Western Washington University Western CEDAR WWU Honors Program Senior Projects WWU Graduate and Undergraduate Scholarship Spring 2020 Sorting Out Sortases: Designing a Better Enzyme and Synthesizing Trapping Peptides Towards Capturing a Bound-State Structure Katherine Johnston Western Washington University Follow this and additional works at: https://cedar.wwu.edu/wwu_honors Part of the Chemistry Commons Recommended Citation Johnston, Katherine, "Sorting Out Sortases: Designing a Better Enzyme and Synthesizing Trapping Peptides Towards Capturing a Bound-State Structure" (2020). WWU Honors Program Senior Projects. 398. https://cedar.wwu.edu/wwu_honors/398 This Project is brought to you for free and open access by the WWU Graduate and Undergraduate Scholarship at Western CEDAR. It has been accepted for inclusion in WWU Honors Program Senior Projects by an authorized administrator of Western CEDAR. For more information, please contact [email protected]. Sorting Out Sortases: Designing a Better Enzyme and Synthesizing Trapping Peptides Towards Capturing a Bound-State Structure By Katherine Johnston June 2020 A Thesis Presented to The Honors Department of Western Washington University In Partial Fulfillment Of the Requirements for University Honors Abstract Sortase A is a powerful protein engineering tool that cleaves proteins and attaches them to an acyl acceptor of choice. However, the most active wild type variants of sortase A only show high activity at a limited number of cleavage motifs, and so work is underway to create a variant of sortase A that shows high activity at a greater variety of cleavage sites. Additionally, current studies attempting to optimize the enzyme require a way to stabilize an unstructured loop for the crystallization, in order to collect X-ray diffraction structural data. Here, preliminary results from the design of an “loop-swapped” sortase A enzyme variants are discussed, as well as the synthesis of two different thiol-containing “trapping” peptides intended to freeze sortase A in its bound conformation via a covalent disulfide bond. The first peptide is a previously described unnatural peptide, and was discontinued before completion, and the second peptide is a natural peptide which was completed and purified. Future work will involve the co-crystallization of this peptide with “loop-swapped” sortase A variants. ii Table of Contents Abstract................................................................................................................................... ii List of Figures......................................................................................................................... iv Introduction............................................................................................................................. 1 Activity of the β-7/β-8 Loop................................................................................................... 4 Fluorescent Peptides............................................................................................................... 5 Synthesis of a Trapping Peptide: Two Routes Unnatural Peptide....................................................................................................... 8 Natural Peptide........................................................................................................... 12 Future Directions.................................................................................................................... 14 Methods.................................................................................................................................. 15 Acknowledgements................................................................................................................. 22 References............................................................................................................................... 24 Supporting Information.......................................................................................................... 25 iii List of Figures Title Page Number Figure 1: Graphical representation of sortase A catalyzed protein ligation 1 Figure 2: Heat map of relative cleavage by sortase A variants 2 Figure 3: Existing structures of bound-state SrtAstaph 3 Figure 4: β-7/β-8 “Loop Swapped” sortase A variants 4 Figure 5: Structure of fluorescent peptides 7 Figure 6: Synthesis scheme of unnatural peptide 8 Figure 7: NMR spectra of unsuccessful reduction 10 Figure 8: NMR spectra of successful reduction 11 Figure 9: Structure of crystallization peptide 13 Figure S1: Workflow of protein expression and purification 25 Figure S2: Simplified scheme of SPPS 26 Figure S3: Workflow of peptide expression and purification 27 iv Introduction Sortase A (SrtA) is