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A binding site hotspot map of the FKBP12–rapamycin–FRB ternary complex by photo-affinity labeling and mass spectrometry-based proteomics Hope A Flaxman, Chia-Fu Chang, Hung-Yi Wu, Carter H Nakamoto, and Christina M Woo J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b03764 • Publication Date (Web): 16 Jul 2019 Downloaded from pubs.acs.org on July 17, 2019

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Journal of the American Chemical Society

A binding site hotspot map of the FKBP12–rapamycin–FRB ternary complex by photo-affinity labeling and mass spectrometry-based proteomics Hope A. Flaxman,1 Chia-Fu Chang,1 Hung-Yi Wu,1 Carter H. Nakamoto,1 Christina M. Woo1,* 1

Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138

Supporting Information Placeholder

Structural characterization of small molecule binding site hotspots within the global proteome is uniquely enabled by photo-affinity labeling (PAL) coupled with chemical enrichment and unbiased analysis by mass spectrometry (MS). MS-based binding site maps provide structural resolution of interaction sites in conjunction with identification of target proteins. However, binding site hotspot mapping has been confined to relatively simple small molecules to date; extension to more complex compounds would enable the structural definition of new binding modes in the proteome. Here, we extend PAL and MS methods to derive a binding site hotspot map for the immunosuppressant rapamycin, a complex macrocyclic natural product that forms a ternary complex with the proteins FKBP12 and FRB. Photo-rapamycin was developed as a diazirine-based PAL probe for rapamycin, and the FKBP12–photo-rapamycin–FRB ternary complex formed readily in vitro. Photoirradiation, digestion, and MS analysis of the ternary complex revealed a McLafferty rearrangement product of photo-rapamycin conjugated to specific surfaces on FKBP12 and FRB. Molecular modeling based on the binding site map revealed two distinct conformations of complex-bound photo-rapamycin, providing a 5.0 Å distance constraint between the conjugated residues and the diazirine carbon and a 9.0 Å labeling radius for the diazirine upon photo-activation. These measurements may be broadly useful in the interpretation of binding site measurements from PAL. Thus, in characterizing the ternary complex of photo-rapamycin by MS, we applied binding site hotspot mapping to a macrocyclic natural product and extracted precise structural measurements for interpretation of PAL products that may enable the discovery of new binding sites in the “undruggable” proteome. ABSTRACT:

Photo-affinity labeling (PAL) is a widely used method for capturing the biomolecular targets of non-covalent

Figure 1. Overview of the application of PAL to the rapamycin ternary complex. A) The structures of rapamycin, photo-rapamycin, and the minimalist tag. B) Workflow used to obtain conjugated peptides from the FKBP12–photo-rapamycin–FRB ternary complex.

ligands. In a PAL experiment, a photo-activatable group embedded in a chemical probe is activated by ACS Paragon Plus Environment

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light to generate a reactive intermediate that can form a stable covalent bond to the interacting proteins.1–3 The combination of PAL with chemical enrichment and detection by mass spectrometry (MS) enables target identification on the proteome-wide scale and structural elucidation of the binding site if the small moleculeconjugated peptide is isolated. Although mapping of the binding site itself has increasingly been used to localize small molecule ligands on their protein targets,4–7 a systematic definition of the distances represented by PAL measurements is needed to improve structural interpretation of the data. Furthermore, extension of binding site mapping to more complex macrocyclic natural products, many of which possess biological activity through unique modes of binding to proteins,8 would enable investigation into the binding preferences of this privileged class of ligands, including macrocycles that have previously been developed as PAL probes for target identification.9,10 Inspired by parallel advances in cross-linking MS that have yielded structural insights from crosslinked peptides,11,12 we sought to enable similar insights from binding site maps of complex macrocyclic natural products and simultaneously derive the structural distances reported by PAL. Herein, we evaluate the structure of the FKBP12–rapamycin–FRB ternary complex using diazirine-based PAL chemistry to characterize macrocycle–peptide conjugates by MS and develop a model to determine a PAL radius based on the binding site map. Rapamycin was one of the earliest examples of a “molecular glue,” exerting its biological effects through the stabilization of a protein–protein interaction between FKBP12 and the FRB domain of mTOR.13–15 The ternary complex has since been characterized by crystallography and biophysical methods.16,17 To understand how PAL measurements can encode structural information and extend binding site mapping methods to macrocyclic natural products, we designed a PAL probe for rapamycin termed photo-rapamycin (Figure 1, Scheme S1). Photo-rapamycin was functionalized with a diazirine-based “minimalist tag”18 at the C40 position, a position amenable to derivatization in prior studies (Figure 1A).19 The diazirine is a minimally-perturbative photo-activatable group that is readily embedded in molecules through functionalized tags.18,20 To gain insight into how photorapamycin-conjugated peptides may fragment by MS, an aliquot of photo-rapamycin was irradiated in methanol to mimic the diazirine insertion event. Fragmentation of the methanolized photo-rapamycin by qTOF MS yielded species resulting from neutral losses and fragmentations in the macrocyclic backbone, such as cleavage of the lactone, fragmentation across the diketone, and a McLafferty rearrangement (Figure S1, Table S1).21 The observed fragmentation was in alignment with previous studies of everolimus, a rapamycin derivative.22

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The formation of the ternary complex from recombinant FKBP12 and FRB (Figure S2) was evaluated by

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Figure 2. Validation of photo-rapamycin as a probe for rapamycin. A. SPR measurement of the binding kinetics of FKBP12–rapamycin and FKBP12–photo-rapamycin with FRB. B. Ternary complex formation with rapamycin and photo-rapamycin in different buffer conditions, monitored by SEC. C. Representative in-gel fluorescence of 10 µM FKBP, 10 µM FRB in PBS + 0.1% Triton + 2.5% EtOH with the indicated treatments. Samples were photoirradiated and the fluorophore was attached using CuAAC. D. Quantification of fluorescent signal across three replicates with standard errors indicated.

