Selective binders of the tandem SH2 domains in Syk and ZAP-70

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Selective binders of the tandem SH2 domains in Syk and ZAP-70 kinases by DNA-programmed spatial screening Michaela Marczynke, Katharina Groeger, and Oliver Seitz Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00382 • Publication Date (Web): 02 Aug 2017 Downloaded from http://pubs.acs.org on August 8, 2017

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Bioconjugate Chemistry

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Figure 1. Crystal structures of (A) Syk and (B) Zap-70 tSH2 domains in complex with dually tyrosine phosphorylated peptides from the (A) CD3ϵ chain of the B cell receptor complex or (B) the zeta chain of the T cell receptor. From pdb 1A81 (A) and 2OQ1 (B). 505x940mm (83 x 83 DPI)


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Figure 2. A) Chemical structure of DNA-peptide linkage in B) ternary DNA-peptide complexes used for the spatial screening of ZAP70-tSH2 and Syk-tSH2. C) Complexes TCn7 and TCn8 were previously used for the spatial screening of Syk-tSH2. (35). The monofunctionalized ternary complexes (chemical linkage shown in Fig. S3) in D) were used for control measurements. Arrow heads indicate the C-terminal end of the peptide. n is the distance between peptides in nucleotides resulting from paired spacer nucleotides and unpaired spacer nucleotides m + 1. The consensus peptide motif recognized by the tSH2 domains is underlined.) 197x428mm (600 x 600 DPI)



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Figure 3. A) DNA-based spatial screening of the tSH2 domain in ZAP-70 using bivalent ternary DNA-peptide conjugates with peptides attached in A) same strand orientation (TCn1a and TC2na) or B) opposing strand orientation (TCn4a and TCn5). For comparison, the distance-affinity-relationship for interactions of Syk tSH2 with ternary complexes TCn7b and TCn8b is included for comparison. (n is the number of paired and number of unpaired spacer nucleotides m+1). 144x186mm (600 x 600 DPI)


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Figure 4. A) Orientation-dependent interactions between bivalent pYETL displays in ternary complexes TC11b, TC13b, TC14b and TC16b and tSH2 domains of Syk tSH2 (red) and Zap-70 (green). 63x52mm (600 x 600 DPI)



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Figure 5. Different binding modes of tSH2 domains from Syk and Zap-70. A) The Syk tSH2 domain has two independent binding modules for synergistic binding of ITAM consensus motifs (pYXXL/I). B) Owing to the flexible interdomain both binding modules remain engaged upon increases of the distance between the two consensus motifs. C) The Zap-70 tSH2 domain comprises one independent binding site and one shared binding site. D) Only one binding module remains engaged when the two consensus motifs are separated by larger distance. 115x124mm (600 x 600 DPI)


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Figure 6. Subtype-specific binders for tSH2 domains of Syk tSH2 (red) and Zap-70 tSH2 (green) through A) sequence-dependent interactions between bivalent pYXXL-PNA-pYXXL conjugates or B) distance-dependent interactions between bivalent pYXXL-oligoethylene glycol conjugates. 152x248mm (600 x 600 DPI)



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Selective binders of the tandem SH2 domains in Syk and ZAP-70 kinases by DNA-programmed spatial screening Michaela Marczynke, Katharina Gröger, Oliver Seitz Institut für Chemie, Humboldt-Unive rs itä t zu Be rlin, Brook-Ta ylor-S tra ß e 2, D-12489 Be rlin, Ge rma ny Members of the Syk family of tyrosine kinases arrange Src homology 2 (SH2) domains in tandem to allow firm binding of immunoreceptor tyrosine-based interaction motifs (ITAMs). While the advantages provided by the bivalency enhanced interactions are evident, the impact on binding specificity is less clear. For example, the Spleen tyrosine kinase (Syk) and the Zeta-chain-associated protein kinase (ZAP-70) recognize the consensus sequence pYXXI/L(X)6-8 pYXXI/L with near identical nanomolar affinity. The non-discriminatory recognition on the one hand poses a specificity challenge for the design of sub-type selective protein binders and, on the other hand, raises the question as to how differential activation of Syk and ZAP-70 is ensured when both kinases are co-expressed. Herein, we identified the criteria for the design of binders that specifically address either the Syk or the Zap-70 tSH2 domain. Our approach is based on DNA-programmed spatial screening. Tyrosine-phosphorylated peptides containing the pYXXI/L motif were attached to oligonucleotides and aligned in tandem on a DNA template by means of nucleic acid hybridization. The distance between the pYXXI/L motifs and the orientation of strands were varied. The exploration exposed remarkably different recognition characteristics. While Syk tSH2 has a rather broad substrate scope, ZAP-70 tSH2 required a proximal arrangement of the phosphotyrosine ligands in defined strand orientation. The spatial screen led to the design of mutually selective, DNA-free binders which discriminate Zap-70 and Syk tSH2 by one order of magnitude in affinity.

