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Structural and Kinetic Analysis of Escherichia Coli Signal Peptide Peptidase A

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Structural and Kinetic Analysis of Escherichia coli Signal Peptide Peptidase A by Apollos C. Kim B.A. (Psychology), Simon Fraser University, 2001 B.Sc. (Biology), Seoul National University, Korea, 1992 Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Department of Molecular Biology and Biochemistry Faculty of Science Apollos C. Kim 2013 SIMON FRASER UNIVERSITY Summer 2013 All rights reserved. However, in accordance with the Copyright Act of Canada, this work may be reproduced, without authorization, under the conditions for “Fair Dealing.” Therefore, limited reproduction of this work for the purposes of private study, research, criticism, review and news reporting is likely to be in accordance with the law, particularly if cited appropriately. Approval Name: Apollos C. Kim Degree: Doctor of Philosophy (Molecular Biology and Biochemistry) Title of Thesis: Structural and kinetic analysis of Escherichia coli signal peptide peptidase A Examining Committee: Chair: Frederic Pio, Associate Professor Mark Paetzel Senior Supervisor Associate Professor Nicholas Harden Supervisor Professor Edgar C. Young Supervisor Associate Professor Dipankar Sen Internal Examiner Professor Ross MacGillivray External Examiner Professor Department of Biochemistry and Molecular Biology University of British Columbia Date Defended/Approved: June 19, 2013 ii Partial Copyright Licence iii Abstract Secretory proteins contain a signal peptide at their N-terminus. The signal peptide functions to guide proteins to the membrane and is cleaved off by signal peptidase. The remnant signal peptides must be removed from the membrane to prevent their accumulation which can lead to membrane destabilization. Escherichia coli signal peptide peptidase A (SppAEC) has been identified as a major enzyme that processes the remnant signal peptide to smaller fragments. SppAEC, however, had remained uncharacterized such that the structure, catalytic mechanism, and substrate preference were unknown. I have cloned, overexpressed, purified and crystallized an active soluble domain of SppAEC. We have determined the structure of SppAEC by X-ray crystallography, which revealed that: 1) SppAEC has two similar alpha-beta domains (despite limited sequence identity), 2) SppAEC forms a tetramer that results in a dome shaped structure with a hydrophobic interior and hydrophilic exterior, 3) the four active sites within the cavity each utilize a Ser-Lys dyad where the general base (Lys209) arrives from the N-terminal domain and the nucleophile (Ser409) from the C-terminal domain. I have further characterized SppAEC using steady-state kinetics and cocrystallization. Using a series of fluorometric peptide substrates, I discovered that leucine is the most preferred residue at the P1 substrate position. I cocrystallized an SppAEC active site mutant (K209A), in complex with the substrate Z-LLL-MCA. The electron density within the active site of the 1.95 Å resolution structure is consistent with the carbonyl carbon of the substrate’s C-terminal leucine being covalently linked via an ester bond to the Ser409 O, thus revealing an acyl-enzyme complex in SppAEC. This is direct evidence that Ser409 O serves as a nucleophile in the SppAEC catalyzed reaction and confirms the identity of the S1 and S3 substrate specificity pockets. Lastly, I measured the pH dependence of both WT and S431A enzymes (Ser431 Ois hydrogen-bonded to Lys209 Nζ). The pH-rate profiles are consistent with S431 playing a role in lowering the pKa of the lysine general base, which would be critical for activity at iv physiological pH. We propose that the active site architecture of SppAEC may be best described as a Ser-Lys-Ser triad. Keywords: Protein translocation; signal peptide processing; signal peptide peptidase; Ser-Lys-Ser triad catalysis; steady-state kinetics; X-ray crystallography. v Dedication To God who is the reason for my life vi Acknowledgements First of all, I thank my senior supervisor, Dr. Mark Paetzel, who provides the best environment and guidance for doing and learning science. He has taught me how to enjoy science not just as a job but as a calling. Also, my two other supervisory members, Dr. Nicholas Harden, who has first hired me as a teaching lab technician, has trained me, and has been my “big brother”, and Dr. Edgar Young, who knows how to spread the passion for science and is always willing to help. It has been such a blessing that I could work under this wonderful group of mentors. I thank Dr. Ross MacGillivray for taking time to be my external examiner at the last- minute request. I thank Dr. Dipankar Sen for evaluating my work as an internal examiner and Dr. Frederic Pio for chairing my dissertation defense. I thank my current and former lab-mates, Deidre de Jong-Wong, Chuanyun Luo, Jae- Yong Lee, Ivy Chung, Kelly Kim, Charles Stevens, Sung-Eun Nam, Daniel Chiang, Suraaj Aulakh, Linda Zhang, Minfei Fu, Michael Ungerer and Zohreh Sharafianardekani. I am indebted to Deidre, Chuanyun, and Jae-Yong who have been my friends. Sung- Eun and Daniel have been excellent shareholders in this S49 peptidase family project. I thank Dr. Bruce Brandhorst and Dr. Lynne Quarmby who were wonderful bosses. I also thank Jenny Lum, Duncan Napier, Christine Beauchamp, Nancy Suda, Mimi Fourie, and Livleen Diwana who have been great to work with. Finally, I know I could not finish this without my family who have always supported me and have believed in me. Thank you all. vii Table of Contents Approval .......................................................................................................................... ii Partial Copyright Licence ............................................................................................... iii Abstract .......................................................................................................................... iv Dedication ...................................................................................................................... vi Acknowledgements ....................................................................................................... vii Table of Contents .......................................................................................................... viii List of Tables .................................................................................................................. xi List of Figures................................................................................................................ xii Glossary ........................................................................................................................xiv 1. Signal peptide peptidase A .................................................................................. 1 1.1. The function of signal peptides in protein translocation ........................................... 1 1.2. The structure of signal peptides .............................................................................. 5 1.3. Discovery of E. coli signal peptide peptidase A ....................................................... 8 1.4. S49 family proteases ............................................................................................ 10 1.5. Catalytic mechanism of serine proteases ............................................................. 11 1.5.1. Principles of catalysis ................................................................................ 16 1.5.2. pH dependence ......................................................................................... 17 1.5.3. Temperature dependence ......................................................................... 19 1.6. Substrate specificity .............................................................................................. 22 1.7. Aims of this thesis ................................................................................................ 24 2. Crystallographic analysis of E. coli signal peptide peptidase A with an empty active site ................................................................................................ 26 2.1. Introduction .......................................................................................................... 27 2.2. Methods and materials ......................................................................................... 27 2.2.1. Cloning...................................................................................................... 27 2.2.2. Overexpression and purification of SppAEC proteins .................................. 28 2.2.3. Crystallization and data collection ............................................................. 30 2.2.4. Structure determination and analysis ........................................................ 32 2.2.5. Figure preparation and Protein Data Bank accession codes ..................... 34 2.3. Results and discussion ......................................................................................... 34 2.3.1. Protein expression and purification ........................................................... 35 2.3.2. The protein fold of SppAEC ........................................................................ 35 2.3.3. The shape, dimensions, and surface features of the SppAEC tetramer .................................................................................................... 37 2.3.4. The assembly of Ser-Lys dyad active sites from adjacent domains and monomers .........................................................................................
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