Discovering Selective Binders for Photoswitchable Proteins Using

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Discovering selective binders for photoswitchable proteins using phage display Jakeb M. Reis, Xiuling Xu, Sherin McDonald, Ryan Woloschuk, Anna Jaikaran, Frederick Vizeacoumar, G. Andrew Woolley, and Maruti Uppalapati ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00123 • Publication Date (Web): 11 Sep 2018 Downloaded from http://pubs.acs.org on September 13, 2018

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ACS Synthetic Biology

Discovering selective binders for photoswitchable proteins using phage display

Jakeb M. Reis1*, Xiuling Xu1*, Sherin McDonald2*, Ryan M. Woloschuk1, Anna S.I. Jaikaran1, Frederick S. Vizeacoumar2, G. Andrew Woolley†1, Maruti Uppalapati2† 1. Department of Chemistry, University of Toronto, Toronto, ON, Canada. 2. Department of Pathology and Laboratory Medicine, University of Saskatchewan, Saskatoon, SK, Canada. * These authors contributed equally †

Corresponding authors

NMR instrumentation at the Centre for Spectroscopic Investigation of Complex Organic Molecules and Polymers was supported by the CFI (Project number: 19119) and the Ontario Research Fund. The work is supported by the University of Saskatchewan (MU), and NSERC (GAW).

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ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Nature provides an array of proteins that change conformation in response to light. The discovery of a complementary array of proteins that bind only the light-state or dark-state conformation of their photoactive partner proteins would allow each light-switchable protein to be used as an optogenetic tool to control protein-protein interactions. However, as many photoactive proteins have no known binding partner, the advantages of optogenetic control – precise spatial and temporal resolution – are currently restricted to a few well-defined natural systems. In addition, the affinities and kinetics of native interactions are often sub-optimal and are difficult to engineer in the absence of any structural information. We report a phage display strategy using a small scaffold protein that can be used to discover new binding partners for both light and dark states of a given light-switchable protein. We used our approach to generate binding partners that interact specifically with the light state or the dark state conformation of two light-switchable proteins: PYP, a test case for a protein with no known partners, and AsLOV2 a well-characterized protein. We show that these novel light-switchable proteinprotein interactions can function in living cells to control subcellular localization processes.

Keywords: optogenetics; phage display; photoactive yellow protein; PYP; LOV; blue light; photoswitchable


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Optogenetic tools that enable fine spatial and temporal control of protein-protein interactions are revolutionizing experimental approaches in cell biology.1-4 Most current optogenetic tools have been created by taking naturally-occurring light-dependent proteinprotein interactions and fusing the interacting partners to proteins of interest.3 For several of these cases (e.g. Cry/CIB1, FKF1/Gigantea, phytochromes/PIFs)2, 3, 5, 6 the proteins involved are large, or multimeric, and this may alter the behavior of the protein they are attached to. In addition, there are very few naturally occurring cases of light-induced heterodimeric proteinprotein dissociation (as opposed to association). The absence of structural data makes efforts to alter natural photoswitchable protein affinity or the kinetics of association/dissociation difficult.7 For the structurally well-characterized photoswitchable protein AsLOV2, synthetic binding partners for the light state (TULIPS) have been designed8 by using PDZ domains to recognize mutated versions of the Jα-helix, which dissociates from the AsLOV2 core upon irradiation. A similar strategy has been employed to engineer light-induced dimers (iLIDS) based on the interaction of AsLOV2 mutants with the SspB protein.9 However, this approach is not generalizable to other light-switchable proteins. In the first example of designed light-induced dissociation, Wang et al, recently reported LOVTRAP, a modified Z domain protein that was selected using mRNA display methods to bind to the dark state of AsLOV2.10 The selection procedure in this study utilized a mutated version of AsLOV2 that is locked in the dark-state. Development of such mutants needs significant protein engineering effort, which is not possible in the absence of structural information. In this study, we describe a generalizable approach to find binding partners for specific states of switchable proteins that does not require structural characterization of the target.