Bivalent or multivalent engagement is used by nature to increase the strength of weak receptor-ligand interactions.1 This principle has frequently been studied in the context of multivalent carbohydrate ligands that bind multiple carbohydrate binding sites on cell surface lectins.2-4 Yet, the affinity-increase-by-multivalence approach is by no means restricted to the cell exterior. Repeats of interaction modules are the hallmark of a number of intracellular adaptor proteins and protein regulatory domains. For example, WW domains and Src homology 3 (SH3) domains (recognize proline-rich sequences),5-10 Src homology 2 (SH2) domains (bind consensus peptides containing phosphotyrosine, vide infra) and epigenetic readers11, 12 are often arranged in tandem. The engagement of repeat interaction modules provides affinity advantages. However, the distinct bivalent recognition systems are frequently comprised of closely related binding modules. As a result, two different proteins may have near identical affinity for shared consensus ligands. This issue on the one hand poses specificity issues, which complicates the design of specific protein inhibitors and on the other hand raises the biological question how differential activities are achievable when both proteins are co-expressed. One such case has been documented for tyrosine kinases of the Syk family, the Spleen tyrosine kinase (Syk) and the Zeta-chain-associated protein kinase (ZAP-70). The tandem-SH2 domains (tSH2) of the Syk and ZAP-70 kinases both bind with nanomolar affinity to diphosphorylated activation motifs comprising the consensus sequence pYXXI/L(X)6-8 pYXXI/L.13-15 The single SH2 domains bind

with less than 1% of the affinity which indicates a cooperative binding of the two SH2 modules in the tSH2 domains of Syk and ZAP-70.13, 16-18 The Syk kinase plays a key role in maturation/proliferation of B-cells. The ZAP-70 kinase is considered to act as the T-cell counterpart of Syk and plays a crucial role in activation and development of T cells.19-23 Syk is expressed and widely spread in hematopoietic cells and non-hematopoietic cells.24-26 The distribution pattern of ZAP-70 is narrower. Initially, its expression was believed to be restricted to T cells and natural killer cells19. Yet, recent reports have described Zap-70 expression in some populations of normal and malignant B cells, and both Syk and ZAP-70 are expressed during T cell development.27, 28 Given their co-expression, how is differential activation of Syk and ZAP-70 ensured? In a cell, full activation of Syk and ZAP-70 by autophosphorylation requires the two tSH2 domains to bind tyrosine phosphorylated cytoplasmatic segments (immunoreceptor tyrosine-based interaction motifs, ITAMs) within the engaged B-cell and T-cell receptor complexes.29-31 Therefore, selective recognition of specific ITAM sequences may contribute to the differential activation of the two kinases. Crystal structure analyses of the Syk and the Zap-70 tSH2 domains in complex with dual phosphorylated ITAM-peptides revealed, at a first glance, very similar binding modes (Figure 1).14, 32 However, the asymmetric unit of Syk tSH2 complexes showed six different complexes, which differed in the relative orientation of the two SH2 domains.32 This, the results of calorimetry and


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surface plasmon resonance measurements as well as the results of a previous DNA-programmed spatial screen hinted at a high conformational flexibility of the linker domain that connects two independently binding SH2 domains.33-36 As a result, the Syk tSH2 domain is able to adopt a variety of arrangements and bind diverse ITAM motives with high affinity. By contrast, the phosphotyrosine binding sites in the ZAP-70 tSH2 domain seem more restrained. One of the two phosphotyrosine binding sites is created by amino acids from both the N-terminal and the C-terminal SH2 domain.14 Of note, this C-terminal domain (green in Fig. 1B) has no affinity for ITAM peptides and requires the presence of the N-terminal domain.37 The binding mode may limit the flexibility of the Zap-70 tSH2 domain. We, therefore, surmised that a defined spatial arrangement of the two phosphotyrosine-containing consensus motifs within the ITAM peptide will be the key to specificity. The previous studies on ITAM analogues have revealed the criteria for high affinity binding of the Syk tSH2 domain.3436, 38, 39 However, the criteria for obtaining preferential recognition of the Zap-70 over the Syk tSH2 domain, or vice versa, have not been explored yet. The key challenge is how selective binding of the ZAP-70 tSH2 domain can be achieved without concomitant binding of the more promiscuous Syk-tSH2 domain. Previously, we35, 40-44 and others45-53 showed that DNA and PNA complexes are suitable scaffolds to precisely arrange binding motifs in space. We assumed that a DNA-based spatial screening will expose the requirements for discriminatory recognition of Syk and Zap-70 tSH2 domains. We decided to equip oligonucleotides pYXXI/L with peptide motifs found in ITAM peptide sequences of B cell and T cell receptor complexes (Fig. 2). After hybridization of two phosphopeptide-DNA conjugates with DNA templates the tyrosine phosphorylated peptides will be displayed in defined distances and orientation. Herein we describe the different distance-affinity relationships for bivalent recognition of Syk and Zap-70 tSH2 domains. The results clearly exposed the different recognition properties for each tSH2 protein and guided the design of binders, which specifically address either the Syk or the ZAP-70 tSH2 domain.

