High‐resolution structural analysis shows how different crystallographic environments can induce alternative modes of binding of a phosphotyrosine peptide to the SH2 domain of Fer tyrosine kinase

Abstract

Fes and Fes‐related (Fer) protein tyrosine kinases (PTKs) comprise a subfamily of nonreceptor tyrosine kinases characterized by a unique multidomain structure composed of an N‐terminal Fer/CIP4 homology‐Bin/Amphiphysin/Rvs (F‐BAR) domain, a central Src homology 2 (SH2) domain, and a C‐terminal PTK domain. Fer is ubiquitously expressed, and upregulation of Fer has been implicated in various human cancers. The PTK activity of Fes has been shown to be positively regulated by the binding of phosphotyrosine‐containing ligands to the SH2 domain. Here, the X‐ray crystal structure of human Fer SH2 domain bound to a phosphopeptide that has D‐E‐pY‐E‐N‐V‐D sequence is reported at 1.37 å resolution. The asymmetric unit (ASU) contains six Fer‐phosphopeptide complexes, and the structure reveals three distinct binding modes for the same phosphopeptide. At four out of the six binding sites in the ASU, the phosphopeptide binds to Fer SH2 domain in a type I β‐turn conformation, and this could be the optimal binding mode of this phosphopeptide. At the other two binding sites in the ASU, it appears that spatial proximity of neighboring SH2 domains in the crystal induces alternative modes of binding of this phosphopeptide.

1. INTRODUCTION

The Src homology 2 (SH2) domain is a phosphotyrosine (pY)‐binding domain that functions as a highly versatile component of many cell signaling mechanisms.1 At least 120 unique SH2 domains have been identified in human proteins including kinases, phosphatases, transcription factors, and adaptor proteins.2 At present, over 300 structures of SH2 domains have been determined and released in the Protein Data Bank (PDB), covering approximately 50% of all human SH2 domains.2 All of these structures share a conserved domain structure that consists of a central antiparallel β‐sheet made of seven strands (βA‐βG) flanked on either side by two α‐helices (αA and αB).3, 4 The SH2 domains bind to short stretches of amino acids that contain pY residues and thereby mediate protein–protein interactions during cell signaling. Typically, SH2 domains contain two major pockets on their surface for ligand binding: a pY binding pocket, and a second pocket (a so‐called specificity‐determining pocket) to accommodate adjacent residues located C‐terminal to the pY residue in the ligand.3, 4 Although the pY binding pocket is highly conserved, the second pocket is more variable and determines the binding specificity that is crucial for the accuracy of signal transduction.1

The SH2 domain was first described as a noncatalytic domain of Fes (also known as Fps) that can profoundly influence the adjacent kinase domain in the context of P130^gag‐fps viral oncoprotein.5 Fes and Fes‐related (Fer) protein tyrosine kinases (PTKs) comprise a distinct subfamily of nonreceptor PTKs characterized by a multidomain structure composed of an N‐terminal Fer/CIP4 homology‐Bin/Amphiphysin/Rvs (F‐BAR) domain, a central SH2 domain, and a C‐terminal PTK domain.6 Fes is primarily expressed in hematopoietic cells of myeloid lineage and is involved in hematopoiesis and cytokine signal transduction.7 In contrast to Fes, Fer is ubiquitously expressed and regulates diverse processes including cell adhesion, migration, and proliferation.7, 8, 9, 10, 11 Fer is activated upon growth factor stimulation,12, 13 and is a key target of c‐Src in oncogenic signaling.14 Upregulation of Fer has been implicated in various human cancers, and Fer is receiving a great deal of attention as a promising target for cancer therapy.14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32

The crystal structures of a C‐terminal fragment of Fes containing both the SH2 domain and the PTK domain have been determined in both active and inactive conformations,33, 34 and it has been shown that the SH2 domain interacts with the PTK domain in such a way that the binding of pY‐containing ligands to the SH2 domain positively regulates the PTK activity.33 Because the key residues involved in the SH2‐PTK interactions are highly conserved in Fes/Fer, it seems likely that this allosteric activation mechanism also applies to Fer. However, it has remained elusive how pY‐containing ligands bind to the SH2 domain of Fes/Fer. Although specific sequence motifs recognized by the SH2 domains of Fes/Fer have been studied in vitro using high‐throughput array‐based approaches, these studies have yielded somewhat controversial results for Fes/Fer.35, 36 Using a degenerate phosphopeptide library, Songyang et al. showed that the Fes SH2 domain recognizes the pY‐E‐x‐V/I motif (where x denotes any natural amino acid), with no apparent selectivity for position pY + 2.35 By contrast, Huang et al. used an oriented peptide array library (OPAL) approach and found that both Fes and Fer have strong preference for Asn at position pY + 2.36 To explore the nature of ligand binding, especially in terms of the selectivity for positions pY + 2 and pY + 3, I have determined the crystal structure of Fer SH2 domain bound to a synthetic phosphopeptide containing pY‐E‐N‐V sequence.