surface plasmon resonance (SPR). The binding of FRB to immobilized GST-FKBP12 displayed similar binding kinetics in the presence of rapamycin or photorapamycin (Figures 2A, S3) that are comparable to previously reported binding kinetics for rapamycin (Kd = 12 ± 0.8 nM).17 Despite similar binding constants for ternary complex formation observed by SPR, differences in solubility at µM concentrations were observed (Figure S4). We therefore optimized ternary complex formation by size exclusion chromatography (SEC). In the presence of rapamycin, ternary complex formation occurred quantitatively with each component at 10 μM in PBS, 0.2% EtOH, as compared to 12% complex

formation with photo-rapamycin (by relative peak height, Figure 2B). Upon buffer optimization, the FKBP12–photo-rapamycin–FRB ternary complex was formed at 51%. The ternary complex was formed quantitatively upon increasing the photo-rapamycin concentration (3 equiv). The FKBP12–photorapamycin–FRB complex was photo-irradiated and successful conjugation of photo-rapamycin to FKBP12 or FRB was visualized by in-gel fluorescence, using a fluorophore attached via copper-catalyzed azide-alkyne cycloaddition (CuAAC). In-gel fluorescence showed photo-rapamycin-dependent labeling of both FKBP12 and FRB that was competitively displaced by rapamycin (Figures 2C, 2D, S5). The photo-rapamycin ternary complex was then prepared for structural characterization by MS (Figure 1B). As we expected the internal fragmentation pathways of rapamycin to add analytical complexity, an isotopically-coded handle6 was installed by CuAAC to improve confidence in the assignment of modified peptides. Isotopic recoding produces recognizable markers in the full scan mass spectrum (MS1) that improve detection and validation of chemically-



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Figure 3. Photo-rapamycin-conjugated peptides identified by LC-MS/MS. A. Photo-rapamycin-conjugated peptides were observed following an internal fragmentation to afford the McLafferty rearrangement product (RP) as the primary modification on conjugated peptides. B. Representative assignment of a conjugated peptide from FKBP12. C. Representative assignment of a conjugated peptide from FRB. Predicted amino acid modification sites shown in grey.


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Figure 4. Structural analysis of photo-rapamycin labeling of FKBP12 and FRB. A. Labeling intensity as a function of residue number on each protein (bars) aligned with distance from each residue to the rapamycin C40-OH (black solid lines). Residues to which modifications were assigned are highlighted (vertical dotted lines). Relative labeling is calculated based on the summed precursor species intensities of RP-modified PSMs including each residue. Residues are numbered according to Sequence S3. B. Labeling measured by streptavidin blotting following CuAAC attachment of a biotin-azide to wild-type (WT) FKBP12 and FRB or mutant (mut) FKBP12 D79A and FRB E18A treated with rapamycin or photo-rapamycin and irradiated, quantified across three replicates with standard errors indicated. C. Conformation leading to observed labeling on FKBP12. D. Conformation leading to observed labeling on FRB. E. Distance from the diazirine carbon to FKBP12 D79 (blue) or FRB E18 (orange) side chains over the course of a 1 ns MD simulation of the FKBP12–photo-rapamycin-FRB ternary complex. Simulations were performed with restraints to starting position on all non-solvent components except the cyclohexyl ring and its substituents on photo-rapamycin.



modified species.23 Following installation of the isotopically-coded handle, the ternary complex was digested with trypsin using a S-Trap.24,25 Digested samples were analyzed on an Orbitrap Fusion Tribrid by higher-energy collision induced dissociation (HCD) fragmentation. Photo-rapamycin-conjugated peptides were identifiable based on the embedded isotopic code in the MS1 spectra. Clusters of isotopically-coded peaks were readily observed with mass differences corresponding to differences in oxidation state which were likewise observed with rapamycin and photo-rapamycin when analyzed directly (Figure S6). Investigation of the tandem MS (MS2) spectra derived from isotopicallycoded precursors revealed that the conjugated peptides did not carry intact photo-rapamycin as a modification but were instead modified by photo-rapamycin fragments that formed prior to, or during, the MS1 analysis. Evaluation of these spectra revealed that the major modification masses on conjugated peptides resulted from the same fragmentations observed with methanolized photo-rapamycin, particularly cleavage of the lactone and a McLafferty rearrangement,21 and variations corresponding to addition and elimination of water or methanol yielding several rearrangement products (RPs) as modifications (Figure 3A, S7). Database searching by SEQUEST HT was therefore performed by (1) searching the HCD MS2 spectra against a database of unmodified peptides resulting from the semi-tryptic digest of FKBP12, FRB, and common contaminant proteins, then (2) searching unassigned spectra against a database of peptides resulting from a semi-tryptic digest of FKBP12 and FRB with RPs as variable modification on any amino acid. MS2 spectra assigned to photo-rapamycin-conjugated peptides were validated by manual examination for isotopic coding of the MS1 precursor. RP-conjugated peptides were identified on both FKBP12 and FRB (Figures 3B, 3C). In a representative experiment, a total of 26 RP-modified peptide spectral matches (PSMs) were identified (Tables S2, S3). By comparison, treatment with the minimalist tag (10 equiv) yielded a broader and largely orthogonal array of labeled regions at a lower overall labeling frequency (Figure S5, S8, Table S4, S5). RP conjugation was exclusively observed on residues 75–110 of FKBP12 and residues 10–22 of FRB (Figures 4A, 4B). The MS2 spectra displayed fragments of the underlying peptide, but fragment ions with conjugated RPs were not observed, potentially due to the large modified peptide mass, absence of charge localized to the modification, or neutral loss of the modification from the peptide backbone analogous to a labile post-translational modification.26 Sequenced fragment ladders therefore provided poor localization of RP modifications. Additional parent and peptide ions resulting from MS-