Figure 1. Crystal structures of (A) Syk and (B) Zap-70 tSH2 domains in complex with dually tyrosine phosphorylated peptides from the (A) CD3ϵ chain of the B cell receptor complex or (B) the zeta chain of the T cell receptor. From pdb 1A81 (A) and 2OQ1 (B).

from the T cell receptor complex (Table 1). Syk tSH2 preferred, as expected, interactions with a peptide (4) related to the B cell receptor complex. However, binding was not mutually selective as both, the ZAP-70 and the Syk tSH2 domains recognized the BCR- and TCR-derived ITAM peptides with rather similar affinity. We concluded that the three sequence motifs are a suitable start point to assess how the spatial arrangement of two phospho-

Results Interactions of Syk and ZAP-70 tSH2 with ITAM peptides. Prior to the investigation of distance-affinity relationships we explored the affinities of Syk and ZAP-70 tSH2 domains for doubly phosphorylated ITAM peptides. We chose a solution-based binding assay to avoid potential perturbation by cross-linking of the bivalent proteins. Nterminal GST fusions of the Syk and ZAP-70 tSH2 domains and the fluorescence-labeled reference binder 1 (FamKpYTGLNTRSQETpYETLG) were used in a competitive fluorescence polarization (FP) assay. From the three ITAM peptides tested, the ZAP-70 tSH2 domain showed the highest affinity (IC50 = 33 nM) for a ligand (2) derived

Table 1. IC50-values for the interaction of native peptides with the tSH2 domains in Syk and ZAP-70 kinasesa

2

Sequence

Syk

Zap-70

(source)

IC50 [nM]

IC50 [nM]

AcKpYNELNLGREEpYDVLG

85

33

97

45

50

67

(TCRζ TAM1) 3

AcPDpYEPIRKGQRDLpYSGLNQRG

(TCRɛ TAM) 4

AcKpYTGLNTRSQETpYETLG

(FcεRIγ TAM)

a IC50 values were determined by measurements of fluorescence anisotropy in a competition experiment with FAMKpYTGLNTRSQETpYETLG. The consensus motifs are underlined.


2


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Design of DNA-peptide conjugates for the spatial screening of tSH2-domains. DNA scaffolds were used to control the spatial arrangement of ITAM phosphopeptides. We conjugated the pYXXL motifs in solution with thiolmodified oligonucleotides by means of a maleimide unit hanging from the side chain of N- or C-terminal lysine residues (Figure 2A, Figure S2). We designed the oligonucleotide sequences to form bipartite ITAMs upon annealing of two 12-14 nt long oligonucleotide-peptide conjugates on 24-34 nt long DNA templates (Figure 2B-D). For distance alterations, spacer nucleotides were included between the double helical segments of the resulting ternary complexes TC1 – TC8 (Figure 2B,C). Single stranded oligonucleotide spacers gain flexibility with increased length. To avoid blurring of the distance-affinity relationship at pYXXLpYXXL distances beyond 10 nucleotides, the ternary complexes TC2 and TC5 were included in the study. In these complexes the spacer was comprised of a 12 nt long double strand segment and a 0−8 nt long single strand. For binding measurements at short distances we considered four different conjugation modes. Complexes TC1 and TC7 contained two N-terminally attached phosphopeptides. In complexes TC3, both pYXXL motifs were linked via a C-terminal lysine and in complexes TC4, TC5 and TC6 both conjugation modes were combined. Particularly the combination of C-terminal attachment at the left double helix segment with N-terminal attachment at the right double helix in complexes TC4 seemed to emulate best the arrangement of the pYXXL motifs within a contiguous ITAM. For control measurements, we also prepared ternary complexes TC9 and TC10 which either contained only a single pYXXL motif (negative control; TC9b, TC10c) or two pYXXL motifs (positive control; TC9c, TC10d) within one contiguous ITAM-like sequence (Figure 2D).

Figure 2. A) Chemical structure of DNA-peptide linkage in B) ternary DNA-peptide complexes used for the spatial screening of ZAP70-tSH2 and Syk-tSH2. C) Complexes TCn7 and TCn8 were previously used for the spatial screening of Syk-tSH2. 35. The monofunctionalized ternary complexes (chemical linkage shown in Fig. S3) in D) were used for control measurements. Arrow heads indicate the C-terminal end of the peptide. n is the distance between peptides in nucleotides resulting from paired spacer nucleotides and unpaired spacer nucleotides m + 1. The consensus peptide motif recognized by the tSH2 domains is underlined.)

tyrosine-containing consensus motifs (pYXXL) affects the synergistic binding interactions between the two SH2 domain modules.

DNA-based spatial screening of the Zap-70 tSH2-domain. The complexes TCn1a and TCn2a featured the pYNEL and pYDVL motifs found in the high affinity peptide 2 derived from the natural ZAP-70 substrate TCRζ TAM1. Here, both monophosphorylated maleimide-peptide sequences were linked via the N-terminus (referred to as NN-conjugation). The distance between the phosphopeptide attachment sites was varied from 1−20 nucleotides. However, none of the complexes showed proof for avid binding (IC50 > 10 µM) via bivalent recognition of the ZAP70 tSH2 domain (Figure 3A). Control measurements with the ITAM-DNA duplex conjugates TC9c and TC10d revealed IC50 = 0.043 µM and 0.058 µM, respectively, which confirmed that the DNA duplex scaffold does not impose sterical hindrance to bivalent recognition. For a comparison, Figure 3A includes the distance-affinity profile previously measured for interaction of complexes TCn7b and TCn8b with the Syk-tSH2 domain.35 The assemblies presented the pYETL motifs of peptide 4 from the BCR complex in 2−20 nucleotides distance. Obviously, the Syk-tSH2 domain adopted to a wide range of distances until a threshold of 10-13 nucleotides above which binding was severely affected. The comparison of the two


3

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Figure 4. A) Orientation-dependent interactions between bivalent pYETL displays in ternary complexes TC11b, TC13b, TC14b and TC16b and tSH2 domains of Syk tSH2 (red) and Zap-70 (green).

