for his humor, rigor, and generosity with his time in working with me on X-ray crystallography – even joining me on my graveyard shifts at the beamline. I thank the Kuriyan, Marletta, and Marqusee labs for untold amounts of shared chemicals, shakers, media tubes, crystallography materials, instrument time, and endless amounts of advice. I thank the two spectacular undergraduates, Jimmy Ton and Ahmed Alkholeidi, who worked for me through thick and thin, and contributed a great deal to the final chapter of this thesis. I also thank the newer members of the lab: Alex, Zhijuan, Aleks, Hagit, and Wenshu, for making lab feel more and more like family. I’d also like to thank the members of the Science, Technology, and Engineering Policy (STEP) group for giving me the courage to explore a new professional dream. ii Table of Contents Chapter 1: Introduction to sigma54-dependent transcriptional regulation 1 1.1 Transcription 1 1.2 The σ70 class of σ factors 1 1.3 The σ54 factor 4 1.4 Structural understanding of σ54 4 1.5 σ54-dependent transcriptional activators 5 1.6 Structure and function of σ54 transcriptional activators 7 1.7 Overview of two-component signal transduction. 8 1.8 Thesis outline 9 Chapter 2: DNA-recognition by a σ54 transcriptional activator from Aquifex aeolicus 10 2.1 Summary 10 2.2 Introduction 10 2.2.1. Structures of σ54-activator DNA-binding domains 10 2.2.2. NtrC and Fis are homologues 10 2.3 Materials and Methods 11 2.3.1 Protein expression 11 2.3.2. Protein Purification 12 2.3.3. NMR of 1DBD and 4DBD complexes 12 2.3.4. Complex preparation for crystallography 13 2.3.5. Crystallography of 4DBD-complex 13 2.3.6. Binding affinity assays 14 2.4 Results 14 2.4.1. NMR of 1DBD and 4DBD complexes 14 2.4.2. Crystal structure of free-4DBD 15 2.4.3. Determination of the DNA-binding site for NtrC4 19 2.4.4. Structure determination of the 4DBD-site_1 complex 19 2.4.5. 4DBD-complex: protein-DNA interactions 19 2.4.6. 4DBD-complex: DNA conformation overview 21 2.4.7. Biochemical analysis of DNA-binding site of NtrC4 22 2.5. Discussion 22 2.5.1. The relevance of lpxC activation to NtrC4 activity 22 2.5.2. Comparison of 4DBD free protein to Fis 22 2.5.3. Comparison of the 4DBD and Fis complex structures: protein interactions 23 2.5.4. Comparison of the 4DBD and Fis complex structures: DNA distortion 26 2.5.5. Conclusions on the mode of NtrC4 DNA-binding/bending 27 iii Chapter 3: NtrC-like C-terminal DNA-binding domains: structure, function, and flexible tethering to the central domain 29 3.1 Summary 29 3.2 Introduction 29 3.2.1 DNA-binding domains of σ54 activators 29 3.2.2 Functions of the DNA-binding domain 29 3.2.3 CD-linker regions 30 3.3 Materials and Methods 32 3.3.1 Constructs 32 3.3.2 Protein Expression and Purification 33 3.3.3 Analytical ultracentrifugation 33 3.3.4 Structural comparisons 33 3.4.
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  • Two Tandemly Arranged Ferredoxin Genes in the Hydrogenobacter Thermophilus Genome: Comparative Characterization of the Recombinant [4Fe–4S] Ferredoxins

    Two Tandemly Arranged Ferredoxin Genes in the Hydrogenobacter Thermophilus Genome: Comparative Characterization of the Recombinant [4Fe–4S] Ferredoxins

    Biosci. Biotechnol. Biochem., 69 (6), 1172–1177, 2005 Two Tandemly Arranged Ferredoxin Genes in the Hydrogenobacter thermophilus Genome: Comparative Characterization of the Recombinant [4Fe–4S] Ferredoxins Takeshi IKEDA,1 Masahiro YAMAMOTO,1 Hiroyuki ARAI,1 y Daijiro OHMORI,2 Masaharu ISHII,1 and Yasuo IGARASHI1; 1Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 2Department of Chemistry, School of Medicine, Juntendo University, Inba, Chiba 270-1695, Japan Received February 14, 2005; Accepted March 17, 2005 A thermophilic, obligately chemolithoautotrophic has been reported that Hydrogenobacter and Aquifex hydrogen-oxidizing bacterium, Hydrogenobacter ther- assimilate carbon dioxide via the reductive tricarboxylic mophilus TK-6, assimilates carbon dioxide via the acid cycle.6,7) Two key enzymes of this cycle, pyruvate: reductive tricarboxylic acid cycle. A gene encoding a ferredoxin oxidoreductase (POR) and 2-oxoglutarate: ferredoxin involved in this cycle as an electron donor ferredoxin oxidoreductase (OGOR), catalyze carbox- (HtFd1) was cloned and sequenced. Interestingly, an- ylation of acetyl-CoA and succinyl-CoA respectively, other ferredoxin gene (encoding HtFd2) was found in which should require strong reducing power.8) Hence tandem with the HtFd1 gene. These two ferredoxin characterization of low-potential electron carriers, such genes overlapped by four bp, and transcriptional analy- as ferredoxins, which can act as electron donors for sis revealed that they are co-transcribed as an operon. these carboxylation reactions, is of particular importance The deduced amino acid sequences of HtFd1 and HtFd2 for a deeper understanding of the cycle. were 42.9% identical and each contained four cysteine In our previous studies, a ferredoxin (designated residues that serve as probable ligands to an iron-sulfur HtFd1) was purified from H.