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cleavage of the modification from the peptide were also assigned in some spectra. Therefore, initial prediction of the conjugated residues was performed based on distance and geometry from the rapamycin C40-OH and emerging evidence for the amino acid selectivity for alkyl diazirines.16,27,28 The primary conjugation sites were predicted to be FKBP12 D79 and FRB E18, which are, respectively, 10.5 Å and 10.8 Å from the rapamycin C40-OH (Figure 4A). Mutation of these residues to alanine resulted in a decrease in labeling (60% for FKBP12, 42% for FRB, Figure 4B, S9–11), demonstrating that conjugation at these residues contributes significantly to the species observed by MS. The additional labeling on FRB E18A may arise from conjugation to the adjacent E19, which is 12.1 Å from the rapamycin C40-OH but was not amenable to point mutation. A model of the photo-rapamycin complex, based on guided molecular dynamics (MD) simulations starting from the crystal structure of the FKBP12–rapamycin– FRB ternary complex,16 yielded possible conformations of photo-rapamycin that would lead to labeling at each of the observed residues with minimal perturbation of the overall ternary complex structure (Figures 4C, 4D). Importantly, these data predict that rapamycin is conformationally flexible about the cyclohexyl ring resulting in at least two distinct conformations in solution. In the modeled structures, the diazirine carbon is 4.5 Å (FKBP12) and 5.0 Å (FRB) from the labeled residue. A 5.0 Å distance can thus be used as a restraint in developing docked models of novel small molecule– protein interactions formed by PAL. To improve prediction of surfaces that are labeled by PAL, we also performed a 1 ns unguided MD simulation that showed the minimalist extension of photo-rapamycin exhibited conformations in which the diazirine carbon was within 9 Å of each of the labeled residues (Figure 4E). However, conjugation of a region on FKBP12 (residues 51–60, Figure 4A) that is within 10 Å of the rapamycin C40-OH was not observed. The presented binding site map of FKBP–photo-rapamycin–FRB therefore reflects the conformational preferences of the functionalized photo-rapamycin as well as potential underlying preferences in ionization, fragmentation, and residue selectivity. In conclusion, analysis of the FKBP12–rapamycin– FRB ternary complex by PAL and MS yielded, to our knowledge, the first binding site hotspot map of a complex macrocyclic natural product. Data obtained with photo-rapamycin provides a 5.0 Å minimum distance restraint and a 9.0 Å predicted labeling radius for interpretation of binding site maps from PAL. The labeling of specific regions of FKBP12 and FRB reflects two conformations that were modeled by MD, illustrating the potential of PAL to profile dynamic interactions. The distance restraints from these results


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will inform structural analysis of binding sites derived from PAL coupled with MS, both in vitro and in complex cellular environments. This extension of PAL may further expand the profiling of binding sites on protein surfaces beyond canonical liganded space and yield insights into the “undruggable” proteome. ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website. Supplemental data and procedures (PDF) Tables S2-S5 summarizing MS data (Excel) The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD014319. (Username: [email protected], Password: HoCfKcHb).

AUTHOR INFORMATION Corresponding Author

*[email protected] Funding Sources

Support from the Burroughs Wellcome Fund (C.M.W.), Ono Pharma Foundation (C.M.W.), Sloan Research Foundation (C.M.W.), National Science Foundation (H.A.F.), and Harvard University are gratefully acknowledged.

ACKNOWLEDGMENT We thank Bogdan Budnik and the Harvard University Mass Spectrometry and Proteomics Resource Laboratory for proteomics support and Paul Boudreau and the Balskus Lab for assistance with small molecule MS.

REFERENCES (1) Das, J. Aliphatic Diazirines as Photoaffinity Probes for Proteins: Recent Developments. Chem. Rev. 2011, 111 (8), 4405– 4417. (2) Sumranjit, J.; Chung, S. Recent Advances in Target Characterization and Identification by Photoaffinity Probes. Molecules 2013, 18 (9), 10425–10451. (3) Flaxman, H. A.; Woo, C. M. Mapping the Small Molecule Interactome by Mass Spectrometry. Biochemistry 2018, 57 (2), 186– 193. (4) Niphakis, M. J.; Lum, K. M.; Cognetta III, A. B.; Correia, B. E.; Ichu, T.-A.; Olucha, J.; Brown, S. J.; Kundu, S.; Piscitelli, F.; Rosen, H.; Cravatt, B. F., A Global Map of Lipid-Binding Proteins and Their Ligandability in Cells. Cell 2015, 161 (7), 1668–1680. (5) Parker, C. G.; Galmozzi, A.; Wang, Y.; Correia, B. E.; Sasaki, K.; Joslyn, C. M.; Kim, A. S.; Cavallaro, C. L.; Lawrence, R. M.; Johnson, S. R.; Narvaiza, I.; Saez, E.; Cravatt, B. F., Ligand and Target Discovery by Fragment-Based Screening in Human Cells. Cell 2017, 168 (3), 527–541.e29. (6) Gao, J.; Mfuh, A.; Amako, Y.; Woo, C. M. Small Molecule Interactome Mapping by Photoaffinity Labeling Reveals Binding Site Hotspots for the NSAIDs. J. Am. Chem. Soc. 2018, 140 (12), 4259– 4268.