Indeed, given a dense arrangement (1 nt distance) and appropriate orientation (NC- or CN-attachment in TC16b and TC14b, respectively, rather than NN- or CC-attachment in TC11b and TC13b) ZAP-70-tSH2 recognized the bipartite pYETL displays with submicromolar affinity (Figure 4). Figure 3. A) DNA-based spatial screening of the tSH2 domain in ZAP-70 using bivalent ternary DNA-peptide conjugates with peptides attached in A) same strand orientation (TCn1a and TC2na) or B) opposing strand orientation (TCn4a and TCn5). For comparison, the distance-affinity-relationship for interactions of Syk tSH2 with ternary complexes TCn7b and TCn8b is included for comparison. (n is the number of paired and number of unpaired spacer nucleotides m+1).

tSH2 domains suggests that a defined arrangement of the pYXXL motifs is more important for the Zap-70 than for the Syk tSH2 domain. This is in agreement with the crystal structure analysis hinting at the spatial constraints imposed by the tight contacts between the SH2 domains of ZAP-70.14 To better mimic the orientation within an ITAM, the pYNEL motif of the TCR ζ-chain was displayed via C-terminal attachment at the 5’-end of the oligonucleotide while pYDVL was connected at the N-terminus with the 3’or 5’-end of the second oligonucleotide (referred to CNorientation). By using complexes TC4a the pYNEL-pYDVL distance was varied in a range between 1 and 11 nucleotides. Complexes TC5 offered longer distances up to 21 nucleotides. Of note, the affinity for ZAP-tSH2 was highest for TC1 4a and decreased significantly with an increasing number of spacer nucleotides between the two phosphorylated recognition peptides (Figure 3B). Complexes with more than three unpaired nucleotides showed little affinity for tSH2 domain of ZAP70. The lack of bivalency enhancement prevailed in studies of complexes TCn 5, in which the peptide attachment sites were separated by 12 paired plus maximum 8 unpaired nucleotides. We concluded that high affinity binding of ZAP-tSH2 requires a close proximity of the pYXXL motifs. To assess whether bipartite recognition by ZAP-70 tSH2 also extends to ITAMs from the BCR complex, we investigated the DNA-programmed display of pYETL motifs (used previously for spatial screening of Syk-tSH2).

The DNA-controlled spatial screening of tSH2 proteins from ZAP-70 and Syk exposed the different recognition properties of these important kinase regulatory domains. Previous reports indicate that the two SH2 domains of Syk function independently and that the linker connecting the two SH2 domains remains flexible even with the doubly phosphorylated ITAM peptide bound.34, 35, 38 This explains why Syk-tSH2 readily interacts with bipartite ITAM displays and tolerates a range of distances between the pYXXL/I motifs (see also Figures 5A and B). By contrast, the data in Figure 3 and Figure 4 suggest that avid recognition by the ZAP-70 tSH2 domain requires a

Figure 5. Different binding modes of tSH2 domains from Syk and Zap-70. A) The Syk tSH2 domain has two independent binding modules for synergistic binding of ITAM consensus motifs (pYXXL/I). B) Owing to the flexible interdomain both binding modules remain engaged upon increases of the distance between the two consensus motifs. C) The Zap-70 tSH2 domain comprises one independent binding site and one shared binding site. D) Only one binding module remains engaged when the two consensus motifs are separated by larger distance.


