Identifying Peptides Compatible with AsLOV2 Caging

To identify protein-binding peptides well suited for caging with the AsLOV2 domain we searched the protein database (PDB) for peptide sequences similar to portions of the Jα helix. The similarity score for each position in the alignment was weighted based on how likely a match or mismatch was to disrupt caging or peptide binding. For instance, if a position in the alignment mapped to residues important for both the AsLOV2-Jα helix interaction and the peptide-protein interaction, then a large favorable score was given if the amino acids were identical, and a large unfavorable score was given if the amino acids were dissimilar. The importance of a residue to the AsLOV2-Jα helix interaction or the peptide-protein interaction was assigned based on how buried the residue was; residues that were more buried were considered more important. There was little reward or penalty for conserving amino acids on the surface of the Jα helix. Using a sliding window of 6 residues, the sequence of the Jα helix was aligned with over 3000 peptide sequences taken from crystal structures of peptides co-crytallized with their protein-binding partners. Peptides from several hundred structures were identified as candidates for caging with the AsLOV2 domain. It is worth noting that this approach can be expanded to also consider known protein-binding peptides that have not been crystallized with their binding partners.

We picked out two peptide sequences for experimental testing and optimization: a vinculin binding peptide from the invasin protein ipaA (Izard, et al., 2006) and the SsrA peptide fromE. Coli, which binds the protease delivery protein SspB (Levchenko, et al., 2003). These sequences were chosen for a variety of reasons. First, at functionally important residues they align well with the Jα helix (Figure 2A,B). Indeed, several alternative alignments were identified for the SsrA peptide. Second, they adopt alternative conformations when binding their targets, ipaA adopts a helix while SsrA binds in an extended conformation (Figure S1). By testing both peptides, we examine if our approach can be used to cage peptides regardless of the conformation they adopt when bound to their target protein. Third, the peptides have different intrinsic affinities for their target proteins. IpaA binds the D1 domain of vinculin very tightly, K_d < 1 nM, while SrrA binds SspB with an affinity of ~30 nM.

Finally they have complementary advantages for use in controlling cell biology. The SsrA and SspB sequences are specific to bacteria, and therefore it is expected that they will not interact with other proteins and peptides in higher organisms. Fusing proteins of interest to a photoactivable SsrA and SspB should provide a general approach for light-induced heterodimerization, which can be used to localize proteins in the cell and activate cell-signaling pathways. Vinculin and ipaA-like peptides are found in mammals, and therefore caged ipaA should only be useful as a general tool for co-localization in orthogonal systems such as yeast. However, caged ipaA may be useful for probing vinculin biology in mammalian cells. Vinculin is a protein that connects integrin binding proteins such as talin (Cohen, et al., 2006) to the actin cytoskeleton at focal adhesions (Saunders, et al., 2006) and adherens junctions (Carisey and Ballestrem, 2010), cellular structures important for determining cell shape and motility. The ipaA peptide is from the IpaA protein of theShigella flexneri bacterium, and binds to the talin binding site on vinculin (Izard, et al., 2006). It has been proposed that IpaA binding prevents vinculin from binding talin, and thus linking integrin signaling to the actin cytoskeleton. Selectively photo-controlling the binding of ipaA to vinculin through the LOV-ipaA photoswitch could render it useful as a dominant negative inhibitor of vinculin in mammalian cells, and an integral tool for studying the role of vinculin dynamics in cell motility.

Design of LOV-ipaA

In our search of peptides in the protein database, the first ten residues of the ipaA VBS1 helical peptide were identified as a close match to the last ten residues of the AsLOV2 Jα helix (Figure 2A). Five of the ten positions are identical, and the hydrophobic residues on the Jα helix that make critical contacts with the AsLOV2 domain β-sheet at residues 539 542, and 543 are conserved in the alignment with ipaA. Additionally, residues in the ipaA sequence that make extensive contacts with vinculin are conserved in the alignment (Ile 612, Ala 615, Ala 616, and Val 619 in ipaA). This analysis suggests that it should be possible to design a chimeric sequence for the Jα helix that is compatible with the AsLOV2 structure and will bind vinculin when undocked from the AsLOV2 structure.

