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Structure-Function and Substrate-Specificity Studies Of Structure-Function and Substrate-Specificity Studies of Escherichia coli YidC Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Balasubramani Hariharan Graduate Program in Biophysics The Ohio State University 2018 Dissertation Committee Professor Ross E. Dalbey, Advisor Professor Natividad Ruiz Professor Charles Bell Professor Karin Musier-Forsyth 1 Copyrighted by Balasubramani Hariharan 2018 2 Abstract This dissertation examines the substrate properties of M13 Procoat-Lep protein that determine the pathway it takes for membrane insertion and describes Electron paramagnetic resonance (EPR) methods that explore the solvent exposure of YidC greasy slide residues, the microenvironment of the hydrophilic groove to probe the role of the strictly positively charged residue, and the distance(s) between two strategic positions within YidC that were not resolved in the structure. Chapter one of this dissertation reviews membrane protein targeting and protein translocation pathways in prokaryotes and eukaryotes. Secretory and membrane proteins are synthesized in the cytoplasm and targeted to their destined membranes in multiple ways. Co-translationally targeting is achieved either by a direct interaction of ribosomes to the translocases or by ribosome nascent chain complexes that are targeted to the Sec translocase by signal recognition particle and the FtsY. Post-translationally targeting to the SecYEG translocase embedded in the membrane is achieved by cytosolic chaperones and SecA. The heterotrimeric SecYEG inserts the majority of the substrates through the membrane. The cytosolic motor ATPase SecA works along with the trimeric Sec YEG complex providing energy for protein translocation. The ancillary proteins SecDF/YajC also associate with SecYEG to improve translocation efficiency. The substrates that are ii independent of SecYEG insert via the YidC membrane insertase, which is the primary focus of this dissertation. YidC functions within the cell, as an independent insertase, to insert membrane proteins or function in concert with the Sec translocon to insert, fold and assemble proteins into the membrane. In addition to the Sec translocase and YidC insertase systems, proteins can be exported by the twin arginine translocation pathway (Tat), in which proteins are folded prior to export. In this chapter, the structural components and mechanism of insertion will be reviewed for each of the export pathways. Chapter two of this dissertation focuses on the insertion components YidC and SecYEG in the inner membrane of E. coli. Both of these insertases are essential and universally conserved, and together insert most proteins into the plasma (cytosolic) membrane in bacteria. The structure of SecYEG revealed an hourglass channel structure with a central pore ring and a lateral gate. Proteins enter the SecYEG complex through the cytoplasmic region and the hydrophilic regions of the substrate are translocated across the channel through the pore ring while the transmembrane segments of membrane proteins exit the channel to integrate into the lipid bilayer through the lateral gate. The structure of YidC revealed it possesses a novel hydrophilic groove exposed to both the cytoplasm and the lipid bilayer. Based on structure-function studies of a single-spanning membrane protein, YidC was proposed to catalyze membrane insertion by recruiting the substrate hydrophilic region into hydrophilic groove via an electrostatic interaction. This mechanism would effectively reduce the membrane crossing distance for translocation. Exactly how YidC would insert more complicated membrane proteins and how it would cooperate with the Sec translocase was not clear based on the structure. Previously, the iii polarity and charge of the periplasmic regions of membrane proteins was proposed to determine the YidC and Sec translocase requirements for insertion. We have further tested this polarity/charge hypothesis using the bacteriophage M13 coat protein, which is synthesized in a precursor form called procoat with two hydrophobic regions. We found that M13 procoat becomes increasingly YidC/Sec dependent by making the periplasmic loop increasingly polar in the absence of charged residues. In addition, we discovered that increasing the loop hydrophilicity beyond a certain threshold blocked translocation even with Sec/YidC. However, translocation can be restored by adding hydrophobic residues to the transmembrane segment to increase the driving force for membrane insertion. We also demonstrated that the length of the Procoat-Lep loop is a determinant for Sec dependence. Based on these results we envision that insertion of proteins is occurring at the interface of SecY and