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Crystallographic Analysis of Bacillus Subtilis Signal Peptide Peptidase (Sppa)

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Crystallographic Analysis of Bacillus Subtilis Signal Peptide Peptidase (Sppa) Crystallographic analysis of Bacillus subtilis signal peptide peptidase (SppA) by Sung-Eun Nam B.Sc., Simon Fraser University, 2006 Thesis Submitted in Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Department of Molecular Biology and Biochemistry Faculty of Science Sung-Eun Nam, 2012 SIMON FRASER UNIVERSITY Fall 2012 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: Sung-Eun Nam Degree: Doctor of Philosophy Title of Thesis: Crystallographic analysis of Bacillus subtilis signal peptide peptidase (SppA) Examining Committee: Chair: Michael Silverman, Associate professor Mark Paetzel Senior Supervisor Associate professor Nicolas Harden Supervisor Professor Edgar Young Supervisor Associate professor Jack Chen Internal Examiner Associate professor SFU/Department of Molecular Biology & Biochemistry J. Thomas Beatty External Examiner Professor, Department of Microbiology and Immunology University of British Columbia Date Defended/Approved: December 6, 2012 ii Partial Copyright Licence iii Abstract Secreted proteins are initially synthesized in a precursor form that contains an N- terminal signal peptide for targeting the proteins to the cytoplasmic membrane. Upon translocation across the membrane via the Sec machinery, the signal peptide is cleaved off by signal peptidase. The remnant membrane embedded signal peptide is then cleaved within its hydrophobic core by signal peptide peptidase A (SppA). SppAs are membrane bound peptidases found in archaea, plant chloroplasts and bacteria. SppAs utilize a serine nucleophile and a lysine general base as catalytic residues. My PhD research has focused on the Bacillus subtilis SppA (SppABS). During my PhD study, I solved the three-dimensional structure of SppABS using X-ray crystallography, allowing us to study the difference and similarities between Gram- positive SppABS and the previously solved Gram-negative Escherichia coli SppA (SppAEC). Both proteins form similarly sized dome-shaped multi-subunit structures where the catalytic residues reside inside the concave portion of the dome. However, each subunit of SppABS is half the size of the SppAEC subunit, thus SppABS is an octameric complex while SppAEC is a tetramer. The structure revealed eight active sites in SppABS where three of the protomers come together to form one complete active site whereas SppAEC contains only four active sites. Structural analysis on the substrate binding pockets S1 and S3, along with activity assays using a range of peptide substrates, revealed that SppABS prefers substrates with leucine, arginine or tyrosine at the P1 position – a significantly different profile from SppAEC which prefers leucine but not arginine or tyrosine at the P1 position. I also solved the crystal structure of SppABS in complex with its own C-terminus bound within its active site. This structure has provided information on how the enzyme recognizes its substrates, confirms our previous SppABS substrate preference analysis and has posed a new question “Why does SppABS cleave its own C-terminus?” We show that SppABS cleaves its own C-terminus in an intra-complex fashion and mutational analysis shows that Tyr331 is important for self-cleavage. I also showed that SppABS is able to digest folded proteins which suggests that SppABS possibly has a membrane quality control function. iv Keywords: Signal peptide peptidase A; self-compartmentalized protease; Ser/Lys protease; v Dedication To my husband, Younghoon & my daughter, Heebin vi Acknowledgements I would like to express my deep gratitude to Dr. Mark Paetzel for taking me under his wing. The knowledge, experiences and accomplishments I have achieved would have not happened without him. I am grateful for your enthusiasm, encouragement and patience. I would like to thank my supervisory committee, Dr. Nick Harden and Dr. Edgar Young, for their advice and guidance over the years. Special thanks to Dr. Jack Chen for being my internal examiner, Dr. Michael Silverman for being the chairman and Dr. Tom Beatty for being my external examiner. Deidre, what can I say? You have been my friend, mother, teacher, advisor, listener, proof reader and lastly the best lab manager ever! I don’t think I could have survived my PhD career without you. Hopefully you will still be there forever for me. Many thanks go to my second family (the Paetzel lab members): Kelly Kim – You have been a wonderful lab sister, thanks; Apollos Kim – Thank you for sharing your passion for the research; Linda Zhang & Suraaj Aulakh – You guys are the best bus buddies ever; Dan Chiang – Thanks for all your entertainment in the lab; Yun Luo – Thank you for always being optimistic; Charlie Stevens – Thank you for your great computer skills; Ivy Cheung – You are great talker; and special thanks to Jae Lee who gives me such insightful advice. And finally, a warm welcome to our new members, Mike and Minfei. I hope for you all the best! And most importantly to my first family Thank you to my husband, Younghoon Oh, for supporting me and to my daughter, Heebin, for laughter at home. Thank you Mom and Dad for your endless love and support for me. 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 Acronyms ......................................................................................................................xiv Glossary ........................................................................................................................ xv 1. Introduction .......................................................................................................... 1 1.1. Bacteria cell envelope ............................................................................................ 1 1.1.1. Outer membrane and peptidoglycan layer ................................................... 2 1.1.2. Cytoplasmic membrane .............................................................................. 3 1.2. Protein secretion .................................................................................................... 3 1.2.1. Signal peptide ............................................................................................. 4 1.2.2. Protein secretion pathway ........................................................................... 7 1.2.3. Signal peptide released by Type I and II signal peptidase ........................... 8 1.2.4. Signal peptide hydrolysis .......................................................................... 11 1.2.4.1. Escherichia coli signal peptide peptidase A ................................ 11 1.2.4.2. Bacillus subtilis signal peptide peptidase A ................................. 12 1.3. Other self-compartmentalized proteases in bacteria ............................................. 15 1.4. SppA utilizes a Ser/Lys catalytic dyad mechanism ............................................... 18 1.4.1. Ser/Lys dyad protease .............................................................................. 18 1.4.2. S49 family of proteases ............................................................................. 21 1.5. Research objectives ............................................................................................. 23 2. Crystal Structure of Bacillus subtilis SppA ...................................................... 24 2.1. Overview .............................................................................................................. 25 2.2. Materials and methods ......................................................................................... 26 2.2.1. Cloning and mutagenesis .......................................................................... 26 2.2.2. Expression and purification ....................................................................... 27 2.2.3. Limited proteolysis .................................................................................... 28 2.2.4. Amino-terminal sequencing analysis ......................................................... 28 2.2.5. Analytical size exclusion chromatography (SEC) and multi-angle light scattering (MALS) analysis ................................................................ 28 2.2.6. Crystallization...........................................................................................
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