ENZYME-CATALYZED EXPRESSED PROTEIN LIGATION by Samuel Henager A dissertation submitted to The Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland August, 2017 Abstract Expressed protein ligation involves the chemoselective reaction of recombinant protein thioesters produced via inteins with N-Cys containing synthetic peptides and has proved to be a valuable method for protein semisynthesis. Expressed protein ligation requires a cysteine residue at the ligation junction which can limit its use. Here we employ subtiligase, a re-engineered form of the protease subtilisin, to ligate a range of synthetic peptides, without the requirement of an N-terminal cysteine, to a variety of recombinant protein thioesters in rapid fashion. We have further broadened the scope of subtiligase-mediated protein ligations by employing a second-generation form (E156Q/G166K subtiligase) and a newly developed form (Y217K subtiligase) for ligation junctions with acidic residues. We have applied subtiligase-mediated expressed protein ligation to the generation of tetraphosphorylated, monophosphorylated, and non-phosphorylated forms of the tumor suppressor lipid phosphatase PTEN. In this way, we have demonstrated that the natural sequence around the ligation junction produced by subtiligase rather than cysteine-mediated ligation is necessary to confer the dramatic impact of tail phosphorylation on driving PTEN's closed conformation and reduced activity. We thus propose that subtiligase-mediated expressed protein ligation is an attractive traceless technology for precision analysis of protein post- translational modifications. Thesis Advisor: Dr. Philip Cole Second Reader: Dr. Jungsan Sohn ii To my family and friends, without whom none of this would have been possible. iii Acknowledgements I am especially grateful to Dr. Nam Chu, who carried out the experiments with ubiquitin and subtiligase Y216K described in chapter 2 and who has been very helpful throughout my thesis research, and Stephanie Henriquez, who assisted with expressing and purifying semisynthetic PTEN. I would also like to thank Amy Weeks, from the Wells lab at UCSF who provided the subtiligase M222A described in chapter 3. I am also grateful to the other members of the Cole lab for all their help and friendship throughout my time at Hopkins. iv Contents Abstract .......................................................................................................................................................... ii Acknowledgements .................................................................................................................................. iv List of Tables .............................................................................................................................................. vii List of Figures ........................................................................................................................................... viii Chapter 1: Introduction .......................................................................................................................... 1 Phosphorylation and Protein Semisynthesis ..................................................................... 1 Serine Proteases and Subtiligase ............................................................................................. 7 Phosphorylation and Signal Transduction ........................................................................ 13 Phosphatases ................................................................................................................................. 14 The PTEN/PI3K/AKT Signaling Pathway .......................................................................... 15 PTEN and Cancer .......................................................................................................................... 17 Structure of PTEN ........................................................................................................................ 20 Regulation of PTEN ..................................................................................................................... 23 PTEN Phosphorylation .............................................................................................................. 23 Summary .......................................................................................................................................... 28 Chapter 2: Protein Semisynthesis with Subtiligase .................................................................. 30 Introduction .................................................................................................................................... 30 Methods ............................................................................................................................................ 33 Results ............................................................................................................................................... 46 Discussion ........................................................................................................................................ 61 Chapter 3: Creating Wild Type Semisynthetic Phospho-PTEN ............................................ 63 v Introduction .................................................................................................................................... 63 Methods ............................................................................................................................................ 64 Results ............................................................................................................................................... 72 Discussion ........................................................................................................................................ 95 Bibliography ............................................................................................................................................... 90 Curriculum Vitae ................................................................................................................................... 108 vi Tables Table 1. ......................................................................................................................................................... 35 Primers used for GST cloning and mutagenesis. Table 2. ......................................................................................................................................................... 36 Primers used for ubiquitin mutagenesis. Table 3. ......................................................................................................................................................... 37 Primers used for subtiligase mutagenesis. Table 4. ......................................................................................................................................................... 44 Peptides used for GST and ubiquitin ligations. Table 5. ......................................................................................................................................................... 53 Ubiquitin-thioester ligations catalyzed by subtiligase. Table 6. ......................................................................................................................................................... 60 GST-thioester ligations catalyzed by subtiligase. Table 7. ......................................................................................................................................................... 66 Primers used for PTEN and subtiligase mutagenesis Table 8. ......................................................................................................................................................... 68 Peptides used for PTEN ligations Table 9. ......................................................................................................................................................... 94 Catalytic activity and tail phosphorylation sensitivity to alkaline phosphatase for semisynthetic PTEN constructs. vii Figures Figure 1. ......................................................................................................................................................... 5 Mechanism of Expressed Protein Ligation Figure 2. ......................................................................................................................................................... 8 Serine protease mechanism Figure 3. ....................................................................................................................................................... 11 Subtiligase ligation mechanism Figure 4. ....................................................................................................................................................... 12 Crystal structure of subtiligase with a protein inhibitor Figure 5. ....................................................................................................................................................... 16 The PTEN/PI3K/AKT pathway Figure 6. ....................................................................................................................................................... 21 Partial crystal structure of PTEN. Figure 7. ......................................................................................................................................................