Proximity- and Affinity- Based Labeling Methods for Interactome Mapping

Proximity- and affinity- based labeling methods for interactome mapping

Beryl X. Li March 13th, 2019 Proximity- and affinity-based labeling methods significance

Human protein-protein interactome Classical methods contains a quater million interactions yeast two-hybrid assay between 22,000 proteins host incompatibility low through-put

affinity-complex purification non-physiological conditions limited to high affinity interactions

ribonuclease A and inhibitor (1DFJ) Proximity labeling

spatial resolution temporal resolution native enviroment

Protein-protein and protein ligand interactions elucidate biological processes and facilitate drug-target identification

For Y2H see: Juliana, F.; Vieira, S.J.; Margarida, F. J. Proteomics 2018, 171, 127-40. For ACP see: Puig, O. et al. Methods 2001, 24 (3), 218-29. Proximity- and affinity-based labeling methods general mechanism

proximity-based affinity-based labeling labeling X bait (protein or small catalyst molecule ligand) isolate prey among a mixture of proteins

prey (target protein)

covalent linkage X

catalyst tags near catalyst catalyst are activated Proximity- and affinity-based labeling methods outline

Prevalent enzymatic methods

BioID: (Biotin IDentification) – Split BioID X – BioID2, BASU, TurboID

APEX (Enhanced Ascorbate PeroXidase) isolate prey among a – temporal resolution mixture of proteins

Trinkle-Mulcahy, L. F1000 Research 2019, 8, 135. Kim, D.I.; Roux, K.J. Trends Cell Biol. 2016, 26 (11), 804–7.

Affinity-guided catalysts prey Ligand-tethered DMAP

MoAL method (MOdular Affinity Labeling) X Local SET catalysis

Survey of photo-affinity labeling agents tags

Chen, Y.; Topp, E.M. J. Pharm. Sci. 2019, 108 (2), 791–7. bait Murale, D.P.; Hong, S.C.; Haque, M.; Lee, J.-S. Proteome Science 2017, 15:14. catalyst Chen, Y.; Hu, C. Tetrahedron Lett. 2015, 56, 884–8. Proximity- and affinity-based labeling methods Biotin IDentification (BioID)

HN NH BirA* tags

HO S O NH2

Lamin-A (bait) ATP O NH biotinoyl AMP intermediate filament 2 N (t1/2 ~1 min) PPi HN NH protein on nuclear lamina N prey O N N O P O S O O O OH

AMP hydrolysis O (diffusion radius 10–50 nm) HN NH BirA* H N Biotin ligase biotinylation occurs in proximity to S R118G mutant to prematurely BirA* activity, which leads to selectivity O release biotinoyl AMP

Hamachi, I. et al. J. Am. Chem. Soc. 2008, 130, 245–51. Proximity- and affinity-based labeling methods Biotin IDentification (BioID)

BirA*

H N

Lamin-A (bait) intermediate filament protein on nuclear lamina biotinylated prey

BirA* is 35 kDa, might impact native binding long incubation times lack temporal resolution cells can express bait–ligase conjugate in vivo identify SLAP75, novel nuclear envelope constituent

no predicted transmembrane domain no clues in sequence motif Hamachi, I. et al. J. Am. Chem. Soc. 2008, 130, 245–51. Proximity- and affinity-based labeling methods BioID to map inhibitory postsynaptic density proteome

biotin HN NH BirA*

HO S O

inhibitory synapse dampen neuronal activity by postsynaptic hyperpolarization genetic perturbations strongly implicated in developmental brain disorders molecular basis to synapse regulation is poorly understood

Soderling, S.H. et al. Science 2016, 353, 1123–8. Proximity- and affinity-based labeling methods variations of BioID

Split-BioID BioID2

Ago protein plays at least two roles in gene-silencing biotin ligase from Aquifex aeolicus which 1) represses mRNA in miRISC complex naturally lacks N-terminus 2) load miRNA in RISC-loading complex 27 kDa (vs. original 35 kDa) how to assign novel identified proteins to a specific step? Roux, K.J. et al. Mol. Biol. Cell 2016, 27 (8),1188–96.

protein fragment complementation assay BASU

biotin ligase from Bacillus subtilis removing N-terminus did not impact activity; 28 kDa

Khavari, P.A. et al. Nat. Methods 2018, 15 (3),207–212.

