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Translocation of an Intracellular Protein via Peptide-Directed Ligation Christiane Stiller, Dennis M Krüger, Nicolas Brauckhoff, Marcel Schmidt, Petra Janning, Hazem Salamon, and Tom N. Grossmann ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b01013 • Publication Date (Web): 21 Dec 2016 Downloaded from http://pubs.acs.org on December 24, 2016

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ACS Chemical Biology

Translocation of an Intracellular Protein via Peptide-Directed Ligation Christiane Stiller1,2, Dennis M. Krüger1,2, Nicolas Brauckhoff1, Marcel Schmidt1, Petra Janning3, Hazem Salamon1, Tom N. Grossmann1-4,*

1

Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Str. 15, 44227 Dortmund, Germany.

2

Technical University Dortmund, Department of Chemistry and Chemical Biology, Otto-Hahn-Str. 6,

44227 Dortmund, Germany. 3

Max Planck Institute of Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany.

4

VU University Amsterdam, Department of Chemistry & Pharmaceutical Sciences, De Boelelaan 1108,

1081 HZ, Amsterdam (The Netherlands).

* Correspondence to Dr. Tom N. Grossmann, Professor of Organic Chemistry, VU University Amsterdam, Department of Chemistry & Pharmaceutical Sciences, De Boelelaan 1108, 1081 HZ, Amsterdam (The Netherlands), Email: [email protected]

Abstract Ligand-directed reactions allow chemical transformations at very low reactant concentrations and can thus provide access to efficient approaches for the post-translational modification of proteins. The development of these proximity-induced reactions is hampered by the number of appropriate ligands and the lack of design principles. Addressing these limitations, we report a proximity-induced labeling system which applies a moderate affinity peptide ligand. The design process was structure-guided and supported by molecular dynamics simulations. We show that selective protein labeling can be performed inside living cells enabling the subcellular translocation of a protein via ligand-directed chemistry for the first time.

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Introduction Approaches that allow the chemical modification of intracellular proteins can promote the understanding of crucial biological processes and they can provide access to novel therapeutic agents.1–4 Most current labeling strategies require the genetic modification of target cells which can interfere with cellular functions. Such approaches involve the fusion of target proteins with fluorescent proteins,5 self-labeling tags,6 short peptide tags or the incorporation of non-natural amino acids,7,8 of which the latter techniques often utilize subsequent bioorthogonal reactions.7,9 An approach that in principle would allow the intracellular modification of unmodified proteins involves proximity-induced labeling reactions.10–14 This technique requires a ligand that selectively binds the protein target. The ligand bears a reactive group that triggers chemical reactions with amino acid side chains in proximity to the corresponding binding site. When designed appropriately, the desired proximity-induced reaction proceeds with significantly higher rates than undesired background reactions, which is a prerequisite for selective modifications.12–15 As reactive proteinogenic groups, nucleophilic amino acids have been targeted, e.g. cysteine, lysine and histidine.3 The lack of general design principles for efficient proximityinduced reactions and the identification of appropriate ligands represent the limiting factors for the development of labeling systems. So far, mainly small molecules have been used as ligands limiting this strategy to targets with corresponding binding pockets.3 In a cellular context, only a few examples of peptide-directed modifications have been reported all of them targeting extracellular domains.16–21 However, due to their enhanced surface recognition properties, the use of peptide ligands for intracellular proteins could vastly expand the scope of proximity-induced reactions.22,23 For many protein targets, short peptides tend to show low affinity,22 and it is unclear how that affects intracellular labeling reactions. Herein, we report the structure-based design of a proximity-induced labeling system (Figure 1a) employing a peptide ligand. Supported by molecular dynamics (MD) simulations, we gain insight into the structural factors that influence reactivity. Though the peptide ligand exhibits only moderate affinity, we observe very high proximity-induced acceleration of the reaction rate. Compared to the non-covalent system, reactive protein/ligand-pairs show dramatically improved efficiency in cell lysate and in live cells. We demonstrate that selectivity and robustness of the labeling system enable the translocation of an intracellular target protein from the cytosol to membranes.