2. RESULTS AND DISCUSSION

2.1. Overall structure of human Fer SH2 domain bound to a phosphopeptide

The human Fer SH2 domain (residues 453–552) bound to a 7‐residue phosphopeptide (D‐E‐pY‐E‐N‐V‐D; I refer to this peptide as DEpYENVD) crystallized in the monoclinic space group P2₁ with six Fer‐peptide complexes in the asymmetric unit (ASU) (Figure S1), giving a Matthews coefficient of 2.17 å³/Da and a solvent content of 43%. The structure was refined at 1.37 å resolution to free and working R‐factor values of 18.55 and 16.46%, respectively, and the final model had excellent stereochemistry (Table 1).

Table 1.

Crystallographic statistics

Data collection
Space group	P21
Unit cell dimensions
a, b, c (å)	34.70, 186.66, 50.58
α, β, γ (degree)	90.00, 96.96, 90.00
Wavelength (å)	0.98
X‐ray source	Photon factory BL‐17A
Resolution range (å)a	25.87–1.37 (1.39–1.37)
No. of measured reflectionsa	409,258 (16,283)
No. of unique reflectionsa	130,076 (5,919)
Completeness (%)a	97.7 (89.8)
R merge (%)a	4.4 (11.6)
Mean I/σ(I)a	14.8 (6.5)
Mean I half‐set correlation CC(1/2)a	0.997 (0.981)
Multiplicitya	3.1 (2.8)
Wilson B factor (å2)	10.7
Refinement
Resolution range (å)a	25.87–1.37 (1.39–1.37)
R work (%)a	16.46 (19.37)
R free (%)a	18.55 (21.01)
No. of atoms
Protein	5,326
Water	1,075
No. of amino acids	641
Mean B factor (å2)
Fer	18.3
Phosphopeptide	28.8
Water	30.3
RMSD from ideality
Bond lengths (å)	0.004
Bond angles (degree)	0.752
Protein geometryb
Ramachandran favored (%)	97.66
Ramachandran outliers (%)	0
Rotamer outliers (%)	0
Cβ outliers (%)	0
MolProbity score (percentile)	1.18 (97th)
PDB code	6KC4

^aValues in parentheses are for the highest‐resolution shell.

^bMolProbity was used to analyze the structure.

In the crystal structure, the Fer SH2 domain adopts the canonical SH2 fold (Figure S2), as observed in the solution structure of its apo‐form (PDB code, 2KK6; determined by Tang et al.) and in the crystal structures of Fes.33, 34 Throughout this report, the standard nomenclature for the SH2 domain secondary structures37 is adopted, as labeled in Figure S2. For clarity, the amino acid residues of the Fer SH2 domain will be referred to using three‐letter code, whereas those of the DEpYENVD peptide will be referred to using single‐letter code. The central core of the Fer SH2 domain is constructed by a three‐stranded anti‐parallel β‐sheet composed of βB (Asp479‐Gln484), βC (Tyr492‐Ser498), and βD (Gln501‐Ile506), which is flanked on one side by helix αA (Arg467‐Leu473) and on the other side by helix αB (Ile524‐Thr534). The βD is followed by a short strand βD′ (Gln508‐Val510) that forms a small antiparallel β‐sheet with βE (Met513‐Arg515), which packs against the helix αB. A short β‐strand βG (Asn548‐Pro549) forms hydrogen bonds with βB and caps the core of the SH2 domain on the side opposite to the phosphopeptide‐binding surface. Short β‐strands βA and βF observed in many other structures of SH2 domains3, 4 are not present in the Fer SH2 domain. The intervening loops connecting the secondary structures are named after their adjacent secondary structural elements.