(7) Gertsik, N.; am Ende, C. W.; Geoghegan, K. F.; Nguyen, C.; Mukherjee, P.; Mente, S.; Seneviratne, U.; Johnson, D. S.; Li, Y.M. Mapping the Binding Site of BMS-708163 on γ-Secretase with Cleavable Photoprobes. Cell Chem. Biol. 2017, 24 (1), 3–8. (8) Villar, E. A.; Beglov, D.; Chennamadhavuni, S.; Porco Jr, J. A.; Kozakov, D.; Vajda, S.; Whitty, A. How Proteins Bind Macrocycles. Nat. Chem. Biol. 2014, 10 (9), 723–731. (9) MacKinnon, A. L.; Garrison, J. L.; Hegde, R. S.; Taunton, J. Photo-Leucine Incorporation Reveals the Target of a Cyclodepsipeptide Inhibitor of Cotranslational Translocation. J. Am. Chem. Soc. 2007, 129 (47), 14560–14561. (10) MacKinnon, A. L.; Taunton, J. Target Identification by Diazirine Photo-Cross-linking and Click Chemistry. Curr. Protoc. Chem. Biol. 2009, 1, 55. (11) Yu, C.; Huang, L. Cross-Linking Mass Spectrometry: An Emerging Technology for Interactomics and Structural Biology. Anal. Chem. 2018, 90 (1), 144–165. (12) Sinz, A. Cross-Linking/Mass Spectrometry for Studying Protein Structures and Protein–Protein Interactions: Where Are We Now and Where Should We Go from Here?. Angew. Chem., Int. Ed. Engl. 2018, 27, 966. (13) Brown, E. J.; Albers, M. W.; Shin, T. B.; Ichikawa, K.; Keith, C. T.; Lane, W. S.; Schreiber, S. L. A Mammalian Protein Targeted by G1-Arresting Rapamycin-Receptor Complex. Nature 1994, 369 (6483), 756–758. (14) Sabatini, D. M.; Erdjument-Bromage, H.; Lui, M.; Tempst, P.; Snyder, S. H. RAFT1: A Mammalian Protein That Binds to FKBP12 in a Rapamycin-Dependent Fashion and Is Homologous to Yeast TORs. Cell 1994, 78 (1), 35–43. (15) For a review of small molecules as a “molecular glue” see: (15a) Fischer, E. S.; Park, E.; Eck, M. J.; Thomä, N. H. SPLINTS: Small-Molecule Protein Ligand Interface Stabilizers. Curr. Opin. Struct. Biol. 2016, 37, 115–122; (15b) Schreiber, S. A Chemical Biology View of Bioactive Small Molecules and a Binder-Based Approach to Connect Biology to Precision Medicines. 2018, 1–10; (15c) Stanton, B. Z.; Chory, E. J.; Crabtree, G. R. Chemically Induced Proximity in Biology and Medicine. Science. 2018, 359 (6380). eaao5902. (16) Liang, J.; Choi, J.; Clardy, J. Refined Structure of the FKBP12-Rapamycin-FRB Ternary Complex at 2.2 Å Resolution. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999, 55 (4), 736–744. (17) Banaszynski, L. A.; Liu, C. W.; Wandless, T. J. Characterization of the FKBP·Rapamycin·FRB Ternary Complex. J. Am. Chem. Soc. 2005, 127 (13), 4715–4721. (18) Li, Z.; Hao, P.; Li, L.; Tan, C. Y. J.; Cheng, X.; Chen, G. Y. J.; Sze, S. K.; Shen, H.-M.; Yao, S. Q. Design and Synthesis of Minimalist Terminal Alkyne-Containing Diazirine Photo-Crosslinkers and Their Incorporation into Kinase Inhibitors for Cell- and TissueBased Proteome Profiling. Angew. Chemie Int. Ed. 2013, 52 (33), 8551–8556. (19) Sedrani, R.; Cottens, S.; Kallen, J.; Schuler, W. Chemical Modification of Rapamycin: The Discovery of SDZ RAD. Transplant. Proc. 1998, 30 (5), 2192–2194. (20) Chang, C.-F.; Mfuh, A.; Gao, J.; Wu, H.-Y.; Woo, C. M. Synthesis of an Electronically-Tuned Minimally Interfering Alkynyl Photo-Affinity Label to Measure Small Molecule–Protein Interactions. Tetrahedron 2018, 74 (26), 3273. (21) McLafferty, F. W. Mass Spectrometric Analysis: Molecular Rearrangements. Anal. Chem. 1959, 31 (1), 82–87. (22) Boernsen, K. O.; Egge Jacobsen, W.; Inverardi, B.; Strom, T.; Streit, F.; Schiebel, H. M.; Benet, L. Z.; Christians, U. Assessment and Validation of the MS/MS Fragmentation Patterns of the Macrolide Immunosuppressant Everolimus. J. Mass Spectrom. 2007, 42 (6), 793–802. (23) Woo, C. M.; Felix, A.; Byrd, W. E.; Zuegel, D. K.; Ishihara, M.; Azadi, P.; Iavarone, A. T.; Pitteri, S. J.; Bertozzi, C. R. Development of IsoTaG, a Chemical Glycoproteomics Technique for Profiling Intact N- and O-Glycopeptides from Whole Cell Proteomes. J. Proteome Res. 2017, 16 (4), 1706–1718. (24) Ludwig, K. R.; Schroll, M. M.; Hummon, A. B. Comparison of In-Solution, FASP, and S-Trap Based Digestion



Methods for Bottom-Up Proteomic Studies. J. Proteome Res. 2018, 17 (7), 2480–2490. (25) HaileMariam, M.; Eguez, R. V.; Singh, H.; Bekele, S.; Ameni, G.; Pieper, R.; Yu, Y. S-Trap, an Ultrafast SamplePreparation Approach for Shotgun Proteomics. J. Proteome Res. 2018, 17 (9), 2917–2924. (26) Woo, C. M.; Iavarone, A. T.; Spiciarich, D. R.; Palaniappan, K. K.; Bertozzi, C. R. Isotope-Targeted Glycoproteomics (IsoTaG): A Mass-Independent Platform for Intact N- and O-Glycopeptide Discovery and Analysis. Nat. Methods 2015, 12 (6), 561–567. (27) Iacobucci, C.; Goetze, M.; Piotrowski, C.; Arlt, C.; Rehkamp, A.; Ihling, C. H.; Hage, C.; Sinz, A. Carboxyl-PhotoReactive MS-Cleavable Cross-Linkers: Unveiling a Hidden Aspect of Diazirine-Based Reagents. Anal. Chem. 2018, acs.analchem.7b04915. (28) West, A.; Woo, C.M. Unpublished results.