Binding partners of 1:1 stoichiometry and selectable affinity can be developed for photoswitchable proteins for which no natural binding partner has been identified. As a test case, we use our approach to find binders for photoactive yellow protein (PYP), a protein that has been the subject of extensive photochemical and biophysical studies for over 30 years,11 but for which no natural binding partners have been conclusively identified.12, 13 Using a novel codon set and

negative selection on the opposite (light vs. dark)

conformation, we used a modified phage display approach to enable selection for small stable protein domains that bind to the light state or the dark state of the target protein of interest. We generated binding partners that interact specifically with the light state or the dark state conformation of two light-switchable proteins: PYP, our test case for a protein with no known partners, and AsLOV2 a well-characterized protein for which partners have been developed using other approaches. The novel binding partners were expressed, purified and protein-protein interactions were tested in vitro using ELISA, UV-Vis and NMR spectroscopy. The binding partners were then tested in living cells for their ability to mediate light-driven subcellular localization processes. This is the first demonstration of using phage display methods to generate small, disulfide free, protein domains that specifically recognize conformational changes within the same protein. This combinatorial library can potentially be used to generate partners for other light-switchable proteins to create an array of light switchable protein-protein interactions, easily customizable for different optogenetic applications.


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Results Library design and screening of protein binders to light-switchable domains We chose the GA domain scaffold14 for making combinatorial phage display libraries. In our previous work,15 this scaffold consistently performed well against a set of diverse targets. The structures of the GA domain and the surface positions chosen for randomization are shown in Figure 1. These positions for randomization were chosen from a set of contiguous surface exposed residues that are within 4 Åof the interaction partner, human serum albumin in crystal structure (pdb 1TF0).

Figure 1. Structures of the proteins used in this study. (a) The GA domain (1TF0) with the randomized surface residues colored red; (b) Photoactive yellow protein (structure of cPYP based on 1NWZ) (22) in the dark state (left) and a snapshot of the light state ensemble (2KX6). (c) AsLOV2 in the dark state (2V0U) and light state (model based on 2V0W with the J-α helix undocked).



A custom codon (N1)(N2)(K1) was used to introduce random mutations in the 12 selected positions. Here (N1),(N2),(K1) are defined mixes of nucleotides (see Methods) that allow for a bias towards Tyr, Ser and Phe residues while allowing all 20 amino acids to occur. The mix was selected to minimize the frequency of Pro, Cys and Gly residues, which can disrupt secondary structure or introduce spurious folds, while maintaining a frequency of Tyr and Ser close to 14%. Our strategy for phage-based selection was as follows: The scaffold library (GA domain) was initially displayed fused to the M13 coat protein pVIII, using a custom 8+8 type phagemid (see Methods). Since this protein is present in multiple copies per phage (~10-60 copies for proteins of similar size

16

), avidity effects were anticipated to enable selection of even low

affinity binders from naïve libraries.17, 18 The target light-switchable protein was expressed as a fusion to GST with an AviTag sequence to permit site-specific biotinylation during expression. We opted to use variants of PYP (designated cPYP19) and AsLOV2 in which the thermal relaxation to the dark state was slower than wild type to facilitate complete switching to the light state without requiring very bright light sources. The purified proteins were immobilized on neutravidin-coated plates and three rounds of selection were carried out. In each round, the library was first depleted for unwanted binders (e.g. the library was incubated with light-adapted target protein to remove light-state binders, then the non-binding supernatant was exposed to dark-adapted target protein to select for dark-state binders). After three rounds of selection, 48 single clones were isolated from each pool and tested for selective binding to target protein using an ELISA (Figure 2).


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Figure 2. Phage-based selection of binders. ELISA detected binding of selected clones after three rounds of pVIII-based selection from the GA-domain library. Binding targets are indicated in the legend. Clones selected for expression are indicated (BoPL and BoLD, see text)

At this stage, several clones were moved to expression vectors and the proteins characterized. As expected when expressed as isolated proteins, not all GA variants were wellbehaved since a large fraction of the protein surface has been altered. Nevertheless, well-behaved binders for the light-state of cPYP and the dark-state of AsLOV2 were obtained (vide infra). We designate these proteins as BoPL (for binder of cPYP-light state), and BoLD (binder of AsLOV2-dark state). We transferred the enriched phage pools from third round of selection to a 3+3 type phagemid for display as a pIII fusion. Since the number of copies of pIII displayed proteins is 1 mM. The light state affinity may be estimated by measuring the effect of BoPL on the photocycle kinetics of cPYP.20 Fig. 3e shows the effect of added BoPL on the thermal relaxation rate. These data fit well to a 1:1 binding curve from which a Kd of 3.2 µM for BoPL binding to the light state of cPYP was determined - a change in Kd of >300-fold. Adding a substoichometric amount of cPYP to the BoPL sample produced a spectrum with two sets of resonances, indicating bound and free states are in slow exchange on the NMR timescale.21 From a ZZ-HN-HSQC-exchange experiment (Fig. 3c, Supplementary Fig. S5),22 the rate constants for association and dissociation were estimated as ~1 x106 M-1s-1 and 1 s-1 respectively.