The six Fer‐peptide complexes in the ASU can be viewed as two trimers in which the three Fer‐peptide complexes are related by pseudo three‐fold rotational symmetry (Figure S1). The six complexes in the ASU provide six crystallographically independent views of the Fer‐peptide interactions. The structures of all molecules of Fer (chains A, C, E, G, I, and K) in the ASU are very similar, with the C_α root mean square deviation (r.m.s.d.) between any two of the Fer chains being smaller than 0.45 å. Interestingly, however, not all chains of the DEpYENVD peptide complexed with Fer in the ASU adopt the same conformation. Although four (chains B, F, J, and L) out of the six chains (chains B, D, F, H, J, and L) of the DEpYENVD peptide in the ASU have essentially identical conformation (Figures 1a and 2a; referred to as binding mode 1), chain H (Figures 1b and 2b; referred to as binding mode 2) and chain D (Figures 1c and 2c; referred to as binding mode 3) adopt distinctly different conformations (Figure 1d) as described in detail below. Although conformational diversity of phosphopeptides bound to Src SH2 domain was previously proposed by a molecular dynamics simulation study,38 the present study is, to my knowledge, the first experimental demonstration that an SH2 domain can have the versatility to recognize multiple conformations of the same ligand.

Figure 1.

The Fer SH2 domain bound to the DEpYENVD peptide. (a–c) overall structure of human Fer (ribbon representation) bound to the DEpYENVD peptide (stick representation) in three distinct binding modes (a, binding mode 1; b, binding mode 2; c, binding mode 3). The omit F _O–F _C map covering the phosphopeptide is shown in blue mesh (contoured at 3.0σ) with the refined model of the peptide. (d) Overlay of the three binding modes of the DEpYENVD peptide. The tubes represent the main‐chain traces of the DEpYENVD peptide (orange, binding mode 1; green, binding mode 2; blue, binding mode 3)

Figure 2.

Stereo diagrams of the interactions between the Fer SH2 domain (ribbon representation) and the DEpYENVD peptide (stick representation) in three distinct binding modes (a, binding mode 1; b, binding mode 2; c, binding mode 3). Water molecules are depicted as cyan spheres. Dashed lines represent hydrogen bonds or salt bridges

2.2. Recognition of the phosphopeptide: Binding mode 1

In the binding mode 1, all residues except for the C‐terminal aspartate (D7) of the DEpYENVD peptide are well defined in the electron density map (Figure 1a). The side chain of the N‐terminal aspartate (D1) at position pY‐2 is involved in crystal packing interactions and forms hydrogen bonds with the side chains of Tyr509 and Asn512 of an adjacent Fer molecule in the ASU (Figure S1). Essentially the same interactions between the D1 side chain and Tyr509 and Asn512 of an adjacent Fer molecule are observed for all DEpYENVD peptides (including those in binding modes 2 and 3) in the ASU, and may help stabilize the symmetrical trimers of Fer‐peptide complexes in the crystal. The other residues of the DEpYENVD peptide make direct contacts only with the Fer SH2 domain to which the peptide is directly bound (Figure 2a), and this is also the case in binding modes 2 and 3. The side chain of E2 at position pY‐1 projects into solvent and makes no specific interactions with Fer, but the main‐chain carbonyl of E2 is hydrogen bonded to the side chain of Arg467. The side chain of pY inserts into the conserved pY binding pocket formed by the central β‐sheet, helix αA, and the BC loop. This pocket is highly basic (Figure S3a). The phosphate group of pY forms hydrogen bonds with main‐chain amide groups of Fer residues His486 and Gly487. Furthermore, the phosphate group forms salt bridges with the guanidinium side‐chain groups of Arg467 and Arg483. The oxygen atom bridging the phosphate group and the aromatic ring of pY forms a hydrogen bond with the side chain of Ser485. The aromatic ring of pY is cradled in a hydrophobic pocket formed by the nonpolar side chains of Val493, Ile506 and the aliphatic portions of the side chains of Lys488 and His504. The four residues from pY to pY + 3 adopt a type I β‐turn conformation with a hydrogen bond made between the main‐chain carbonyl of the pY and the main‐chain amide of V6 at position pY + 3, and pack against the exposed edge of the strand βD. The side chain of E4 at position pY + 1 lies across the aromatic ring of Phe505 and interacts with the main‐chain amide of His504 through water‐bridged hydrogen bonding. In addition, the carboxyl group of E4 is located about 4 å from the guanidinium group of Arg503, indicating a somewhat weak hydrogen bond. The main‐chain amide of E4 forms a hydrogen bond with the main‐chain carbonyl of His504. The side chain of N5 at position pY + 2 makes bidentate hydrogen bonds with the main‐chain amide and carbonyl of Ile506. The hydrophobic side chain of V6 at position pY + 3 contacts the side chain of Ile506. Thus, four residues of the peptide (pY, E(pY + 1), N(pY + 2), and V(pY + 3)) appear to contribute to specific recognition of this peptide by Fer SH2 domain in this binding mode.