Results and discussion Protein/peptide complex and probe design We aimed for the use of a biocompatible reaction allowing the covalent modification of natural amino acids. Due to the unique reactivity of thiols, cysteine (C) was chosen as target residue.24–26 Previous studies identified α-chloroacetamide as suitable electrophile for conjugation reactions with cysteine.27,28


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To test the feasibility of both reaction partners for peptide-directed modifications in complex biological samples, we selected a structurally well-characterized protein/ligand-pair (Figure 1b): The globular protein domain KIX from human CBP (CREB binding protein) served as target protein (P, white) while the transactivation domain of MLL (mixed lineage leukemia) constitutes the peptide ligand (L, grey).29 Peptide L shows only moderate affinity for protein P rendering this protein/ligand-pair a representative system for peptide-directed modifications. Importantly, the wild-type sequence of protein P does not contain cysteines which allows the construction of single cysteine-containing variants of P. Focusing on one α-helix of P in proximity to the L binding site, we selected six amino acids for the replacement by cysteine with distances to the N-terminus of L ranging from 12 Å to 22 Å (Figure 1b).

Figure 1. (a) Scheme of proximity-induced reactions. (b) NMR-structure (PDB 2LXS)29 of protein P (white) with ligand L (grey). A cysteine (orange) was introduced at six different sites, respectively. Amino acid position and mean distances (l) between thiol 30

and N-terminus of L are given (l was derived from P/L NMR-structure (PDB 2LXS) with the software Pymol).

Protein variants were obtained by heterologous expression from Escherichia coli. The dissociation constants (Kd) of all P variants with a fluorescently labeled 17-mer peptide ligand L (Figure 1b) were determined in fluorescence polarization assays revealing moderate affinities for all complexes (Kd = 0.2‒ 2.4 µM, Figure S9). Based on L, reactive probes were designed that contain a C-terminal tag and an N-terminal spacer which is capped with α-chloroacetamide (Figure 2a). To investigate the influence of

the linker length on ligation reactions, five different spacers (Figure 2b, n = number of main chain atoms) were selected involving lengths from 6.5 Å (n = 4) to 37 Å (n = 38). This provides spacer lengths that cover the range of expected distances between thiols and the N-terminus of the bound peptide (l = 12‒22 Å, Figure 2b). 3 ACS Paragon Plus Environment


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Figure 2. (a) General structure of reactive probes (Cl-nL-tag). The methionine residue is replaced by norleucine (B). (b) Spacer structures are given including distances between the N-terminus of L and the α-carbon of the attached α-chloroacetamide (calculated with Chem3D31). (c) Left: Heat map representation of reaction rates between protein variants and peptides (with varying linker length, Cl-nL-f). Initial reaction rates (v) were determined via analytical HPLC (for details see Figure S11). Right: Heat map representation of the probability of occurrence (P) of the reacting atoms (cysteinic sulfur and α-carbon of 32

α-chloroacetamide) within a distance of 5 Å. Data obtained from 500 ns MD simulations using AMBER14

(for details see

Supporting Methods).

Proximity-induced reactions and MD simulations

To evaluate reaction rates of various P/L combinations, we added a C-terminal fluorescein tag (f) to L allowing sensitive HPLC-based readout of product formation. P variants were incubated with electrophile-modified

ligands

(Cl-nL-f)

at

concentrations

that

ensure

sufficient

binding

(c(Cl-nL-f) =10 µM, c(P) = 20 µM). Product formation after 1 h and 6 h was determined (Table S7) and initial reaction rates (v) were calculated revealing a high dependency on cysteine position and spacer length (Figure 2c, left heat map). Cysteine positions 626, 630, 642 and 648 show low reactivity while cysteines at position 634 or 638 exhibit high reaction rates. Highest reactivity is observed for P(C638) with ligands bearing medium sized spacers (n = 9, 13 and 19). In these cases, quantitative yields were observed after 1 h (v > 2 nM·s-1). Apparently, the distance between thiol and N-terminus of the ligand is not the only crucial parameter (e.g. Cl-13L-f (l = 17 Å) shows high v for P(C634) (l = 13 Å), but very low v for P(C642) (l = 15 Å)). Also, reaction rates do not correlate with the dissociation constants of the corresponding L/P complexes (Figure S9). For that reason, we hypothesized that accessibility of the 4 ACS Paragon Plus Environment