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Figure 1. Overview of the application of PAL to the rapamycin ternary complex. A) The structures of rapamycin, photo-rapamycin, and the minimalist tag. B) Workflow used to obtain conjugated peptides from the FKBP12–photo-rapamycin–FRB ternary complex.


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Figure 2. Validation of photo-rapamycin as a probe for rapamycin. A. SPR measurement of the binding kinetics of FKBP12–rapamycin and FKBP12–photo-rapamycin with FRB. B. Ternary complex formation with rapamycin and photo-rapamycin in different buffer conditions, monitored by SEC. C. Representative in-gel fluorescence of 10 µM FKBP, 10 µM FRB in PBS + 0.1% Triton + 2.5% EtOH with the indicated treatments. Samples were photo-irradiated and the fluorophore was attached using CuAAC. D. Quantification of fluorescent signal across three replicates with standard errors indicated.





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Table of Contents


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A binding site hotspot map of the FKBP12–rapamycin–FRB ternary complex by photo-affinity labeling and mass spectrometry-based proteomics Hope A. Flaxman,1 Chia-Fu Chang,1 Hung-Yi Wu,1 Carter H. Nakamoto,1 Christina M. Woo1,* 1

Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138

Supporting Information Placeholder ABSTRACT: Structural characterization of small mole-

cule binding site hotspots within the global proteome is uniquely enabled by photo-affinity labeling (PAL) coupled with chemical enrichment and unbiased analysis by mass spectrometry (MS). MS-based binding site maps provide structural resolution of interaction sites in conjunction with identification of target proteins. However, binding site hotspot mapping has been confined to relatively simple small molecules to date; extension to more complex compounds would enable the structural definition of new binding modes in the proteome. Here, we extend PAL and MS methods to derive a binding site hotspot map for the immunosuppressant rapamycin, a complex macrocyclic natural product that forms a ternary complex with the proteins FKBP12 and FRB. Photo-rapamycin was developed as a diazirine-based PAL probe for rapamycin, and the FKBP12–photorapamycin–FRB ternary complex formed readily in vitro. Photo-irradiation, digestion, and MS analysis of the ternary complex revealed a McLafferty rearrangement product of photo-rapamycin conjugated to specific surfaces on FKBP12 and FRB. Molecular modeling based on the binding site map revealed two distinct conformations of complex-bound photo-rapamycin, providing a 5.0 Å distance constraint between the conjugated residues and the diazirine carbon and a 9.0 Å labeling radius for the diazirine upon photo-activation. These measurements may be broadly useful in the interpretation of binding site measurements from PAL. Thus, in characterizing the ternary complex of photo-rapamycin by MS, we applied binding site hotspot mapping to a macrocyclic natural product and extracted precise structural measurements for interpretation of PAL products that may enable the discovery of new binding sites in the “undruggable” proteome.

Photo-affinity labeling (PAL) is a widely used method for capturing the biomolecular targets of non-covalent

Figure 1. Overview of the application of PAL to the rapamycin ternary complex. A) The structures of rapamycin, photo-rapamycin, and the minimalist tag. B) Workflow used to obtain conjugated peptides from the FKBP12– photo-rapamycin–FRB ternary complex.

ligands. In a PAL experiment, a photo-activatable group embedded in a chemical probe is activated by light to generate a reactive intermediate that can form a stable covalent bond to the interacting proteins.1–3 The combination of PAL with chemical enrichment and de-



tection by mass spectrometry (MS) enables target identification on the proteome-wide scale and structural elucidation of the binding site if the small moleculeconjugated peptide is isolated. Although mapping of the binding site itself has increasingly been used to localize small molecule ligands on their protein targets,4–7 a systematic definition of the distances represented by PAL measurements is needed to improve structural interpretation of the data. Furthermore, extension of binding site mapping to more complex macrocyclic natural products, many of which possess biological activity through unique modes of binding to proteins,8 would enable investigation into the binding preferences of this privileged class of ligands, including macrocycles that have previously been developed as PAL probes for target identification.9,10 Inspired by parallel advances in crosslinking MS that have yielded structural insights from crosslinked peptides,11,12 we sought to enable similar insights from binding site maps of complex macrocyclic natural products and simultaneously derive the structural distances reported by PAL. Herein, we evaluate the structure of the FKBP12–rapamycin–FRB ternary complex using diazirine-based PAL chemistry to characterize macrocycle–peptide conjugates by MS and develop a model to determine a PAL radius based on the binding site map. Rapamycin was one of the earliest examples of a “molecular glue,” exerting its biological effects through the stabilization of a protein–protein interaction between FKBP12 and the FRB domain of mTOR.13–15 The ternary complex has since been characterized by crystallography and biophysical methods.16,17 To understand how PAL measurements can encode structural information and extend binding site mapping methods to macrocyclic natural products, we designed a PAL probe for rapamycin termed photo-rapamycin (Figure 1, Scheme S1). Photo-rapamycin was functionalized with a diazirine-based “minimalist tag”18 at the C40 position, a position amenable to derivatization in prior studies (Figure 1A).19 The diazirine is a minimally-perturbative photo-activatable group that is readily embedded in molecules through functionalized tags.18,20 To gain insight into how photo-rapamycin-conjugated peptides may fragment by MS, an aliquot of photo-rapamycin was irradiated in methanol to mimic the diazirine insertion event. Fragmentation of the methanolized photorapamycin by qTOF MS yielded species resulting from neutral losses and fragmentations in the macrocyclic backbone, such as cleavage of the lactone, fragmentation across the diketone, and a McLafferty rearrangement (Figure S1, Table S1).21 The observed fragmentation was in alignment with previous studies of everolimus, a rapamycin derivative.22 The formation of the ternary complex from recombinant FKBP12 and FRB (Figure S2) was evaluated by