The way the DEpYENVD peptide is specifically recognized by the Fer SH2 domain in the binding mode 1 is strikingly similar to the way pYxNx peptide is recognized by Grb2 SH2 domain in a type I β‐turn conformation (Figure S4).39 In the case of Grb2 SH2 domain, the classical explanation for the recognition of the peptide in this conformation is that the bulky side chain of Trp121 prevents the phosphopeptide from adopting an extended conformation and forces the peptide to adopt a β‐turn, with the side chain of asparagine at position pY + 2 forming hydrogen bonds with the exposed edge of the strand βD (Figure S4).39 By contrast, in the case of Fer SH2 domain, there is no such structural element (corresponding to Trp121 of Grb2) that forces the peptide to bind only in the β‐turn conformation, and the peptide can also bind Fer in alternative conformations as described below.

2.3. Recognition of the phosphopeptide: Binding mode 2

In the binding mode 2, residues 1–6 of the DEpYENVD peptide are well defined in the electron density map (Figure 1b). In this binding mode, the interactions in the pY binding pocket are essentially the same as those described for the binding mode 1 (Figure 2b). The differences between the binding modes 1 and 2 are in the details of the interactions between Fer and the peptide residues from pY + 1 to pY + 3 [Figure 2b]. In the binding mode 2, the side chain of E4 at position pY + 1 adopts a conformation different from that in the binding mode 1 and makes bidentate salt bridges with Arg503. The backbone of the peptide in the binding mode 2 is twisted at pY + 2 but it is not in a β‐turn conformation. In contrast to the binding mode 1 in which the main‐chain amide and carbonyl of Ile506 form bidentate hydrogen bonds with the N5 (pY + 2) side chain, the main‐chain amide of Ile506 makes a hydrogen bond with the main‐chain carbonyl of E4 (pY + 1) in the binding mode 2. The main‐chain carbonyl of Ile506 is hydrogen bonded to the N5 (pY + 2) side chain, which makes another hydrogen bond with the main‐chain carbonyl of Phe516. The side chain of N5 (pY + 2) also interacts with Glu517 and Thr539 through water‐bridged hydrogen bonding. The position of N5 (pY + 2) is further stabilized by a hydrogen bond made between its main‐chain carbonyl and the side chain of Gln508. In the binding mode 2, the side chain of V6 (pY + 3) is exposed to solvent and does not make direct contact with Fer. This is in stark contrast to the binding mode 1, in which the V6 side chain is involved in hydrophobic interaction with Fer. Thus, three residues of the peptide (pY, E(pY + 1), and N(pY + 2)) appear to be important for specific recognition of this peptide by Fer SH2 domain in this binding mode.

2.4. Recognition of the phosphopeptide: Binding mode 3

In the binding mode 3, all residues of the DEpYENVD peptide are well defined in the electron density map (Figure 1c). In this binding mode, the peptide adopts a fully extended conformation, as observed previously in the majority of the structures of SH2 domain‐phosphopeptide complexes,3, 4 and binds perpendicular to the central β‐sheet of the Fer SH2 domain. The interactions in the pY binding pocket is again essentially identical with those described for the binding mode 1 (Figure 2c). Analogous to the binding mode 2, the side chain of E4 (pY + 1) makes a salt bridge with Arg503 in the binding mode 3. A unique feature of the binding mode 3 is that the side chain of N5 at position pY + 2 does not make specific interactions with Fer and, instead, makes an intramolecular hydrogen bond with the D7 (pY + 4) side chain. This hydrogen bond, together with hydrogen bonding networks formed between the main chain atoms of the peptide and Fer residues (Ile506, Glu517, and Thr539), may help stabilize the extended conformation of the phosphopeptide. In this conformation of the peptide, the side chain of V6 (pY + 3) faces toward Fer and fits into an electrostatically neutral pocket formed by Phe505, Ile506, Gln508, Phe516, Glu517, and Thr539 (Figures 2c and S3c). This is reminiscent of the classical structure of Src SH2 domain bound to a high affinity peptide containing pY‐E‐E‐I sequence, with the side chain of isoleucine at position pY + 3 inserted into a hydrophobic pocket (Figure S5).40 Thus, three residues of the peptide (pY, E(pY + 1), and V(pY + 3)) appear to contribute to specific recognition of the DEpYENVD peptide by Fer SH2 domain in the binding mode 3.