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cysteine is the crucial factor. To address this question and to provide an additional rational for the future design of probes, the use of accelerated MD simulations was considered. Given the limitations associated with MD simulations of such large biological systems, we decided for the following simplifications: A pre-formed peptide/protein complex was used as starting point not considering the affinity of the binding partners or binding kinetics. In addition, the relative distance between both reacting atoms during the simulation was considered a measure for reactive encounters, not taking into account that the trajectory of the encounter also determines reactivity. For MD simulations, all 30 P/Lcomplexes including spacer and functional groups were investigated. These complexes were simulated allowing the spacer to sample its surrounding space while monitoring the distance between the two reacting atoms (cysteinic sulfur and α-carbon of α-chloroacetamide). Simulations (500 ns per P/Lcomplex) were executed in explicit water using AMBER14 as a basis to calculate the mean probability of the two reacting atoms to be present within a distance of 5 Å.33 Notably, these calculations (Figure 2c, right heat map) are in good agreement with the experimentally determined reaction rates (v, Figure 2c, left) showing highest probability for encounter in complexes that include P(C634) or P(C638). The largest discrepancy between both data sets is observed for P(C648) in combination with Cl-19L-f and Cl-38L-f. In these cases, MD simulations suggest very low reactivity while the experiment shows efficient product formation. This may be due to factors that are not considered by MD simulations (e.g. changed reactivity due to differences in the microenvironment). Overall, we observe a very good correlation (Pearson’s r = 0.75, Figure S12) which suggests that reactivity is mainly determined by the probability of encounter of reactive groups in the preformed complex and that MD simulations are a useful tool to support the design process. To challenge the good correlation between MD-based distance probabilities and ligation rates, we aimed for the testing of an additional electrophile. For this purpose, peptide 9L-f bearing the 9 atom spacer was equipped with dimethylamino acrylamide and reacted with all protein variants using gel electrophoreses in combination with a fluorescence imager as readout (Figure S13). Most notably, again highest reactivity was observed for proteins P(C634) and P(C638) which are the only two variants that show efficient protein labeling during the first two reaction hours. This is in line with the results obtained from MD simulations and with the reactivity of α-chloroacetamide-modified peptide bearing the same linker (Cl-9L-f) (Figure 2c).

Rate and selectivity of ligand-directed ligation

To determine accurate reaction rates for the most reactive P/L pairs (P(C638) with Cl-nL-f, n = 9, 13, 19), kinetic measurements were performed using HPLC as readout (Table S10, Figure S14). We observed similar initial rates for all three combinations (v = 7.6‒34 nM·s-1) with P(C638)+Cl-9L-f being the fastest pair (v = 34 nM·s-1). Due to its high reactivity, this P/L pair was selected for further investigations. HPLCMS experiments already indicated the formation of the expected covalent adduct (Figure S15). To verify



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the actual modification site, we performed HPLC-coupled high-resolution (HR) MS experiments. After incubation of P(C638) with or without Cl-9L-f, samples were subjected to tryptic digestion and analyzed. Cl-9L-f treatment resulted in the formation of a tryptic fragment with a monoisotopic mass of 1010.8448 ([M+3H]3+) which was absent in the untreated sample (Figure 3a). MS/MS sequencing of this peak (Figure S16) identified a fragment consisting of two peptides linked by a thioether via C638 (Figure 3a) which verifies this cysteine as modification site.