To design the chimeric sequence the Rosetta molecular modeling program was used to assess the impact of altering the AsLOV2-Jα and vinculin-ipaA complex sequences (Das and Baker, 2008). Side chain optimization simulations were used to thread the first ten residues of ipaA onto the last ten residues of the Jα helix. The Rosetta scores of individual residues were examined to determine if particular residues in the ipaA sequence packed unfavorably against the AsLOV2 domain β-sheet. A single position showed unfavorable scores, the mutation of aspartic acid 540 to tyrosine. To search for an alternative amino acid to place at this position, Rosetta was used to perform a sequence optimization simulation in which position 540 was allowed to adopt alternative identities and neighboring side chains were free to adopt new side chain conformations. One of the best scoring residues in the 540 position on the Jα helix was isoleucine. A working model for LOV-ipaA was then created by using the threaded AsLOV2-Jα design and adding the remaining 11 residues of ipaA onto the C-terminal end of Jα using the fragment insertion capability of Rosetta’s domain assembly protocol (Figure 2C).

Dark and Lit state binding between LOV-ipaA and vinculinD1

We developed a fluorescence polarization competition assay to measure the binding affinity and kinetics of LOV-ipaA to the vinculinD1 subdomain under dark as well as blue light irradiation conditions (Figure 3A). LOV-ipaA was titrated into a mixture of the vinculinD1 subdomain bound to ipaA peptide labeled with the dye 5-(and-6)-Carboxytetramethylrhodamine (TAMRA). As LOV-ipaA bound to vinculin the dye-labeled peptide was competed off and its fluorescence polarization signal decreased. Initial experiments showed that the slow intrinsic off-rate of the ipaA peptide and LOV-ipaA from vinculin made it possible to monitor both the kinetics and thermodynamics of binding over a time course of several hours. Experiments with varying amounts of LOV-ipaA were fit simultaneously for the on rate and binding affinity of LOV-ipaA to vinculinD1 in Matlab using a numerical integration protocol.

The same concentrations of LOV-ipaA under either blue light irradiation or dark state conditions yielded different kinetic curves (Figure 3B,C). The binding affinity of LOV-ipaA WT in the light is 3.5 nM, while it is 69 nM in the dark, a 19-fold change (Table 1). The change in binding affinity upon light activation is primarily due to changes in the on rate for binding, in the dark the rate constant is 1.3×10³ M⁻¹ s⁻¹ and in the light it is 1.4×10⁴ M⁻¹ s⁻¹. Similar changes were observed for LOV-ipaA mutants that abolish FMN-thiol bond formation (C450A) and lead to a pseudo dark state or mutants that destabilized the Jα helix (A532E I536E), causing a pseudo lit state conformation. These results are consistent with the proposed mechanism of caging. In the dark, the Jα helix is primarily docked against the AsLOV2 domain and the peptide is presented less frequently to the target binding site, slowing the on rate for binding. In general, the on rates for binding are slow both in the lit and dark state. Peptides and proteins often havek_on values greater than 1×10⁵ M⁻¹ s⁻¹ (Alsallaq and Zhou, 2008). The slow rates observed here are consistent with the helix addition mechanism of ipaA binding to vinculin, wherein the vinculin D1 4-helix bundle is rearranged into a 5-helix bundle through the addition of ipaA (Tran Van Nhieu and Izard, 2007).

Design and Optimization of LOV-SsrA

We extended the design strategy used to cage ipaA in order to create a second photoswitchable peptide, LOV-SsrA. The SsrA peptide interacts with the protease delivery protein SspB fromE. coli as a linear epitope using the seven residue sequence AANDENY (Levchenko, et al., 2003). Three possible alignments between SsrA and the Jα helix were identified in our PDB-wide search, one towards the N-terminal side of the helix (LOV-SsrAN), one near the C-terminal end (LOV-SsrAC), and one in the middle of the helix (LOV-SsrAM) (Figure 2B). We first tested the C-terminal alignment, LOV-SsrAC.