Figure 3. (a) HN-HSQC spectrum of BoPL alone (red), after the addition of cPYP (black), and during irradiation (blue). (b) Trp –indole region of HNHSQC spectrum of BoPL showing the effect of binding on Trp47. The bound state is indicated as W47’. (c) ZZ-exchange spectrum



(250 ms mixing time) showing a set of W47 bound, W47 free, and exchange (xch) peaks for BoPL. (d) HN-HSQC spectrum of BoLD alone (red), after the addition of cPYP (black), and during irradiation (blue) (e) Rate constant for thermal relaxation of cPYP as a function of BoPL fitted to a 1:1 binding equation.

The NMR HN-HSQC spectrum of

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N-labelled BoLD alone is shown in Fig. 3d (green

peaks). Again, this is typical of a well-folded monomeric protein. In this case, adding darkadapted AsLOV2 to this sample in a 1:1 mole ratio (~100 µM of each protein) causes a subset of resonances to shift (black peaks). Irradiating the sample with blue light (455 nm) causes the spectrum to revert to that of the spectrum of BoLD alone, indicating greatly reduced interaction with AsLOV2 in the light (blue peaks). The absence of an interaction in the light at these concentrations indicates a Kd of > 1 mM. Adding sub-stochiometric amounts of AsLOV2 again resulted in two sets of peaks (bound/free) indicating a slow exchange process.21 A ZZ-HNHSQC-exchange analysis (Supplementary Fig. S6) gave a lifetime of the bound state ~1.3 s, an on-rate constant of 3.7 x105 M-1s-1 and a Kd ≈ 20 µM. We confirmed the wild-type GA sequence does not bind AsLOV2 in either dark or irradiated states (Supplementary Fig. S7). We also characterized the clones BoPD and BoLL, which were derived from pIII selections. As expected, these exhibited higher affinities to their target proteins. Given that these are high-affinity binders, we did not observe switching of binding in light vs. dark states at the high concentrations (~100 µM) required for NMR spectroscopy. We therefore used competitive phage ELISA and size exclusion chromatography to characterize the relative binding affinity to light vs. dark states. The light state binder for AsLOV2, BoLL showed a modest difference in binding affinity to light vs dark state as measured using a competitive phage ELISA. The IC50 of AsLOV2 binding to BoLL was 380 nM in light-state and 720 nM in the dark-state (Supplementary Fig. S8). More complete light-dependent switching of binding of purified BoLL


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to AsLOV2 was observed using SEC analysis (Supplementary Fig. S9). However, since several light state binders for AsLOV2 have been developed previously using structure-based approaches,8 we did not pursue this target further.

Figure 4. (a) ELISA-detected binding of phage-displayed BoPD to immobilized cPYP in the presence of increasing concentrations of free cPYP (dark-adapted – black line, irradiated – blue line). (b) Thermal recovery of cPYP with increasing concentrations of BoPD. A slow and fast component to the relaxation process is observed. (c) Plot of the fraction of the slow process as a function of added BoPD. Curve fitting was performed as described in the methods section (d) Model of the BoPD (ribbons)/dark-adapted cPYP (surface) complex generated using Patchdock (47) followed by Rosetta Dock (48) using NMR constraints. BoPD residues affected by binding are shown as sticks; cPYP residues affected by binding are colored red.

In the case of dark-state binding of BoPD to cPYP, competitive phage ELISA measurements showed an IC50 of 7.2 nM compared to > 10 µM in the light-state (Fig. 4a). The binding measured in this manner can be affected by the presence of a small fraction of phage with multiple copies of the binder. We therefore also analyzed binding in vitro using purified proteins. Light-dependent binding between purified BoPD and cPYP was observed using SEC analysis (Supplementary Fig. S10). These data allow an estimate of the light-state Kd as >10 µM, and a dark state Kd of

MKLATIDQWLLKNAKEDAIAELKKAGITSDFYFNAINKAKTVEEVNALKNEILKAHAGSSGLEHHHHHH

BoPL> MKLATIDQWLLKNAKEDAIAELKKAGITSDEEFNFINSAFDVADVNWYKNNILKAHAGSSGLEHHHHHH BoLD> MKLATIDQWLLKNAKEDAIAELKKAGITQDYLFNRINEAHFVNQVNVRKNVILKAHAGSSGLEHHHHHH BoPD> MKLATIDQWLLKNAKEDAIAELKKAGITSDESFNSINYAHSVKAVNYFKNKILKAHAGSSGLEHHHHHH BoLL> MKLATIDQWLLKNAKEDAIAELKKAGITSDFYFNNINAARSVLLVNSQKNYILKAHAGSSGLEHHHHHH Calc’d MWs: BoPL 7684 Da, BoLD 7901 Da, BoPD 7746 Da, BoLL 7761 Da