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close proximity of the displayed pYXXL/I motifs (Figure 5C). This is in agreement with investigations reporting that binding of the doubly phosphorylated ITAMs involves tight interactions between the two SH2 domains of ZAP70.32, 37 Selective Syk tSH2 binders. The results of the DNAprogrammed spatial screening provided a guideline for the design of mutually selective binders. Given the tolerance for a spatially distant arrangement of the two pYXXL motifs, the design of exclusive Syk tSH2 binders appeared straightforward. To prevent recognition by the ZAP-70 tSH2 domain a bipartite display should either involve the NN- or CC-terminal connection of the pYXXL motives and/or the spacer in between the two pYXXL motifs should be sufficiently long. The former hypothesis was tested with the complexes TC1 1b, TC1 3b, TC1 4b and TC1 6b, in which the two pYETL motifs protruded from the DNA scaffold in 1 nt distance via NN-, CC-, CN- and NC-terminal connection, respectively (Fig. 4). Regardless of peptide strand orientation, each complex provided for strong interactions with the Syk-tSH2 domain (IC50 = 0.12-1.18 µM). As told earlier, binding of the Zap-70 tSH2 domain was restricted to the complexes TC1 4b and TC1 6b with CN- and NCterminal connection, respectively. The highest binding specificity was provided by complex TC1 1b (IC50 = 0.48 µM) which bound Syk tSH2 with > 40-fold higher affinity than Zap-70 tSH2. We concluded that the orientation of the consensus recognition motifs (pYXXL/I) is an important parameter, which controls the affinity for the Zap-70 kinase. We next aimed for the construction of a unimolecular binder. The ‘orientation criterion’ is difficult to apply in the design of a unimolecular Syk-tSH2 binder. Here it seems more promising to separate the two pYETL motifs by a long spacer. However, most peptide-based spacers provide enough flexibility to allow back folding. Liskamp et al created high affinity ligands for the Syk-tSH2 domain by using propynyl benzoic acid and switchable azobenzene building blocks as rigid spacers34, 36, 54. However, our attempts to separate the two pYETL motifs by long, benzoic acid-based rigid spacers were plagued by solubility issues. We, therefore, took recourse to a peptide nucleic acid (PNA) spacer. Both, peptides and PNA sequences were prepared by solidphase peptide synthesis in one continuous run, which enables conjugate synthesis without post-synthetic conjugation chemistry. In conjugate 5b the two pYETL motifs are separated by a 10mer PNA segment (Figure 6A). This spacer length was sufficient to induce preferential recognition of the Syk tSH2 domain (Syk: IC50 = 0.3 µM; Zap-70: IC50 = 3.0 µM). Hybridization with complementary DNA in complex 6b increases the rigidity of the PNA spacer. The discrimination of the Zap-70 tSH2 protein remained unaffected (Syk: IC50 = 0.9 µM; Zap-70: IC50 = 8.1 µM). Interestingly, selectivity for interactions with Syk was not obtained with the TCR-derived ITAM motifs pYNEL and pYDVL in 5a. These findings suggest that the recognition of ITAMs by the tSH2 domains is influenced by distance, orientation and sequence of the consensus motifs.

Figure 6. Subtype-specific binders for tSH2 domains of Syk tSH2 (red) and Zap-70 tSH2 (green) through A) sequence-dependent interactions between bivalent pYXXL-PNA-pYXXL conjugates or B) distance-dependent interactions between bivalent pYXXL-oligoethylene glycol conjugates.

Selective Zap-70 tSH2 binders. The rational design of a unimolecular compound that preferentially interacts with ZAP-70 tSH2 appears challenging given the wide scope of Syk tSH2 interactions. Guided by the results of the DNAbased spatial screen we assumed that a close vicinity between the pYXXL motifs will be the key for ZAP-70selective tSH2 binding. We explored short oligoethyleneglycol units as linkers. This spacer is shorter and spatially less demanding than the DNA- or PNA-based spacers. Furthermore, we envisioned that the high flexibility of ethyleneglycol-type spacers will facilitate the spatially tight arrangement of the two pYXXL motifs required for high affinity interactions with both SH2 domains in Zap-70. Bivalent peptide-oligoethylenegycol conjugates (7a, 7b, Figure 6B) with recognition motives based on TCRζ TAM1derived peptide 2 and FcεRIγ-derived 4 were synthesized, each containing 1-3 aminoethylethoxyethyloxy acetic acid (aeea) spacer units. Conjugates involving a single aeea spacer unit (71a) were not recognized; neither by Syk tSH2 nor by ZAP-70 tSH2 (Figure 6B). Thus, one aeea-spacer was not sufficient to bridge the two SH2 domains. Interestingly, the desired selectivity for ZAP-tSH2 was obtained with peptides 72a/b, which comprise two aeea-units as a spacer between the two binding motives. The combination of two aeea-units with the TCR-derived ITAM recognition


5

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motives ‘pYNEL’ and ‘pYDVL’ in peptide 72a afforded a 15fold higher binding affinity for ZAP-tSH2 compared to SyktSH2. Peptides 73a/b containing a longer and more flexible linker of three aeea-units were not able to distinguish between the tSH2-domain ZAP-70 and Syk.