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Figure 2. Validation of photo-rapamycin as a probe for rapamycin. A. SPR measurement of the binding kinetics of FKBP12–rapamycin and FKBP12–photo-rapamycin with FRB. B. Ternary complex formation with rapamycin and photo-rapamycin in different buffer conditions, monitored by SEC. C. Representative in-gel fluorescence of 10 µM FKBP, 10 µM FRB in PBS + 0.1% Triton + 2.5% EtOH with the indicated treatments. Samples were photoirradiated and the fluorophore was attached using CuAAC. D. Quantification of fluorescent signal across three replicates with standard errors indicated.


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surface plasmon resonance (SPR). The binding of FRB to immobilized GST-FKBP12 displayed similar binding kinetics in the presence of rapamycin or photorapamycin (Figures 2A, S3) that are comparable to previously reported binding kinetics for rapamycin (Kd = 12 ± 0.8 nM).17 Despite similar binding constants for ternary complex formation observed by SPR, differences in solubility at µM concentrations were observed (Figure S4). We therefore optimized ternary complex formation by size exclusion chromatography (SEC). In the presence of rapamycin, ternary complex formation occurred quantitatively with each component at 10 μM in PBS, 0.2% EtOH, as compared to 12% complex formation with photo-rapamycin (by relative peak height, Figure 2B). Upon buffer optimization, the FKBP12–photorapamycin–FRB ternary complex was formed at 51%. The ternary complex was formed quantitatively upon increasing the photo-rapamycin concentration (3 equiv).

The FKBP12–photo-rapamycin–FRB complex was photo-irradiated and successful conjugation of photorapamycin to FKBP12 or FRB was visualized by in-gel fluorescence, using a fluorophore attached via coppercatalyzed azide-alkyne cycloaddition (CuAAC). In-gel fluorescence showed photo-rapamycin-dependent labeling of both FKBP12 and FRB that was competitively displaced by rapamycin (Figures 2C, 2D, S5). The photo-rapamycin ternary complex was then prepared for structural characterization by MS (Figure 1B). As we expected the internal fragmentation pathways of rapamycin to add analytical complexity, an isotopicallycoded handle6 was installed by CuAAC to improve confidence in the assignment of modified peptides. Isotopic recoding produces recognizable markers in the full scan mass spectrum (MS1) that improve detection and validation of chemically-




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modified species.23 Following installation of the isotopically-coded handle, the ternary complex was digested with trypsin using a S-Trap.24,25 Digested samples were analyzed on an Orbitrap Fusion Tribrid by higher-energy collision induced dissociation (HCD) fragmentation. Photo-rapamycin-conjugated peptides were identifiable based on the embedded isotopic code in the MS1 spectra. Clusters of isotopically-coded peaks were readily observed with mass differences corresponding to differences in oxidation state which were likewise observed with rapamycin and photo-rapamycin when analyzed directly (Figure S6). Investigation of the tandem MS (MS2) spectra derived from isotopically-coded precursors revealed that the conjugated peptides did not carry intact photo-rapamycin as a modification but were instead modified by photo-rapamycin fragments that formed prior to, or during, the MS1 analysis. Evaluation of these spectra revealed that the major modification masses on conjugated peptides resulted from the same fragmentations observed with methanolized photorapamycin, particularly cleavage of the lactone and a McLafferty rearrangement,21 and variations corresponding to addition and elimination of water or methanol yielding several rearrangement products (RPs) as modifications (Figure 3A, S7). Database searching by SEQUEST HT was therefore performed by (1) searching the HCD MS2 spectra against a database of unmodified peptides resulting from the semi-tryptic digest of FKBP12, FRB, and common contaminant proteins, then (2) searching unassigned spectra against a database of peptides resulting from a semi-tryptic digest of FKBP12 and FRB with RPs as variable modification on any amino acid. MS2 spectra assigned to photo-rapamycinconjugated peptides were validated by manual examination for isotopic coding of the MS1 precursor. RP-conjugated peptides were identified on both FKBP12 and FRB (Figures 3B, 3C). In a representative experiment, a total of 26 RP-modified peptide spectral matches (PSMs) were identified (Tables S2, S3). By comparison, treatment with the minimalist tag (10 equiv) yielded a broader and largely orthogonal array of labeled regions at a lower overall labeling frequency (Figure S5, S8, Table S4, S5). RP conjugation was exclusively observed on residues 75–110 of FKBP12 and residues 10– 22 of FRB (Figures 4A, 4B). The MS2 spectra displayed fragments of the underlying peptide, but fragment ions with conjugated RPs were not observed, potentially due to the large modified peptide mass, absence of charge localized to the modification, or neutral loss of the modification from the peptide backbone analogous to a labile post-translational modification.26 Sequenced fragment ladders therefore provided poor localization of RP modifications. Additional parent and peptide ions resulting from MS-cleavage of the modification from the peptide were also assigned in some spectra. Therefore, initial prediction of the conjugated residues was performed based on distance and geometry from the rapamycin