Ago TurboID and MiniTurbo TNRC6C Ago–TNRC6C (only in miRISC) 14 and mutations from original BirA* MiniTurbo is 28 kDa identified novel regulator GIGYF2 with split BioID original efficient labeling in minutes instead of hours system, which previously AP/MS did not show ideal combination of temporal resolution and non-toxicity association with Ago or TNRC6C

Bethune, J. Nat. Commun. 2017, 8, 15690. Ting, A.Y. et al. Nat. Biotechnol. 2018, 36 (9), 880–7. Proximity- and affinity-based labeling methods APEX (Ascorbic peroxidase)

biotin phenol tags HN NH APEX

H N S OH O HO

bait O H O electron-rich amino acids 2 2 prey (tyr, trp, cys, his) HN NH

H N S Ascorbic peroxidase (27 kDa) O oxidizes biotin phenol in O presence of H O 2 2 biotin phenoxy radical APEX (t1/2 <1 min)

biotin phenol tags HN NH OH ~20 nm diffusion radius H N H2O2 may be harsh for labeling conditions S great temporal resolution, labeling in 1 min O HO

Ting, A.Y. Nat. Biotechnol. 2012, 30 (11) 1143–8. Proximity- and affinity-based labeling methods APEX resolves GPCR networks in vivo

G-protein-coupled receptors (GPCRs) mediates physiological responses to many stimuli (e.g., hormones, neurotransmitters, light, etc.)

stimulus

1 min 5 min 10 min, etc.

How to track the series of cascading protein-protein interactions following agonist binding?

Krogan, N.J. et al. Cell 2017, 169, 350–60. Proximity- and affinity-based labeling methods APEX resolves GPCR networks in vivo

G-protein-coupled receptors (GPCRs) mediates physiological responses to many stimuli (e.g., hormones, neurotransmitters, light, etc.)

stimulus

1 min 5 min 10 min, etc.

How to track the series of cascading protein-protein interactions following agonist binding?

Krogan, N.J. et al. Cell 2017, 169, 350–60. Proximity- and affinity-based labeling methods acylation of lectins by ligand-tethered DMAP catalysts

O lactose (ligand) S OH OH OH O O O HO O O Me HO HO C OH N N 2 OH H

N HO O O

DMAP fluorescein (acyl donor) (catalyst)

sugars-binding proteins (e.g., lectins) play important roles in glycobiology sugar-lectin interactions not sufficiently strong for affinity purification

Congerin II (animal lectin with high lactose affinity) use DMAP as acyl-transfer catalyst to covalently tag sugar-binding proteins

Hamachi, I. et al. J. Am. Chem. Soc. 2008, 130, 245–51. Proximity- and affinity-based labeling methods acylation of lectins by ligand-tethered DMAP catalysts

S O

N N N Me Me Me Nu N Nu N Nu N

N O N O N O H H H

35% yield in 3 h

lactose (ligand) fluorescein (acyl donor)

Hamachi, I. et al. J. Am. Chem. Soc. 2008, 130, 245–51. Proximity- and affinity-based labeling methods acylation of lectins by ligand-tethered DMAP catalysts

S O Tyr 51 N N Me Me Nu N Nu N

N O N O H H

35% yield in 3 h single product minimum thioester hydrolysis in presence of Congerin II reaction inhibited by excess ligand

lactose (ligand) fluorescein (acyl donor)