In LOV-SsrAC, the two alanines from SsrA were aligned with A542 and A543 from the last helical turn of the Jα helix, and the final three residues of the Jα helix were replaced with residues from the SsrA binding sequence (Figure 2D). Except for the last leucine, all buried positions in the Jα helix are conserved with this alignment, which only embeds the peptide in a single helical turn of the Jα helix. In comparison, the ipaA peptide is embedded within two helical turns in LOV-ipaA. To measure the affinity of LOV-SsrA for SspB in the dark and light, a fluorescence polarization competition assay was performed with TAMRA-labeled SsrA peptide. Unlike ipaA, SsrA binds rapidly to SspB and only equilibrium populations could be measured. The initial LOV-SsrA design showed a two-fold change in affinity when activated with light, 31 nM to 57 nM (Table S1). As lit state binding was near native in affinity for SspB, approaches to stabilize the Jα helix and therefore decrease dark state affinity were explored. We tested mutations previously shown to stabilize the Jα helix, G528A and N538E, as well as extensions to the C-terminus of LOV-SsrAC. G528A and N538E had the desired effect and lowered dark state affinity to 570 nM with a 5-fold change upon light activation. Extensions of varying lengths were designed with Rosetta (Kleiger, et al., 2009) with the goal of binding the surface of the AsLOV2 domain, thereby constraining SsrA to a single, non-binding conformation. The target surface on AsLOV2 was a hydrophobic patch immediately adjacent to the C-terminus of the Jα helix. Two extensions were experimentally tested, the addition of a single phenylalanine and the addition of the sequence GYGNL. The longer extension had no detectable effect, while the single phenylalanine increased the dynamic range of the switch to 8-fold (Figure 4A).

The LOV-SsrAN designs incorporating the N-terminal alignment of SsrA, had similar dynamic ranges to their corresponding C-terminal alignments. However, all had drastically reduced affinity for SspB in both the lit and dark state, > 10 μM. The initial N-terminally aligned designs showed an approximate 2-fold increase in affinity after exposure to blue light. The addition of the G528A and N538E mutations slightly weakened the lit state affinity for SspB to 12.6 μM and the dark state affinity to 49 μM (Figure 4B). Because of constraints imposed by the AsLOV2 domain, the SspB binding sequence used in LOV-SsrAN was AANDEAY instead of AANDENY as used in LOV-SsrAC. To determine if the sequence change was responsible for the lower dark and lit-state affinities for SspB, a peptide with the same sequence as LOV-SsrAN was synthesized and binding to SspB was measured. Binding was tight, 38 nM, suggesting that the reduced affinity for SspB was not because of changes to the binding sequence, but rather because of the location within the Jα helix that the peptide was embedded. Results with LOV-SsrAM were more ambiguous, as the sequence embedded in the Jα helix, AANDINY, showed reduced affinity for SspB as a peptide (1.9 μM). However, lit (40 μM) and dark state (156 μM) state affinities were still significantly lower than that the isolated peptide, suggesting again that embedding sequences deeper in the Jα helix lowers accessibility to the peptide in both the dark and light (Figure S3).

The reversibility of LOV-SsrA switches was tested by monitoring their ability to compete with a TAMRA-labeled SsraA peptide for binding to SspB over multiple rounds of irradiation followed by incubation in the dark (Figure 4C). As monitored indirectly from following the fluorescence polarization signal from the SsraA peptide, the fraction of LOV-SsrA molecules switching from the bound to unbound state remained unchanged over multiple rounds of illumination, indicating that the switches are reversible. To check that the observed results were not an artifact due to interaction between the blue light and the TAMRA dye, similar reversion experiments were performed with only SspB and labeled peptide. No polarization changes were observed after incubation with blue light. The reversibility experiments also showed that dark state equilibrium was reached within 15 seconds, quicker than what has) been observed previously for the AsLOV2 domain. Half-times between 27 and 50 seconds have been reported for the LOV2 domain fromAvena sativa (Salomon, et al., 2000;Swartz, et al., 2001). Using absorption spectroscopy, we determined the half-life of LOV-SsrAC’s photocycle to be 2.7 ± 0.4 seconds, with a similar relaxation time in the presence of saturating concentrations of SspB, 2.3 ± 0.1 seconds (Figure S3). At this point it is not clear why LOV-SsrAC has an unusually fast rate of reversion to the dark state, but this could be advantageous in applications where quick switching from the bound to unbound state is critical.