C40-OH and emerging evidence for the amino acid selectivity for alkyl diazirines.16,27,28 The primary conjugation sites were predicted to be FKBP12 D79 and FRB E18, which are, respectively, 10.5 Å and 10.8 Å from the rapamycin C40-OH (Figure 4A). Mutation of these residues to alanine resulted in a decrease in labeling (60% for FKBP12, 42% for FRB, Figure 4B, S9–11), demonstrating that conjugation at these residues contributes significantly to the species observed by MS. The additional labeling on FRB E18A may arise from conjugation to the adjacent E19, which is 12.1 Å from the rapamycin C40-OH but was not amenable to point mutation. A model of the photo-rapamycin complex, based on guided molecular dynamics (MD) simulations starting from the crystal structure of the FKBP12–rapamycin– FRB ternary complex,16 yielded possible conformations of photo-rapamycin that would lead to labeling at each of the observed residues with minimal perturbation of the overall ternary complex structure (Figures 4C, 4D). Importantly, these data predict that rapamycin is conformationally flexible about the cyclohexyl ring resulting in at least two distinct conformations in solution. In the modeled structures, the diazirine carbon is 4.5 Å (FKBP12) and 5.0 Å (FRB) from the labeled residue. A 5.0 Å distance can thus be used as a restraint in developing docked models of novel small molecule–protein interactions formed by PAL. To improve prediction of surfaces that are labeled by PAL, we also performed a 1 ns unguided MD simulation that showed the minimalist extension of photo-rapamycin exhibited conformations in which the diazirine carbon was within 9 Å of each of the labeled residues (Figure 4E). However, conjugation of a region on FKBP12 (residues 51–60, Figure 4A) that is within 10 Å of the rapamycin C40-OH was not observed. The presented binding site map of FKBP–photorapamycin–FRB therefore reflects the conformational preferences of the functionalized photo-rapamycin as well as potential underlying preferences in ionization, fragmentation, and residue selectivity. In conclusion, analysis of the FKBP12–rapamycin– FRB ternary complex by PAL and MS yielded, to our knowledge, the first binding site hotspot map of a complex macrocyclic natural product. Data obtained with photo-rapamycin provides a 5.0 Å minimum distance restraint and a 9.0 Å predicted labeling radius for interpretation of binding site maps from PAL. The labeling of specific regions of FKBP12 and FRB reflects two conformations that were modeled by MD, illustrating the potential of PAL to profile dynamic interactions. The distance restraints from these results will inform structural analysis of binding sites derived from PAL coupled with MS, both in vitro and in complex cellular environments. This extension of PAL may further expand the profiling of binding sites on protein surfaces beyond canonical liganded space and yield insights into the “undruggable” proteome.



ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website. Supplemental data and procedures (PDF) Tables S2-S5 summarizing MS data (Excel) The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD014319. (Username: [email protected], Password: HoCfKcHb).

AUTHOR INFORMATION Corresponding Author

*[email protected] Funding Sources

Support from the Burroughs Wellcome Fund (C.M.W.), Ono Pharma Foundation (C.M.W.), Sloan Research Foundation (C.M.W.), National Science Foundation (H.A.F.), and Harvard University are gratefully acknowledged.

ACKNOWLEDGMENT We thank Bogdan Budnik and the Harvard University Mass Spectrometry and Proteomics Resource Laboratory for proteomics support and Paul Boudreau and the Balskus Lab for assistance with small molecule MS.

REFERENCES (1) Das, J. Aliphatic Diazirines as Photoaffinity Probes for Proteins: Recent Developments. Chem. Rev. 2011, 111 (8), 4405– 4417. (2) Sumranjit, J.; Chung, S. Recent Advances in Target Characterization and Identification by Photoaffinity Probes. Molecules 2013, 18 (9), 10425–10451. (3) Flaxman, H. A.; Woo, C. M. Mapping the Small Molecule Interactome by Mass Spectrometry. Biochemistry 2018, 57 (2), 186– 193. (4) Niphakis, M. J.; Lum, K. M.; Cognetta III, A. B.; Correia, B. E.; Ichu, T.-A.; Olucha, J.; Brown, S. J.; Kundu, S.; Piscitelli, F.; Rosen, H.; Cravatt, B. F., A Global Map of Lipid-Binding Proteins and Their Ligandability in Cells. Cell 2015, 161 (7), 1668–1680. (5) Parker, C. G.; Galmozzi, A.; Wang, Y.; Correia, B. E.; Sasaki, K.; Joslyn, C. M.; Kim, A. S.; Cavallaro, C. L.; Lawrence, R. M.; Johnson, S. R.; Narvaiza, I.; Saez, E.; Cravatt, B. F., Ligand and Target Discovery by Fragment-Based Screening in Human Cells. Cell 2017, 168 (3), 527–541.e29. (6) Gao, J.; Mfuh, A.; Amako, Y.; Woo, C. M. Small Molecule Interactome Mapping by Photoaffinity Labeling Reveals Binding Site Hotspots for the NSAIDs. J. Am. Chem. Soc. 2018, 140 (12), 4259– 4268. (7) Gertsik, N.; am Ende, C. W.; Geoghegan, K. F.; Nguyen, C.; Mukherjee, P.; Mente, S.; Seneviratne, U.; Johnson, D. S.; Li, Y.M. Mapping the Binding Site of BMS-708163 on γ-Secretase with Cleavable Photoprobes. Cell Chem. Biol. 2017, 24 (1), 3–8. (8) Villar, E. A.; Beglov, D.; Chennamadhavuni, S.; Porco Jr, J. A.; Kozakov, D.; Vajda, S.; Whitty, A. How Proteins Bind Macrocycles. Nat. Chem. Biol. 2014, 10 (9), 723–731. (9) MacKinnon, A. L.; Garrison, J. L.; Hegde, R. S.; Taunton, J. Photo-Leucine Incorporation Reveals the Target of a Cyclodepsipeptide Inhibitor of Cotranslational Translocation. J. Am. Chem. Soc. 2007, 129 (47), 14560–14561.

