SIGNAL PEPTIDASE SPECIFICITY AND SUBSTRATE SELECTION: INFLUENCE OF S1 AND S3 SUBSTRATE BINDING POCKET RESIDUES ON SPASE I CLEAVAGE SITE SELECTION DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University Andrew Karla, B.S. ***** The Ohio State University 2005 Dissertation committee: Approved by Professor Ross E. Dalbey, Advisor Professor Ming-Daw Tsai Professor Sean Taylor Professor David J. Hart Advisor Chemistry Graduate Program ABSTRACT Signal peptidase, which removes signal peptides from preproteins, has a substrate specificity for small uncharged residues at -1 (P1) and small or larger aliphatic residues at the -3 (P3) position. Structures of the catalytic domain with a 5S-penem inhibitor and a lipopeptide inhibitor reveal candidate residues that make up the S1 and S3 pockets that bind the P1 and P3 specificity residues of the preprotein substrate. We have used site- directed mutagenesis, mass spectrometric analysis, in vivo and in vitro activity assays as well as molecular modeling to examine the importance of the substrate pocket residues in their ability to promote cleavage of the pro-OmpA-nuclease A substrate with WT and mutant processing regions. Generally, we find that the S1 and S3 binding sites can tolerate changes that are expected to increase or decrease the size of the pocket without large effects on activity. One residue that contributes to the high fidelity of cleavage of signal peptidase is the Ile 144 residue. Changes of the Ile 144 residue to cysteine result in cleavage at multiple sites, as determined by mass spectrometry and Edman sequencing analysis. In addition, we find that signal peptidase is able to cleave after phenylalanine at the -1 residue in a double mutant where both Ile 86 and Ile 144 were changed to an alanine. Also, alteration of the Ile 144 and Ile 86 residues to the corresponding residues found in the homologous Imp1 protease changes the specificity to promote cleavage ii following a –1 Asn residue. This work shows that Ile 144 and Ile 86 contribute to the signal peptidase substrate specificity and that Ile 144 is important for the accuracy of the cleavage reaction. Additionally, studies with corresponding β-lactamase substrate mutants were used to further investigate the altered specificity revealed in the pro-OmpA-nuclease A studies. The use of β-lactamase as a substrate allows for a convenient antibiotic selection method to assay for processing in vivo. In a preliminary study, a two plasmid system was developed to assay combinations of SPase and β-lactamase substrate mutants. This study assayed for growth of the temperature sensitive SPase strain, IT41, that was expressing various combinations of SPase and β-lactamase substrate mutants. Growth under conditions of high ampicillin concentration would suggest efficient processing of β- lactamase. Growth was observed in some cases when the β-lactamase substrate carried a relatively conservative -1V substitution in place of the WT -1A residue. The two-plasmid system utilizing the β-lactamase substrate developed in these studies has potential to be implemented in an unbiased genetic selection technique to further define the substrate specificity determinants of signal peptidase. The β-lactamase substrate was also assayed in a pulse-chase study to investigate processing by select SPase mutants found to affect specificity in the pro-OmpA-nuclease A studies. These studies largely confirm the results observed with the pro-OmpA- nuclease A substrate. One added benefit of using β-lactamase as a substrate is that the iii signal peptide cleavage region possesses no alternate cleavage sites which greatly simplifies data analysis. iv This work dedicated to my parents and my family v ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Ross Dalbey, for his advice and guidance throughout my graduate studies. His continuing enthusiasm and support are to a great degree responsible for my success in these investigations. I would like to thank the other professors that have been involved in these studies directly as collaborators and indirectly as sources of valuable advice. Their continuing involvement has meant much to me: collaborators Dr. Mark Lively, Dr. Mark Paetzel, Dr. Natalie Strynadka; and others Dr. Ming-Daw Tsai, Dr. Dehua Pei and Dr. Martin Caffrey. I would also like to thank my graduate committee for their assistance in the development of this dissertation: Dr. Ming-Daw Tsai, Dr. Sean Taylor and Dr. David Hart. Also, I would like to express gratitude to my friends and coworkers in Johnston Lab. It is the involvement of these people that has made this experience not only an enriching one but also an enjoyable one: Li Zhao, Minyong Chen, Joseph Carlos, Fenglei Jiang, Özlem