LOV-ipaA binding to Full-length Vinculin: Actin co-sedimentation assays

Having established the AsLOV2 domain could be used to cage peptides, we tested if LOV-ipaA could be used to perturb vinculin in a biologically relevant manner. Actin co-sedimentation assays were performed to measure the apparent binding affinity of LOV-ipaA to full-length vinculin. Vinculin naturally forms an autoinhibited conformation stabilized by interactions between the head domain (including the D1 domain) and tail domain. Binding of the ipaA peptide to the head domain competes with the autoinhibited state and releases the tail domain to bind with polymerized F-actin (Izard, et al., 2006). Actin co-sedimentation assays were performed to measure the apparent binding affinity of LOV-ipaA in the dark and lit state to full-length vinculin. In the assay, polymerized actin, vinculin, and LOV-ipaA (lit or dark-state mutants) were incubated together (Figure 5A). Only vinculin that bound to LOV-ipaA should be able to interact and bind to F-actin. The resulting mix of bound and unbound vinculin was centrifuged at high speeds, resulting in fractionation of F-actin polymers out of solution, along with the vinculin-LOV-ipaA complexes that had bound to them. An SDS-page gel was used to monitor the amount of vinculin that fractionated out of solution along with F-actin as a function of the total concentration of vinculin, thereby determining the fraction of full-length vinculin bound to LOV-ipaA. The assay was repeated at several concentrations of LOV-ipaA to create a binding curve and calculate apparent binding affinities (Figure 5B and C). In general, the affinities are expected to be considerably weaker with full-length vinculin than those observed for binding to the isolated D1 domain, because in the full-length protein there is competition with the autoinhibited state of vinculin (Le Clainche, et al., 2010). For instance, peptides from the protein talin bind to the vinculin D1 domain with an affinity of 15 nM, but bind to full-length vinculin with an affinity of ~5 μM (Izard and Vonrhein, 2004;Le Clainche, et al., 2010). LOV-ipaA with the lit state mutant bound with an apparent affinity of 8 μM to vinculin, while the dark state mimetics bound with an affinity of 56 μM (Table S2). The binding of vinculin to ipaA in the presence of polymerized actin also gives us a window into the reactions that occurin vivo, where full-length vinculin must be bound both by talin and by F-actin in order to engage in integrin signaling. The difference in binding affinity between lit and dark states of LOV-ipaA to full-length vinculin in the presence of polymerized actin suggests that LOV-ipaA may be a relevant photoswitch for probing such signalingin vivo.