Page 20 of 22

(10) MacKinnon, A. L.; Taunton, J. Target Identification by Diazirine Photo-Cross-linking and Click Chemistry. Curr. Protoc. Chem. Biol. 2009, 1, 55. (11) Yu, C.; Huang, L. Cross-Linking Mass Spectrometry: An Emerging Technology for Interactomics and Structural Biology. Anal. Chem. 2018, 90 (1), 144–165. (12) Sinz, A. Cross-Linking/Mass Spectrometry for Studying Protein Structures and Protein–Protein Interactions: Where Are We Now and Where Should We Go from Here?. Angew. Chem., Int. Ed. Engl. 2018, 27, 966. (13) Brown, E. J.; Albers, M. W.; Shin, T. B.; Ichikawa, K.; Keith, C. T.; Lane, W. S.; Schreiber, S. L. A Mammalian Protein Targeted by G1-Arresting Rapamycin-Receptor Complex. Nature 1994, 369 (6483), 756–758. (14) Sabatini, D. M.; Erdjument-Bromage, H.; Lui, M.; Tempst, P.; Snyder, S. H. RAFT1: A Mammalian Protein That Binds to FKBP12 in a Rapamycin-Dependent Fashion and Is Homologous to Yeast TORs. Cell 1994, 78 (1), 35–43. (15) For a review of small molecules as a “molecular glue” see: (15a) Fischer, E. S.; Park, E.; Eck, M. J.; Thomä, N. H. SPLINTS: Small-Molecule Protein Ligand Interface Stabilizers. Curr. Opin. Struct. Biol. 2016, 37, 115–122; (15b) Schreiber, S. A Chemical Biology View of Bioactive Small Molecules and a Binder-Based Approach to Connect Biology to Precision Medicines. 2018, 1–10; (15c) Stanton, B. Z.; Chory, E. J.; Crabtree, G. R. Chemically Induced Proximity in Biology and Medicine. Science. 2018, 359 (6380). eaao5902. (16) Liang, J.; Choi, J.; Clardy, J. Refined Structure of the FKBP12-Rapamycin-FRB Ternary Complex at 2.2 Å Resolution. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999, 55 (4), 736–744. (17) Banaszynski, L. A.; Liu, C. W.; Wandless, T. J. Characterization of the FKBP·Rapamycin·FRB Ternary Complex. J. Am. Chem. Soc. 2005, 127 (13), 4715–4721. (18) Li, Z.; Hao, P.; Li, L.; Tan, C. Y. J.; Cheng, X.; Chen, G. Y. J.; Sze, S. K.; Shen, H.-M.; Yao, S. Q. Design and Synthesis of Minimalist Terminal Alkyne-Containing Diazirine Photo-Crosslinkers and Their Incorporation into Kinase Inhibitors for Cell- and TissueBased Proteome Profiling. Angew. Chemie Int. Ed. 2013, 52 (33), 8551–8556. (19) Sedrani, R.; Cottens, S.; Kallen, J.; Schuler, W. Chemical Modification of Rapamycin: The Discovery of SDZ RAD. Transplant. Proc. 1998, 30 (5), 2192–2194. (20) Chang, C.-F.; Mfuh, A.; Gao, J.; Wu, H.-Y.; Woo, C. M. Synthesis of an Electronically-Tuned Minimally Interfering Alkynyl Photo-Affinity Label to Measure Small Molecule–Protein Interactions. Tetrahedron 2018, 74 (26), 3273. (21) McLafferty, F. W. Mass Spectrometric Analysis: Molecular Rearrangements. Anal. Chem. 1959, 31 (1), 82–87. (22) Boernsen, K. O.; Egge Jacobsen, W.; Inverardi, B.; Strom, T.; Streit, F.; Schiebel, H. M.; Benet, L. Z.; Christians, U. Assessment and Validation of the MS/MS Fragmentation Patterns of the Macrolide Immunosuppressant Everolimus. J. Mass Spectrom. 2007, 42 (6), 793–802. (23) Woo, C. M.; Felix, A.; Byrd, W. E.; Zuegel, D. K.; Ishihara, M.; Azadi, P.; Iavarone, A. T.; Pitteri, S. J.; Bertozzi, C. R. Development of IsoTaG, a Chemical Glycoproteomics Technique for Profiling Intact N- and O-Glycopeptides from Whole Cell Proteomes. J. Proteome Res. 2017, 16 (4), 1706–1718. (24) Ludwig, K. R.; Schroll, M. M.; Hummon, A. B. Comparison of In-Solution, FASP, and S-Trap Based Digestion Methods for Bottom-Up Proteomic Studies. J. Proteome Res. 2018, 17 (7), 2480– 2490. (25) HaileMariam, M.; Eguez, R. V.; Singh, H.; Bekele, S.; Ameni, G.; Pieper, R.; Yu, Y. S-Trap, an Ultrafast SamplePreparation Approach for Shotgun Proteomics. J. Proteome Res. 2018, 17 (9), 2917–2924. (26) Woo, C. M.; Iavarone, A. T.; Spiciarich, D. R.; Palaniappan, K. K.; Bertozzi, C. R. Isotope-Targeted Glycoproteomics (IsoTaG): A Mass-Independent Platform for Intact N- and OGlycopeptide Discovery and Analysis. Nat. Methods 2015, 12 (6), 561–567.


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(27) Iacobucci, C.; Goetze, M.; Piotrowski, C.; Arlt, C.; Rehkamp, A.; Ihling, C. H.; Hage, C.; Sinz, A. Carboxyl-Photo-Reactive MS-Cleavable Cross-Linkers: Unveiling a Hidden Aspect of Diazirine-Based Reagents. Anal. Chem. 2018, acs.analchem.7b04915. (28) West, A.; Woo, C.M. Unpublished results.



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A Binding Site Hotspot Map of the FKBP12 ... - ACS Publications

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