Hamachi, I. et al. J. Am. Chem. Soc. 2008, 130, 245–51. Proximity- and affinity-based labeling methods “multivalent” DMAP catalysts for live-cell imaging

fluoroscein DIC

bradykinin B2 (ligand) Arg Arg Pro Hyp Gly Phe Ser Phe Leu Arg HN + catalyst O O

NH N Me O

HN control N O NH O Me N Me N

N N

bradykinin B2 receptor

selectively label bradykinin B2 receptor on cell surface development of fluorescent biosensor to screen potential antagonist ligand binding

Hamachi, I. et al. J. Am. Chem. Soc. 2011, 133, 12220–28. Proximity- and affinity-based labeling methods modular affinity labeling based on catalytic amidation

covalent linkage prey X (target protein)

activatable tag with reactive module catalyst

bait (protein or small catalyst possibly synthetically challenging molecule ligand) ligand activity may alter due to modificaiton Proximity- and affinity-based labeling methods modular affinity labeling based on catalytic amidation

covalent linkage prey X (target protein) reactive module

activatable tag catalyst

bait (protein or small catalyst possibly synthetically challenging molecule ligand) ligand activity may alter due to modificaiton

modular approach, where ligand, tags, and reactive module are completely separated

Nomoto, H. et al. Chem. Commun. 2009, 5597–9. Proximity- and affinity-based labeling methods modular affinity labeling based on catalytic amidation

MeO N OMe O N N HN NH O O O O NMe2 NMe2 H N O Me N S 2 O O Me CDMT ligand (biotin) catalyst O

NaO3S O NH2 N H MeO N OMe

N N HN NH2 MeO N OMe Cl N N reactive module (CDMT) fluorescent tag “Cascade Blue” HN O O O NMe2 NMe2

HN NH2 169% yield after 8 h (four binding sites/avidin) negative control gave 11% yield

Nomoto, H. et al. Chem. Commun. 2009, 5597–9. Proximity- and affinity-based labeling methods photoredox proximity-based labeling

H Ru H Ru N N visible light O O

local SET

Carbonic anhydrase O reactive module Me OH (tyrosyl trapping agent) N Me Me N O NH H N N O N O N O Ru 4 H O N N H N N H N 3 O H S NH O S tag (biotin) O H2N O N H H

ligand (sulfonamide) Ru catalyst

Sato, S.; Nakamura, H. Angew. Chem. Int. Ed. 2013, 52, 8681–4. For review, see: Chen, Y.; Hu, C. Tetrahedron Lett. 2015, 56, 884–8. Proximity- and affinity-based labeling methods photoredox proximity-based labeling

– – – + + + Ru – + + + + + H Ru N – – 15 – 15 15 irradiation time (min)

– – – – – + O 1 2 3 4 5 6 1 2 3 4 5 6

reactive module Me OH (tyrosyl trapping agent) N Me

O NH H O N O N O 4 H

H streptavidin-HRP Coomassie brilliant blue S NH tag (biotin) N O H H selectively label carbonic anhydrase in mouse erythrocyte lysate

reactive under specific activation conditions identification of membrane protein targets

(potentially) mild conditions elucidation of protein structure in solution

covalently modify targets for easy purification characterization of proteins in pharmaceutical solids

Nitrenes

N3 N Carbenes hν N N hν 260 nm CF3 CF3 330-370 nm insertion into C–H, N–H, or O–H bonds ring-expansion with nucleophilic addition N preference for cysteine and aromatic residues N

Benzophenone CF3 O O

hν stable diazo isomer 365 nm insertion into C–C and X–H bonds (X = C, O, N, S) triplet ketyl biradical addition to C=C bonds high affinity towards methionine preference for cysteine and aromatic residues relatively long wavelength activation: less damaging to proteins

Murale, D.P.; Hong, S.C.; Haque, M.; Lee, J.-S. Proteome Science 2017, 15:14. Chen, Y.; Topp, E.M. J. Pharm. Sci. 2019, 108 (2), 791–7.