Discussion The recognition of immunoreceptor tyrosine-based interaction motifs (ITAMs) with the tandem SH2 domains of the Syk and Zap-70 kinases has previously been characterized in vitro by crystal structure analysis,14, 32 NMR,37 surface plasmon resonance,15, 36, 38 mass spectrometry55 and kinetic as well as thermodynamic measurements33, 34, 56. Our study revealed, for the first time, the consequences upon bivalent interactions with constrained ITAM derivatives. Of note, non-constrained ITAM peptides (1-4) are recognized by both Zap-70 and Syk tSH2 domains and therefore do not distinguish between the two closely related protein interaction modules (Table 1). This contradicts a previous report from Ottinger et al, where a TCRζ TAM1 peptide bound Zap-70 tSH2 with 14-fold higher affinity than Syk tSH2.15 We repeated the measurements by using identical peptides as Ottinger et al. (Table S5). However, both the Zap-70 and Syk tSH2 domains interacted equally well. The differential behavior may be a result of the different assays used. Ottmann et al. assessed binding by surface plasmon resonance (SPR). This heterogeneous assay may also respond to bridging of two adjacent peptides on the SPR chip. By contrast, we used a solution-based assay with low concentrations of tSH2 domain (80-120 nM). In this case, bridging is less likely to occur. Opportunities for a distinction between Syk tSH2 and Zap70 tSH2 domains became apparent when the ITAM peptides were constrained by means of bipartite presentation on DNA scaffolds. The DNA-programmed spatial screening revealed the narrow substrate scope of Zap-70 tSH2, which in contrast to Syk tSH2 proved sensitive to the strand orientation of the appended pYXXL motifs. To allow high affinity interactions with Zap-70 tSH2 the distance between the two pYXXL units must not exceed 3 spacer nucleotides (Fig. 3B). This corresponds to a distance < 10 Å. By contrast, the Syk tHS2 domain binds rather promiscuously to various bipartite assemblies until a critical pYXXL-pYXXL distance of 10-13 nt is reached. This knowledge allowed the design of Syk-tSH2 selective binders, in which PNA-based scaffolds were used as long length spacers. For the design of Zap-70 selective binders, the need for proximal arrangement of the pYXXL motifs must be taken into account. But why should this design criterion help penalize recognition by Syk tSH2? It has been reported that the interdomain connecting the two Syk SH2 domains can remain flexible even after bivalent recognition of an ITAM peptide.34 Binding of a constrained ITAM motif may therefore decrease the conformation entropy associated with the interdomain flexibility, which would result in a net decrease of free binding energy. This effect plausibly explains the data obtained in the binding experiments with the oligoethylene glycol conjugates 7la/b. In conjugate 71a/b the spacer is too short to reach to the second SH2 domain and

monovalent binding prevails. The two aminoethylethoxyethyloxy acetic acid (aeea) units long spacer in 72a apparently bridges the two SH2 domains in Zap-70 without perturbing the domain-domain contacts. Syk tSH2 does not rely on domain-domain contacts and recognition of proximal pYXXL motifs in 72a may unfavorably constrain the SH2-SH2-interdomain or cause steric clashes between the two SH2 domains. With an extension of oligoethylene glycol spacer in 73a/b, strain will be released and Syk tSH2 binds 73a/b as efficiently as does Zap-70 tSH2. The data described herein and the work of others45-53 attest to the power of DNA-programmed spatial screening. While previous work by Chaput,48, 57 Winssinger49, 50 and Merkx,52, 58 amongst others, focussed on fostering recognition of and exerting control over protein-based receptors, our work35, 40-42, 44 and notable contributions from Baird46 and Rinker et al47 examine distance-dependent multivalent binding as a means to systematically explore structural properties of the targeted proteins. Based on the results of the described binding experiments, it appears plausible that ‘native’ interaction partners in cells (that is proteins associated with the TCR complex for interactions with Zap-70 and proteins associated with the BCR complex for interactions with Syk) have secondary structure for constraining the orientation of the pYXXL motifs such that interactions with cognate kinase (Zap-70 or Syk) become optimal. This would assure specificity in situations when both kinases are expressed in the same cells. Importantly, DNAprogrammed spatial screening provides guidelines for the design of selective inhibitors.

Conclusion The tSH2 domains of Syk and ZAP-70 kinases recognize immunoreceptor tyrosine-based interaction motifs (ITAMs) comprising the consensus sequence pYXXI/L(X)68 pYXXI/L with near identical nanomolar affinity. By means of DNA-programmed spatial screening we searched for selective binders that distinguish between the two closely related tSH2 domains. The pYXXL motifs were attached to oligonucleotide strands. Hybridization of two phosphopeptide-DNA conjugates with template oligonucleotides provided bipartite ITAM motifs in which the phosphotyrosine-containing peptides were displayed in defined orientation and distance. The DNA-programmed spatial screening revealed remarkably different recognition characteristics of the tSH2 domains. Syk tSH2 has a rather broad substrate scope and accepts an array of ITAM-mimics with varied orientations of and distances between the pYXXL peptides. On the contrary, bivalent binding of ZAP-70 tSH2 requires a proximal arrangement of the phosphotyrosine ligands, which need to protrude from the DNA duplex via a combination of N- and C-terminal appendages rather than alternative parallel conjugation modes. The results likely reflect the different binding mechanisms characterized by distinct roles of SH2-SH2 interdomains (flexible in Syk vs. rigid in Zap-70) and intramolecular SH2-SH2 contacts (important for Zap-70 vs. unimportant for Syk). The results on the one hand suggest a means for differential activation when both Zap-70 and Syk kinases are coexpressed. In spite of similar affinities for ITAM peptides, constraint by secondary structure within the engaged B-


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cell and T-cell receptor complexes may account for specificity. On the other hand, the results guided the design of unimolecular tSH2 binders with subtype specificity. Showcasing the first set of submicromolar, mutually selective binders, ITAM analogues featuring long peptide nucleic acid (PNA) spacers were preferentially bound by Syk-tSH2. The use of a short oligoethylene glycol spacer provided for ZAP-70 selective recognition. We conclude that DNAprogrammed spatial screening is a suitable strategy to unravel opportunities for the design of subtype specific binders of closely related protein-protein interaction modules.