UV-Vis measurements UV-Vis spectra and kinetic measurements were performed using a PE Lambda 35 or 25 spectrophotometer or using a diode array UV–Vis spectrophotometer (Ocean Optics Inc., USB4000) in each case, coupled to a temperature controlled cuvette holder (Quantum Northwest, Inc.). Protein concentrations were determined using an extinction coefficient at λmax (~446 nm) of 29 x 103 for M121E-cPYP, 13.5 x 103 for AsLOV2. Irradiation of the protein sample was carried out by using a Thorlabs M455L3 455 nm high power LED (~50 mW/cm2). To determine the rate constants for thermal relaxation, changes in the absorbance spectrum at 450 nm were



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monitored. Data were fitted to single exponential functions to extract rate constants. Binding constants were determined by fitting rate data to the Morrison equation for tight binding: B = k ∗ A − − −K − B − A − −K − B − A − 4 ∗ A ∗ B / /2/A + k ∗ − −K − B − A − −K − B − A − 4 ∗ A ∗ B / /2/A Where Btot = [cPYP]tot and Atot = [BoPL] tot or [BoPD] tot. NMR measurements All NMR experiments were carried out at CSICOMP (Department of Chemistry, University of Toronto) on an Agilent DD2 700 MHz spectrometer equipped with an HFCN cold probe. Samples were prepared in PBS buffer with 10 % D2O in 3 mm NMR tubes.

15

N NH

HSQC were recorded at 20°C using the Varian ‘Biopack’ Watergate HSQC sequence. For dark state spectra, samples were allowed to fully relax in the instrument prior to acquisition. Lightstate spectra were acquired under constant irradiation with blue light (~ 50 mW/cm2). NMR spectra were processed using the NMRpipe processing suite42 HSQC spectra were recorded with 2048 points spanning 8928 Hz in the 1H dimension and 128 increments 2270 Hz spanning in the

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N dimension. No spinning was used. Irradiation was

performed by connecting a blue mounted LED (M450L3, Thorlabs) to a 1000 µm core, 0.37 NA multimode fiber optic from (BFH37-1000, Thorlabs) with a threaded lens tube (SM1L10, Thorlabs) and adapter (SM1SMA, Thorlabs). The outermost layer of cladding was removed so that it fit into a 3mm NMR tube, and the fiber was secured in the sample with Parafilm. For ZZ exchange experiments, samples containing excess binding partners (BoLD, BoPL) were prepared in 5 mm D2O-matched Shigemi tubes (BSM-3). To decrease total


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acquisition time, but maintain the same resolution of the HSQC spectra,

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N windows were

narrowed to 1843 Hz defined by 104 increments (BoLD) and 2056 Hz defined by 114 increments (BoPL). Longitudinal exchange was allowed to evolve for mixing times of 50, 70, 150, 250, 330, 400, 500, 650 and 800 ms for BoPL and 50, 100, 250, 330, 400, 500, 650 and 800 ms for BoLD. NMR spectra were processed as described above and peak volumes were determined using nmrViewJ (One Moon Scientific). The change in auto and exchange peak volumes with respect to mixing time was fit as described22 to obtain exchange rates. Modeling of interactions between cPYP-M121E and BoPD NH-HSQC spectra were obtained for cPYP-M121E in the presence and absence of equimolar BoPD. PYP residues affected by BoPD binding were identified with reference to published assignments of cPYP-M121E.19 Assignments of resonances of BoPD were made using standard triple resonance NMR methods and BoPD residues affected by binding to cPYP were identified. These sets of residues were used as constraints for input into the program Patchdock.23 Coordinates for the receptor were based on pdb files 1NWZ for PYP and the ‘ligand’ 1TF0 for the GA domain. A clustering RMSD of 4Å was used.