Figure 3 (a) High-resolution (HR) MS after tryptic digestion of ligation product (P(C638)+Cl-9L-f) with corresponding fragment. (b) Course of the reaction between Cl-9L-f and P(C638) as well as GSH, and between Cl-9iL-f and P(C638) (thiol concentrations are given, c(electrophile) = 10 µM, for details see Table S10, Figure S14). (c) Coomassie stained SDS-PAGE of pulldown of P(C638) and P(wt) with ligands H-9L-bt, Cl-9L-bt and Cl-9iL-bt (for details see Figure S17).

Next, we were interested in studying the performance of the selected pair (P(C638)+Cl-9L-f) in detail and to evaluate the usefulness of this system for biological applications. To assess the proximityinduced acceleration of the reaction, we compared reaction rates between Cl-9L-f and P(C638) 6 ACS Paragon Plus Environment

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(c = 20µM) with those between Cl-9L-f and glutathione (GSH, c = 20µM). In this setup, GSH probes the non-accelerated general reactivity of the electrophile. While the reaction with P(C638) was completed after less than 30 min, we did not observe any product formation with GSH after 3 h (Figure 3b). To determine the initial rate, this reaction was prolonged to 12 h allowing the detection of sufficient product. Using the initial rate with GSH as reference, relative initial rates (rel. v) were calculated (Figure 3b). Strikingly, the reaction of Cl-9L-f with P(C638) is more than 9000-fold faster than with 20 µM GSH. Aiming for an intracellular application of this system, we also evaluated the reaction caused by cellular levels of GSH (c ≈ 5 mM).34 Importantly, the background reaction with these high GSH concentrations still performs 220-times slower than the proximity-induced reaction, indicating sufficient selectivity for biological applications. In addition, we designed a reactive peptide that serves as a control in the following biological experiments. For this purpose, a probe with inversed peptide sequence (Cl-9iL-f) was synthesized that shows strongly reduced affinity for P(C638) (Figure S10) and a reaction rate that is 200-fold slower than the corresponding reaction with Cl-9L-f (Figure 3b). Having a selective probe system in hand, we were interested to compare the performance of our reactive probes with non-reactive ones. For this reason, pull-down experiments were employed using a reactive probe that bears a C-terminal biotin (Cl-9L-bt). In addition, we designed an analogous nonreactive probe with an N-terminal acetyl cap (H-9L-bt), and a reactive one with the inverted peptide sequence (Cl-9iL-bt). Initially, probes were immobilized on magnetic streptavidin beads and subsequently incubated with P(C638) (c = 20µM). After washing, samples were analyzed via SDS-PAGE (Figure 3c, left). Apparently, only reactive probe Cl-9L-bt shows efficient pull-down of P(C638) indicating that the affinity of the peptide ligand alone (without covalent linkage) is insufficient under these conditions. To verify this observation, we tested the cysteine-free protein P(wt) in combination with reactive probe Cl-9L-bt which again does not result in protein pull-down (Figure 3c, right). These results clearly demonstrate the advantages of a covalent modification for ligands with only moderate target affinity. Translocation of an intracellular protein

So far, intracellular proximity-induced reactions have mainly been used for the labeling of target proteins with small molecular fluorophores.3 We aimed for a challenging application that would require high intracellular labeling yields: namely the translocation of proteins inside a cell. Proteins have been translocated using self-localizing ligands which are composed of a localizing moiety linked to a nonreactive high affinity small molecular ligand.35,36 We reasoned that a ligand-directed ligation might enable the use of ligands with only moderate affinity. For that purpose, we generated HeLa cells that were transiently transfected with a construct coding P(C638) fused to fluorescent protein Cherry for visualization. To ensure that this protein construct behaves similar to the previously tested smaller



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P(C638) variant, pull-down experiments were performed with above described biotinylated Cl-9L probes (Figure S18) verifying the excellent performance of this system. In addition, we performed labeling reactions with P(C638)-Cherry and peptide Cl-9L-f in cell lysate also showing selective modification of the protein construct (Figure S19). Subsequently, we tested the localization behavior of a membrane anchor (MA) composed of a cationic peptide sequence with fatty acid modification (Figure S20a). After microinjection in HeLa cells, confocal microscopy shows localization of a fluorescently labeled MA derivative to the endomembrane system (Figure S20b).37 Having verified the functionality of this localizer, we synthesized probes with a C-terminal MA label (Cl-9L-MA and Cl-9iL-MA).