Experimentals Peptide and PNA synthesis. The peptides and PNApeptide-conjugates were synthesized on TentaGel R RAM resin from Novabiochem (Schwalbach, Germany) by Fmoc-based solid-phase synthesis and employing 2 mL polypropylene syringe reactors from Multisyntech (Witten, Germany) manually or by using an automatized ResPep synthesizer from Intavis (Koeln, Germany). Peptide synthesis was performed in a 5-20 µml scale, PNA-peptide conjugate synthesis in a 2 µmol scale. Fmoc-protected amino acids were purchased from Iris Biotech (Marktredwitz, Germany). PNA-monomers were purchased from ASM Research Chemicals (Burgwedel, Germany). HCTU was purchased from Carl Roth (Karlsruhe, Germany). DMF was purchased from Biosolve (Valkenswaard, Netherland). Fmoc-8-amino-3,6-dioxaoctanoic acid (Fmoc-aeea-OH) was supplied by Carl Roth. The resin was allowed to swell in DMF for 30 min. For Fmoc removal the resin was treated 2 x 4 min with DMF/piperidine (4:1 [v/v]) and subsequently washed with DMF (5 x). A pre-activation vessel was charged with 4 eq. amino acid in DMF (0.6M), 3.6 eq. HOBT, 10 eq. NMM and 3.6 eq. HCTU in DMF (0.6M) for amino acid couplings or with 4 eq. PNA-monomer in DMF, 3.6 eq. HCTU and 10 eq. NMM for PNA-monomer couplings. After 2 min pre-activation time the solution was transferred to the resin. The resin was incubated for 30 min, then washed with DMF (3 x). The coupling step was repeated after incorporation of the eighth amino acid. The Fmoc-Lys(Mmt)-OH building block - introduced to allow side chain conjugation of 6-maleimidocaproic acid - was incorporated manually. Capping of unreacted and free amines was achieved by using Ac2O/2,6-lutidine/DMF (6:5:89 [v/v/v]). The terminal capping step was performed twice. Prior to TFA cleavage the resin was washed 10 x with dichloromethane. The synthesis of fluorescence labelled peptide 1 and the introduction of the maleimido group required Mmt-removal which was achieved upon treatment with 5 x 1 ml of 5% TFA in DMF [v/v]. After 1 min the resin was washed (10 x DCM, 5 x DMF). To obtain the FAMpeptide 1 a solution of 10 eq. 5,6-carboxyfluorescein, 20 eq. NMM and 10 eq. PyBOP (0.6M) in DMF was transferred to the resin. After 5 h the resin was washed (10 x DMF/piperidine (4:1 [v/v]), 5x DMF, 10 x DCM). For the synthesis of maleimido peptides (14 in Fig. S1) a preactivated solution of 4 eq. 6-maleimidocaproic acid, 8 eq NMM and 3.6 eq HCTU (0.6 M) in DMF was added to the resin. The coupling was performed twice for two hours. Afterwards the resin was washed (5x DMF, 10x CH2Cl2). For final cleavage the peptide-resin was treated with 0.5-2 ml

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TFA/H2O/triisopropylsilane (950:25:25) for 1.5 h. The resin was washed with 0.1-0.5 ml TFA. The eluates were combined and 10 % H20 (vol%) was added. After 16 h cold diethylether was added. The supernatant was discarded and the precipitate washed with cold diethylether twice. The crude peptide was dissolved in a water/MeCN mixture (050% MeCN, max 900µl) and purified by semi preparative HPLC using an Agilent 1100 series instrument (Varian polaris C18 A 5 µ 250x21 nm) and solvents A (98.9% H2O, 1% MeCN, 0.1% TFA) and B (98.9% MeCN, 1% H2O, 0.1% TFA) in a linear gradient. The product was characterized using Acquity-UPLC system from Waters and a BEH130 C18 column (1.7 µm, 2.1 x 50 mm, heater set on 50°C) with a binary mixture of the solvents A (98.9% H2O, 1% MeCN, 0.1% TFA) and B (98.9% MeCN, 1% H2O, 0.1% TFA) as a mobile phase (flow: 0.5 ml/min) in a linear gradient. Oligonucleotide synthesis. Oligonucleotide synthesis was performed on a Bioautomation MerMade 4 synthesizer (Irving, Texas) yielding oligomers carrying the terminal DMTr protective group (“trityl-on”). The SynBaseTM CPG support (1µmol, pore size 500 Å), the 3’-thiol-modifiers DMTr-O-C3H6-S-S-C3H6-O2C2H4CO-CPG and the 5’thiol-modifier Tr-S-C6H12-O-P(NiPr2)2-OCNE for the synthesis of the 3’- and 5’-thiol-modified oligonucleotides (ON3 or ON4-5 in Fig. S2) were purchased from Link Technologies Ltd. (Lanarkshire, Scotland). The DNA synthesis reagents were purchased from Roth (Karlsruhe, Germany) and EMP-Biotech (Berlin, Germany). The phosphoramidites dT, dG(DMF), dC(Bz) and dA(Bz) were obtained from Thermo Fischer Scientific (Waltham, USA). All phosphoramidites were used according to manufacturer’s instructions. The yields of coupling steps were monitored by measuring conductivity of the DMTr-cleavage solution. For removal of the nucleobase protection groups and cleavage from the solid support the solid support was treated for 2 h with aqueous ammonia (28-32 %) at 55°C. After lyophilisation, the crude oligonucleotides were purified by RP-HPLC (DMT-on) using a 1105 HPLC-system from Gilson and a Waters X-Bridge BEH130 C18 (5 µ, 10x150 mm, 55°C) at a flow rate of 8 ml/min with a linear gradient of MeCN in 0.1 M triethylammonium acetate (pH 7.5). The DMTr-group was removed with 50% AcOH for 30 min, at room temperature. The detritylation mixture was treated with 3 M sodium acetate solution and the oligonucleotide precipitated with cold iPrOH. All oligonucleotides were characterized by using a 1105 HPLC-system from Gilson and a Waters X-Bridge BEH130 C18 (5 µ, 4.6x150 mm, 55°C) and MALDI-TOF mass spectrometry using a Shimadzu Axima Confidence. Unmodified oligonucleotides were purchased from Biotez GmbH (Berlin, Germany) or Biomers.net GmBH (Ulm, Germany). Synthesis of DNA-peptide conjugates. To remove the thiol protection group the thiol-modified oligonucleotides were dissolved to a final concentration of 0.5 mM in a degassed buffer (pH 6.5) containing 5 mM TCEP, 10 mM NaCl, and 10 mM NaH2PO4. The mixture was agitated for 1-4 hours. MALDI-TOF analysis was used to monitor the progress of the reaction. After completed deprotection the oligonucleotide was precipitated by the addition of 20%