YidC insertase. This hypothesis was corroborated by photo- crosslinking studies where the interactions of the substrate and the translocases can be detected. Our results on the YidC and SecYEG biomachineries highlight the universal principles that guide membrane protein biogenesis in all walks of life. Chapter three of this dissertation focuses on using the biophysical technique electron paramagnetic resonance (EPR) to study YidC. Employing EPR and site-directed spin labeling, we showed that the greasy slide residues 427, 432 and 434 are at the interface of water and lipid. This corroborates our solvent exposed results based on in vivo NEM alkylation studies of YidC in E. coli but is different than the results from molecular dynamics simulations. Additionally, we used continuous wave EPR and power saturation EPR experiments to probe the spatial hydrophilic environment of YidC residues in the iv aqueous groove. The groove possesses a conserved positive charge (366R in E. coli YidC), which was shown to be essential for translocase activity of YidC in Bacillus subtilis and was proposed to recruit the substrate tail into the groove by an electrostatic attraction mechanism, but the exact function of this positive charge is controversial. Chen et al. provided data that the hydrophilic nature of the positively charged residue is more essential than the electrostatic nature and that the positively charged residue is surprisingly dispensable for its translocase activity. Using continuous EPR and site directed spin labeling, we provide data that suggests that the positively charged residue 366 is essential to maintain the hydrophilic nature of the groove when the groove has an apolar residue at the top of the groove at the 517 position. Lastly, the EPR Double electron electron resonance (DEER) technique was used to estimate distance(s) between residues that are not resolved in the crystal structure of YidC. v Dedicated to my Father T.S. Hariharan vi Acknowledgments First, I would like to thank my advisor, Dr. Ross E. Dalbey, for his patience, guidance, encouragement and trust throughout the course of my graduate studies. I would like to thank our collaborator, Professor Andreas Kuhn, for his help and advice on my projects. I would also like to thank all my graduate study committee members Dr. Charles Bell, Dr. Karin Musier Forsyth and Dr. Natividad Ruiz for their valuable advice and guidance. I am also grateful to my former lab members especially Dr. Seth Hennon, Dr. Lu Zhu and Dr. Raunak Soman for their advice and training during the initial course of my lab research. I would like to thank my current lab members Dr. Yuanyuan Chen, Sri Karthika Shanmugam, Haoze He and Margaret Steward for their advice and camaraderie developed during my stay in the lab. I will always be grateful to my friends in Columbus Dr. Shibi Likhite, Dr. Krishna Patel and back home in Chennai Dr. Aloysius Wilfred Raj, Vinod Ravishankar, Anand Kumar and Deepika Govindarajulu, for their trust and encouragement. Lastly, but most importantly, I would like to thank my family, Kamatchi Hariharan, Sridevi Hariharan and Karthika Hariharan who have been my pillars of strength and my other family members for their love and support. I love you all! vii Vita October,1989 ..................................................Born in Chennai, India 2011................................................................B.S. Industrial Biotechnology, Anna University. 2012 - Present ...............................................Graduate Teaching and Research Associate, Department of Biophysics, The Ohio State University Fields of Study: Biophysics viii Table of Contents Abstract…………………………………………………………………………………….i Dedication…………………………………………………………………………...……vi Acknowledgments……………………………………………………………………….vii Vita……………………………………………………………………………………...viii Table of Contents…………………………………………………………………………ix List of Tables……………………………………………………………………………xiii List of Figures …………………………………………………………………………..xiv Chapter 1……...…………………………………………………………………………..1 1.1 Introduction..…………………………………………………………………..1 1.1 Biological membranes………………………………………...…………….....1 1.2 Membranes in Eukaryotic organisms.………………………....…………….....2 1.3 Membranes in Prokaryotic organisms.………………………...……………....6 1.4 Membrane proteins…………………..………………………...……………....6 1.5 Protein targeting……………………………….………………………………8 1.5.1 Co-translational targeting…………………………………………..10 1.5.2 Post-translational targeting……………………………….………...12 1.6 Sec Translocon……….…………….………………………………….……..16 1.6.1 The SecYEG….……………………………………………….……17 1.6.3 Mechanism of Co-translational Pathway……………………….......20 1.6.4 Molecular motor protein SecA…………………………….………..21 ix 1.6.5 Accessary unit SecDF/YajC………………………………..............22 1.7 Membrane Insertase
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