Doğan Ekici, Liang Yi, Hyunjin Cho, Eunjung Shim, Nil Celebi, Yuxia Dong, Kun Xie, Jijun Yuan and members of the Pei and Tsai research groups. vi Lastly, I would like to thank my parents Paul and Susan, my brother Stephen, my sisters Elizabeth and Margaret, and my fiancée Mary Cerny. I am forever grateful for the love and support they have given me over the years. vii VITA 1975……………………….. Born in Cleveland, Ohio 1998……………………….. B. S. in Biochemistry, University of Dayton, Dayton, Ohio 1998-Present……………….. Department of Chemistry, The Ohio State University, Columbus, Ohio. Fellowship support provided by the Chemistry Biology Interface Training Program and through support as a Graduate Teaching and Research Associate. PUBLICATIONS Karla A, Lively MO, Paetzel M, Dalbey RE (2005). The identification of residues that control signal peptidase cleavage fidelity and substrate specificity. J Biol Chem 280(8): 6731-41. Paetzel M, Karla A, Strynadka NC, Dalbey RE (2002). Signal peptidases. Chem Rev 102(12): 4549-80. Review. Carlos JL, Paetzel M, Brubaker G, Karla A, Ashwell CM, Lively MO, Cao G, Bullinger P, Dalbey RE (2000). The role of the membrane-spanning domain of type I signal peptidases in substrate cleavage site selection. J Biol Chem 275(49): 38813-22 viii FIELDS OF STUDY Major field: Chemistry Specific field: Signal peptidase and membrane protein insertion ix TABLE OF CONTENTS Abstract ……………………………………………………………………………. ii Dedication………………………………………………………………………….. v Acknowledgements………………………………………………………………… vi Vita………………………….……………………………………………………… viii List of Tables……………………………………………..……………………...… xii List of Figures…………………………………………….…………………...…… xiii Chapters: 1. Introduction………………………………….………………………...………… 1 1.1 Signal hypothesis………………………………….……………………….. 1 1.2 SPase I family of proteases………………………………….……….…..… 2 1.3 SPase I substrates: structure of signal peptides………………………...….. 6 1.4 Mechanism of type I SPases………………………………….………….… 7 1.5 Protein engineering: understanding and manipulating enzyme stability and specificity. ………………………………….………………... 12 2. The identification of residues that control signal peptidase cleavage fidelity and substrate specificity………………………………….………..…… 27 2.1 Introduction………………………………….………………………..….… 27 2.2 Results……………………………………………………………………… 29 2.2.1 In vitro analysis of SPase binding site mutants with the wild-type preprotein substrate……………………………………...… 29 2.2.2 In vitro analysis of SPase cleavage of pre-protein substrate mutants…………………………………………………………..…… 30 2.2.3 Mass Spectrometric analysis of signal peptide cleavage……….……. 31 2.2.4 In vivo analysis of signal peptidase binding site mutants………….… 36 2.2.5 Molecular modeling of binding site mutants of signal peptidase in complex with signal peptides…………………………..………..… 37 2.3 Discussion……………………………………………………………..…… 38 2.4 Materials and Methods………………………………………………..…… 45 2.4.1 Bacterials Strains and Plasmids……………………………………… 45 2.4.2 DNA methods……………………………………………………...… 45 2.4.3 Purification of signal peptidase and pro-OmpA-nuclease A…….… 46 x 2.4.4 In vitro and in vivo assay of Signal Peptidase Cleavage……….……. 46 2.4.5 Study to assay ability of mutants to complement the growth defect of the temperature-sensitive SPase strain, IT41(DE3)……..…. 47 2.4.6 Automated Edman Degradation…………………………………...…. 47 2.4.7 Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass spectrometry………………………………...… 48 2.4.8 Prediction of theoretical mass for cleavage products…………...…… 49 2.4.9 Molecular Modeling……………………………………………….… 49 3. Analysis of cleavage fidelity and substrate specificity of signal peptidase S1/S3 mutants with β-lactamase substrate in vivo………………………….…… 67 3.1 Introduction………………………………………………………………… 67 3.2 Results: In vivo growth assays…………………………………………...… 69 3.2.1 WT β-lactamase……………………………………………………… 69 3.2.2 -1V β-lactamase……………………………………………………… 72 3.2.3 -1F β-lactamase…………………………………………………….… 73 3.2.4 -1N β-lactamase ……………………………………..……………..... 73 3.3 Discussion……………………………………………………………….…. 74 3.3.1 Genetic applications………………………………………..………… 78 3.4 Results: Pulse-Chase Studies of in vivo processing……………………...… 79 3.4.1 Processing of WT β-lactamase………………………………….…… 79 3.4.2 Processing of –1V β-lactamase………………………………….…… 80 3.4.3 Processing of –1F β-lactamase……………………………….……… 81 3.4.4 Processing of –1N β-lactamase………………………………….…… 82 3.5 Discussion…………………………………………………………….……. 83 3.6 Materials and Methods………………………………………………...…… 87 3.6.1 Bacterial strains and plasmids……………………………………...… 87 3.6.2 Construction of plasmids…………………………………………..… 87 3.6.3 Purification of β-lactamase……………………………..……………. 88 3.6.4 Pulse-chase assay of β-lactamase processing………………...……… 89 3.6.5 Growth assay for β-lactamase processing………………………….… 90 3.6.6