a transpeptidase enzyme found in gram-positive bacteria which cleaves proteins at the LPXTG motif and ligates the C-terminal of the cleaved protein onto an acyl acceptor, lipid II (Figure 1).1,2 Other acyl acceptors such as the N-terminus of another protein or peptide are also able to accept the cleaved protein.3 This function has been successfully exploited by researchers as a protein engineering tool to modify protein sequences post-translationally, attach protein tags to cells, create cyclic structures, and more. Staphylococcus aureus sortase A (SrtAstaph) represents the most commonly used sortase A, in particular due to its high cleavage activity. Despite its many uses, SrtAstaph is limited by its specificity to the LPXTG cleavage motif. The Streptococcus pneumoniae sortase A (SrtAstrep) recognizes more cleavage motifs, including LPATS and LPATA3, however is less active than the S. aureus version, with approximately 1/3 cleavage activity when compared to SrtAstaph (Figure 2). Figure 1. Graphical representation of sortase A catalyzed protein ligation. Figure obtained from Antos, et al., (2016). Designing a sortase variant which has the promiscuity (recognition of additional cleavage sites) of SrtAstrep while maintaining the high activity of SrtAstaph will be beneficial to the research community as a more flexible protein engineering tool. The activity and promiscuity of new sortase variants can be tested against peptide variants of the LPXTG motif, via fluorescence 1 2assay (Figure 2). This improvement of Sortase A is an ongoing endeavor in the Amacher and Antos labs, and some parts of the project will be discussed in this paper. Additionally, structural characterization of Sortase A while it is in its bound conformation will provide insight into the role of the unstructured β-7/β-8 loop which appears to play a role in substrate specificity and cleavage activity. The Figure 2. Heat map of relative cleavage by sortase A variants as measured by fluorescent activity. The catalytic residues for SrtAstrep are single letter labels represent the amino acid in the X position of the fluorescent peptide Abz-LPATXG- C184, H118, and R192. The cysteine, K(DNP). Figure created by Izzi Piper. histidine, and arginine catalytic triad is conserved in SrtA enzymes of other species. Our hypothesis is that a disulfide bond between the catalytic cysteine and the LPXTG peptide involved in crystallization will facilitate the collection of accurate bound-state structural data by both trapping the enzyme in its bound conformation and preventing peptide shifting. A previous structure of S. aureus SrtA, which lacked such a covalent bond, revealed a shifted LPETG peptide out of the active site, while a structure that did contain a covalent bond between its peptide and the active site of sortase did not have this issue (Figure 3A, B).4,5 The structure PDB ID: 2KID, which is the current best structure of a bound-state sortase A enzyme, includes the unnatural peptide LPAT*, where T* is an unnatural, thiol-containing threonine analogue. The authors of Jung, et al., 2005 developed the LPAT* peptide for the purpose of forming a disulfide bond between sortase and a peptide ligand.1 They showed that a 2 A B Figure 3. A. X-ray crystallography structure of SrtAstaph complexed with shifted LPETG peptide, PDB 1T2W. B. NMR structure of SrtAstaph complexed with (PHQ)LPA(B27) peptide, PDB 2KID. disulfide bond did indeed form between the two when the unnatural peptide was added in excess.1 The goal of the disulfide bond was to allow for the collection of structural data on the bound enzyme. They had previously synthesized other versions of this threonine analogue towards the same goal, and found that the resulting bound enzyme was not optimal for structural studies.1 While their original synthesis of T* began with L-threonine and required nine synthetic steps, involving the use of highly toxic hydrogen sulfide gas (H2S), the authors designed a new route in 2012 that was both shorter, requiring only four synthetic steps, and did not require the 1,6 use of H2S. I initially set out to recreate this unnatural peptide following the 2012 synthetic route, with the ultimate goal of co-crystallizing it with our “loop-swapped” sortase variants in order to solve X-ray crystallography structures of the bound enzymes. When the synthesis proved to be unreasonable to complete with the resources available, I created a similar natural peptide to use towards the same goal. 3 Activity of the β-7/β-8 Loop Previous computational work identified the β-7/β-8 unstructured loop near the active site of sortase A as quite variable amongst SrtA enzymes, therefore, we hypothesized that it may affect substrate recognition. The loop is structurally conserved across class A sortases,