Photoactivatable yeast transcription

To demonstrate that the caged peptides can be used to co-localize proteins and activate signaling events in living cells, we used the LOV-ipaA-vinculinD1 interaction as a heterodimerization switch for controlling gene transcription. The experiments were performed in S. cerevisiae, as it is an orthogonal system, lacking proteins that would cross-react with the LOV-ipaA-vinculinD1 interaction. We linked LOV-ipaA L623A to the GAL4 activation domain (AD), while linking vinculinD1 (vinD1) to the GAL4 binding domain (BD), and monitored the GAL4-induced activation of the transcriptional GAL promoter through a yeast two-hybrid assay. This is a strategy that has been widely used to identify protein-protein interactions. In our case, LOV-ipaA and vinculinD1 should only interact upon irradiation with blue light, thus bringing the GAL4 AD and BD into proximity (Figure 6A). The full GAL4 protein can then activate the expression of reporter genes downstream of the GAL promoter. We tested the ability to activate the reporter gene LacZ under both dark state and lit state conditions through quantification of β-galactosidase activity (Figure 6B). We observed an activity of 5 Miller Units under blue light conditions, and an activity of 20 Miller Units with the LOV-ipaA L623A lit state mimetic and 30 units using the ipaA peptide. Almost no activity (0.4 Miller Units) was seen under dark or dark state conditions, and no activity was seen in empty vector negative controls. We also tested activation of genes MEL1, HIS3, and ADE2 under dark and lit state conditions by replica plating BD-vindD1 colonies mated with AD-LOV-ipaA L623A lit state and dark state mimetic mutants or AD-LOV-ipaA L623A WT (Figure 6C). We saw strong growth of colonies containing LOV-ipaA L623A lit mimetic or WT grown in blue light on plates lacking His as well as those lacking His and Ade, indicating expression of both HIS3 and ADE2 genes. Furthermore, colonies were blue, indicating expression of the MEL1 gene whose product, α-galactosidase, interacted with the x-α-gal substrate for blue screening. Colonies containing LOV-ipaA L623A dark mimetic or WT grown in the dark grew on control plates, but did not grow on plates lacking His or Ade, showing low to no expression of HIS2, ADE2 or MEL1 genes. This indicates that LOV-ipaA-vinculinD1 heterodimerization can be used as a tool to photocontrol yeast gene expression.

The LOV-ipaA L623A mutation was critical to achieving a lit state to dark state change of phenotype in yeast gene expression. When experiments for MEL1 and HIS3 were conducted using LOV-ipaA lacking this mutation, no observable growth change was seen between colonies containing lit state and dark state AD-LOV-ipaA mated with BD-vinD1 (Figure S4). The binding affinity of LOV-ipaA to vinculinD1, was hence, so tight, even in the dark state, as to allow binding events and subsequently yeast gene expression to occur. The L623A mutation shifted the binding affinity of LOV-ipaA for vinculinD1 to a range where there was no binding in the dark, but significant activation in the light. This result highlights the usefulness of being able to tune the switches for specific applications.

Significance

Peptides regulate a variety of biological processes by acting as competitive inhibitors, allosteric regulators, and localization signals. Photocontrol of peptide activity is a powerful tool for precise spatial and temporal control of cellular function. Our work in this paper focuses on designing and characterizing peptides caged by embedding their sequences into the Jα helix of the AsLOV2 domain. The peptides ipaA and SsrA were both effectively caged in this manner, allowing for enhanced peptide-effector binding in the light. Mutations introduced into the AsLOV2 domain increased the difference between lit state and dark state effector binding, while the position where the caged sequence is embedded in the Jα helix and the intrinsic affinity of the peptide for its target tunes the range of affinities of the photoactivatable peptide . We discuss ways in which other peptides may be caged in a similar manner using the AsLOV2 domain. We also apply LOV-ipaA as a tool for photoactivatable gene transcription inS. cerevisiae, and describe potential uses for LOV-SsrA.

Cloning

The LOV-ipaA gene was synthesized with a 6 histidine N-terminal tag (Genscript) and cloned into the pET21b vector. The genes for LOV-SsrA and monomeric SspB were synthesized (Genscript) and cloned into pQE-80L and pTriEX4 vectors respectively. All mutations were performed using site-directed mutagenesis. The VinculinD1 subdomain (residues 1-258) and full-length vinculin (residues 1-1066) were cloned into a pET15b vector. See Supplementary Methods for protein expression and purification.

Peptides

Peptides containing the sequence TANNIIKAAKDATTSLSKVLKNIN, TANNIIKAAKDATTSASKVLNIN, TANNIIKAAKDATTSLSKALKNIN, QIEEAANDENY , LIKKAANDINYAAK, and HVRDAANDEAYMLIK were synthesized at UNC-Chapel Hill and amine labeled using 5-(and-6)-Carboxytetramethylrhodamine (TAMRA) dye (Anaspec). Peptide concentration was determined by measuring absorbance of the TAMRA dye at 555 nm using 65,000 M⁻¹ cm⁻¹ extinction coefficient.