Figure 4 Modification and translocation of intracellular proteins. (a) Top: HeLa cells transiently transfected with P(C638)-Cherry before (left) and after injection (1 h, right) of Cl-9L-MA. Bottom: Relative fluorescence intensity along the yellow line in microscopy image above is plotted. (b) Plot of relative fluorescence intensity in HeLa cells transiently transfected with P(wt)-Cherry before and after injection of Cl-9L-MA (Figure S22). (c) Plot of relative fluorescence intensity for HeLa cells transiently transfected with P(C638)-Cherry before and after injection of Cl-9iL-MA (Figure S23). For details see Supporting Methods and Figure S21‒S23.


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In P(C638)-Cherry-containing HeLa cells, fluorescence is homogeneously distributed throughout the entire cell (Figure 4a, left). 1 h after microinjection of Cl-9L-MA, a redistribution of fluorescence is observed, resulting in increased intensity in a circular intracellular pattern, which correlates with the endomembrane system (Figure 4a, right). Fluorescence intensity plots along a cell-crossing line show two distinct peaks indicating the areas of accumulation. This is in agreement with the previously fluorescently labeled MA-peptide (Figure S20b) and indicates successful translocation. In comparison, HeLa cells expressing P(wt)-Cherry, which lacks the reactive cysteine, do not show changes in the distribution of Cherry-fluorescence upon Cl-9L-MA injection highlighting the necessity of covalent linkage in this protein/ligand system (Figure 4b, Figure S22). In addition, P(C638)-Cherry-expressing HeLa cells were injected with Cl-9iL-MA, harboring the inversed peptide sequence. Again, no change in the pattern of Cherry-fluorescence was observed after 1 h (Figure 4c, Figure S23) verifying the proximity-induced nature of this labeling reaction.

Conclusion We report the structure-guided design of a proximity-induced labeling system that employs a peptide ligand with only moderate affinity for the protein target. The system was thoroughly investigated regarding linker lengths and the distances between reactive groups. A comparison of experimental reaction rates with MD-derived proximity estimations between the reacting atoms shows very good agreement suggesting i) that reactivity is mainly determined by the probability of encounter of reactive groups in the preformed protein/ligand complex and ii) that MD simulations are useful to support future probe design. The most reactive architecture was investigated in detail revealing very high proximityinduced acceleration of the reaction rate (>9000-fold). Reactive probes show excellent efficiency in pulldown experiments in particular when compared to their non-reactive counter parts highlighting the importance of covalent bond formation. Most strikingly, efficient protein labeling can be performed inside living cells facilitating the translocation of target proteins from the cytosol to the endomembrane system. This is the first example of a proximity-induced labeling system that was used to alter the subcellular localization of a protein. Given the robustness of ligand-directed reactions and the good availability of peptide ligands, such peptide-directed modifications hold the potential to provide access to novel diagnostic tools and therapeutic agents.

Acknowledgements This work was supported by the German Research Foundation (DFG, Emmy Noether program GR3592/2-1), AstraZeneca, Bayer CropScience, Bayer HealthCare, Boehringer Ingelheim, Merck KGaA and the Max Planck Society. We appreciate access to microscopes at the Max Planck Institute of Molecular Physiology (P. Bastiaens) and technical support by S.A.H. Müller and M. Schulz. 9 ACS Paragon Plus Environment


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Supporting Information The Supporting Information is available free of charge on the ACS Publications website.

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TOC 208x116mm (150 x 150 DPI)

ACS Paragon Plus Environment


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Figure 1 250x229mm (150 x 150 DPI)


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Figure 2 250x262mm (150 x 150 DPI)



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Figure 3 250x381mm (150 x 150 DPI)


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Figure 4 250x330mm (150 x 150 DPI)


Translocation of an Intracellular Protein via Peptide ... - ACS Publications

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