Top scoring results were visually

inspected using Pymol and refined using RosettaDock (http://graylab.jhu.edu/docking/rosetta/).24 Mammalian cell expression, imaging, and photoactivation Both cPYP and AsLOV2 were codon-optimized for mammalian expression by Biobasic and were then subcloned by Gibson assembly into the pLL 7.0 mammalian cell expression plasmid (addgene: 60415). This plasmid adds tgRFP to the N-terminus of the photoswitchable protein. Binders were subcloned into the pTriEx expression vector (addgene:81010), which places a TOM20 mitochondrial localization tag and the mVenus protein N-terminal to the binder.10



BEAS-2B cells were cultured in DMEM culture media (supplemented with 10% FBS, phenol red, streptomycin, penicillin and amphotericin B) at 37◦C in a 5% CO2 incubator. Prior to transfection, cells were washed with PBS (Mg2+ and Ca2+ free) and treated with 0.25% trypsinEDTA (1x) (GibcoTM) before seeding in a 4 chamber cell imaging coverglass (Eppendorf). Following seeding, cells were allowed to grow for 24 hours before transfection. Transfections were performed using lipofectamine 2000 according to manufacturer’s instructions: 1.5 µL lipofectamine 2000 was first added to 50 µL MEM buffer. 1 µg of each plasmid was then added to a separate 50 µL aliquot of MEM buffer. The lipofectamine and DNA solutions were then mixed and left for 10 minutes at room temperature. The resulting solution was then added to the BEAS-2B cells and the media mixed gently. Cells were then returned to the 37◦C, 5% CO2 incubator for 24 hours prior to photoactivation. For cells expressing PYP constructs a 10 mg/mL stock solution of activated chromophore was first prepared in sterile DMSO. 1 mL of DMEM media was then added to 1 µL of the 10 mg/mL chromophore solution and mixed vigorously. 15 minutes prior to imaging, the media was removed from the BEAS-2B cell culture and replaced with the DMEM chromophore solution. Cells were imaged on a Nikon A1R resonant confocal microscope (Nikon, Melville, NY, USA) using an Apo 60x oil λS DIC N2 objective lens, NA 1.40. tgRFP was monitored with excitation at 561 nm (20 mW diode laser), measuring emission at 595 nm on a GaAsP detector with a 405/488/561 dichroic mirror using a 600/50 filter cube. mVenus was excited with a 514 nm diode laser (20 mW) and the emission collected on a GaAsP detector at 585 nm with a 400457/514 dichroic mirror using a 585/35 filter cube. NIS ElementTM software was used to make an automated imaging sequence. The NIS element sequence measured tgRFPs fluorescence and localization every second for 1 to 5 seconds. The cell or cells of interest were then irradiated with


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445 nm light (20 mW laser at 5% laser power) for 5 seconds. Following 445 nm excitation, tgRFP fluorescence and localization was measured over time. The duration and sampling frequency were varied so as to minimize photobleaching and to observe thermal relaxation. Image processing was carried out using the ImageJ software by modifying the methods outlined by Hallett et al.25 In order to compare mitochondrial to cytoplasmic fluorescence, first a mitochondrial region of interest (ROI) was generated by using the threshold function of ImageJ. Mitochondrial thresholds were generated on the cells pre-excitation distribution for BoPD and BoLD and post-excitation distribution for BoPLand BoLL. In order to generate the cytoplasmic ROI, the threshold generated to make the mitochondrial ROI was used to generate a mask which was then subtracted from the post excitation image of BoPD and BoLD or pre-excitation for BoPL and BoLL. The subtracted image was then used to set a new threshold and a cytoplasmic ROI. Mitochondrial fluorescence was generated by first subtracting the raw cytoplasmic fluorescence from the raw mitochondrial fluorescence.

Supporting Information: Details of phagemid library construction and characterization of binders by SEC, CD, NMR and ELISA. Details of fluorescence imaging analysis of function of binders in living cells.

Authors contributions: MU and GAW jointly conceived of and guided the experiments. JMR, XX, SM, RMW, ASIJ, FSV, GAW and MU designed and carried out experiments and analyzed data. GAW & MU wrote the paper.



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Fig. 1 95x122mm (300 x 300 DPI)



Fig. 2 143x78mm (300 x 300 DPI)


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Fig. 3 75x80mm (300 x 300 DPI)



Fig. 4 189x46mm (300 x 300 DPI)


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Fig. 5 212x105mm (300 x 300 DPI)



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Discovering selective binders for photoswitchable proteins using phage display Jakeb M. Reis1*, Xiuling Xu1*, Sherin McDonald2*, Ryan M. Woloschuk1, Anna S.I. Jaikaran1, Frederick S. Vizeacoumar2, G. Andrew Woolley†1, Maruti Uppalapati2†


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Discovering Selective Binders for Photoswitchable Proteins Using

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