Fluorescence polarization experiments

All fluorescence polarization experiments were conducted using a Jobin Yvon Horiba FluoroMax3 fluorescence spectrometer. TAMRA labeled peptides were excited with polarized light at 555 nm and the polarization of emitted light was measured at 583 nm. See Supplementary Methods for detailed methods.

Surface Plasmon Resonance

Surface plasmon resonance experiments were conducted using a Biacore 2000 machine (GE). VinculinD1 was immobilized through amine coupling to the surface of a CM5 chip (GE) Different concentrations of LOV-ipaA mutants were flown over the immobilized protein and the change in response units over time was recorded. Data were fit simultaneously fork_on andk_off to a pseudo-first order binding model.

Actin co-sedimentation assays

Purified rabbit actin (invitrogen) was polymerized for 30 min at room temperature in 10 mM Tris-HCl pH 7.5 containing 100 mM KCl, 2 mM MgCl2 , 2 mM DTT and 1 mM ATP. 2 uM vinculin and either ipaA peptide or LOV-ipaA mutants were mixed for vinculin:ipaA rations of 1:0, 1:1, 1:2.5, 1:5, 1:10, 1:20, or 1:50 per sample. 12 μM polymerized actin was added per sample, within a volume of 45 μL per sample. Samples were incubated at room temperature for 1 hour. They were then centrifuged in a TLA-100 rotor in a Beckman Coulter Optimax XP ultracentrifuge at an acceleration of 150,000 g for 30 min cooled to 20°C. Samples were split into supernatant and pellet fractions. Pellets were re-suspended into 45 μL 2x tris-glycine SDS buffer. All fractions were denatured and run onto an 8% SDS-page polyacrylamide gel. Gels were coomassie stained and analyzed using ImageJ software to determine the fraction of vinculin present in the pellet versus total vinculin in each sample (Le Clainche, et al., 2010).

Yeast two hybrid assays

LOV-ipaA L623A WT, lit state mutants, dark state mutants, and ipaA were cloned into a pGADT7 vector and transformed intoS. cerevisiae Y187 strain, while the vinculinD1 subdomain was cloned into a pGBKT7 vector and transformed intoS. cerevisiae Y2Hgold strain (Clontech). Empty vectors were also transformed into the appropriate strains. Transformed colonies of Y2Hgold and Y187 were mated at 30°C overnight and plated on synthetic dextrose (SD) -Leu -Trp media. For yeast two-hybrid experiments, mated colonies were serially diluted (1:5, from right to left on plates shown) and replica plated onto SD–Leu–Trp media; SD–Leu–Trp media with auerobasidin A and 5-bromo-4-chloro-3-indolyl-α-d-gactopyranoside (x-α-gal); SD–Leu–Trp–His media with auerobasidin A and x-α-gal; and SD–Leu–Trp–His–Ade media with auerobasidin A and x-α-gal. Plates were grown for 3 days at 30°C. For Miller assays, mated colonies were picked and grown to saturation in SD–Leu–Trp media. Saturated colonies were diluted to low-log phase and grown for 4 hours under dark or blue light conditions. Cells were lysed open and treated with Chlorophenol red-β-D-galactopyranoside (CPRG, Roche) substrate to determine β-galactosidase activity in Miller Units (Kennedy, et al., 2010).

Highlights.

• The AsLOV2 domain can be used as a tool to photocontrol the peptides ipaA and SsrA

• LOV-ipaA and LOV-SsrA bind their effectors with 49-fold and 8-fold enhanced affinity in the light, respectively.

• Mutations that increase the difference between dark and lit state effector binding in both photoswitches are described.

• LOV-SsrA and LOV-ipaA are tuned by varying the location where the caged peptide sequences are embedded in the AsLOV2 Jα helix as well as varying the intrinsic affinity of the peptides for their targets.

• LOV-ipaA is applied to photocactivate gene transcription inS. cerevisia

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