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volume of a 3M NH3OAc solution (pH 5.2) and isopropanol. To prevent disulfide formation the obtained oligonucleotide was dried under argon flow. The dried oligonucleotide was dissolved in a degassed buffer (10 mM NaCl, 10 mM NaH2PO4, pH 6.5) containing 2 equivalents of the corresponding maleimide-peptide to a final concentration of 1 mM. The coupling was completed after 1-2 h. Protein expression. GST-Syk-tSH2 (residue 7-268) was overexpressed and purified as previously described.35 GSTZAP-70 tSH2 (1-257) was obtained from GeneScript (Piscataway, NJ, USA) in a buffer containing 50 mM Tris-HCL, 200 mM NaCl and 15% glycerol (pH 8.0, protein content: 1.5 mg/ml). Determination of IC50 values using fluorescence polarization. Binding activity was assessed in a microtiter plate (black 384 well plate, Viewplate, Perkin Elmer) format by means of fluorescence polarization experiments performed by using a Victor X5 multiplate reader from Perkin Elmer. For binding of Syk tSH2, tested compounds were dissolved in buffer 1 (150 mM NaCl, 50 mM HEPES, 0.01% BSA; pH 7.5). Binding of Zap-70 tSH2 was characterized by using buffer 2 (20 mM NaH2PO4, 100 mM NaCl, 2 mM DTT; pH 7.4. The FAM-labeled reference peptide FamKpYTGLNTRSQETpYETLG (1) was diluted in buffer to a final 20 nM concentration. The measured fluorescence anisotropy provided the value corresponding to the unbound reference peptide. The fluorescence polarisation corresponding to bound Fam-peptide 1 was measured after addition of 80 nM GST-labelled tSH2 domain. Afterwards, samples containing a constant concentration of reference peptide 1 (20 nM) and GST-tSH2 domain (80 nM) and an increasing concentration of peptide 2−4, DNA-peptide complex TCn 1a/b − TCn 8a/b, PNA-peptide conjugate 5a/b and 6a/b or peptide oligoethylene glycol conjugates 7la/b were prepared (dilution series) in Eppendorf tubes at 4°C. The samples were transferred to the microtiter plate, centrifuged (1000 rqm, 1 min) and measured immediately. The normalized anisotropy was plotted against the logarithmic concentration of the conjugates. Data analysis was performed by means of a sigmoidal dose response model with variable slope (Y = 1 / (1+10(log(IC50/c)) slope), where Y = normalized anisotropy; c = concentration of inhibitor) in GraphPad Prism version 4.03. The variable slope accounts for putative cooperative binding and/or complexes formed in other than [1+1] stoichiometries. The sigmoidal shape indicated that the steepness of the binding isotherms was not caused by conditions of active site titration (Figures S4−S6). Each IC50 value was measured at least twice.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Syntheses and characterization of peptides, peptide-PNA conjugates, peptide-oligoethyleneglycol conjugates and peptide-oligonucleotide conjugates, binding isotherms and tabulated IC50-values

for the interaction of peptides conjugates with the tSH2 domains in Syk and ZAP-70 kinases.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

ORCID Oliver Seitz: 0000-0003-0611-4810

Notes The authors declare no competing financial interest.

Author Contributions The manuscript was written through contributions of all authors.

Funding Sources Deutsche Forschungsgemeinschaft, Collaborative Research Center 765.

ACKNOWLEDGMENT We acknowledge support from Deutsche Forschungsgemeinschaft CRC 765.

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Selective binders of the tandem SH2 domains in Syk and ZAP-70

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