2.5. Analysis of the effect of crystal packing on the geometry of ligand binding

Close inspection of the microenvironment surrounding the DEpYENVD peptide at the peptide‐binding sites in the ASU suggests that the binding modes 2 and 3 are most likely influenced by packing of Fer‐peptide complexes in the crystal lattice (Figures S6, S7, and S8; Table 2). At the peptide binding site on chain G of Fer, superposition of the peptide in the binding mode 1 to the peptide in the binding mode 2 (the observed mode of binding at this site) indicates that the C‐terminal residue (D7) of this peptide would sterically clash with an adjacent molecule of Fer in the crystal if the peptide adopted the conformation of the binding mode 1 (Figure S6). Thus, a peptide with the conformation of the binding mode 1 cannot be accommodated at this site in the crystal (Table 2). Likewise, a peptide with the conformation in the binding mode 3 cannot bind to this site due to steric clash at the C‐terminus of the peptide with another neighboring molecule of Fer in the crystal (Figure S6). Thus, packing of molecules in the crystal is such that, among the three binding modes, only the binding mode 2 is sterically allowed at this site.

Table 2.

Sterically allowed binding and observed binding at each of the six peptide‐binding sites in the asymmetric unit of the crystal

	Chain		Mode 1		Mode 2		Mode 3
Site	Fer	Peptide	Sterically allowed	Observed	Sterically allowed	Observed	Sterically allowed	Observed
1	A	B	Yes	Yes	Yes	No	Yes	No
2	C	D	No	No	Yes	No	Yes	Yes
3	E	F	Yes	Yes	Yes	No	Yes	No
4	G	H	No	No	Yes	Yes	No	No
5	I	J	Yes	Yes	Yes	No	Yes	No
6	K	L	Yes	Yes	Yes	No	Yes	No

At the peptide binding site on chain C of Fer, similarly to the binding site on chain G, steric clash with an adjacent molecule of Fer in the crystal would prevent the DEpYENVD peptide from adopting the β‐turn conformation (the mode 1 conformation) when bound to this site (Figure S7). At this site, the C‐terminus of the peptide in binding mode 3 is located close to adjacent molecules of Fer in the crystal, making a long‐range electrostatic interaction with Lys552 and a hydrogen bonding network via bridging water molecules with His462 and Tyr461 (Figure S8). These additional interactions may help stabilize the extended conformation of the peptide bound to this site. Although a peptide with the conformation of the binding mode 2 can also be sterically allowed at this site, the potentially stabilizing interactions with neighboring molecules specific to the binding mode 3 as depicted in Figure S8 could account for the reason why the binding mode 3 is favored over the binding mode 2 at this site.

At the other four binding sites (binding sites on chains A, E, I, and K of Fer), a peptide with any of the mode 1, mode 2, or mode 3 conformation can be accommodated without incurring steric clash with neighboring molecules (Table 2). Interestingly, however, only the binding mode 1 was observed at these four sites in the crystal structure (Table 2), suggesting that the binding mode 1 is the most energetically favorable binding mode for this phosphopeptide.

3. CONCLUSIONS

In summary, the high‐resolution crystal structure reported here suggests that the Fer SH2 domain can specifically recognize the DEpYENVD peptide in at least three distinctly different ways, and indicates that all three residues C‐terminal to pY can contribute to specific recognition in diverse ways. Analysis of crystal packing suggests that, among the three binding modes, the binding mode 1 is most likely the optimal binding mode of this phosphopeptide, and the other two binding modes appear to be suboptimal binding modes, which are induced because of spatial proximity of neighboring molecules in the crystal. The structural information obtained in the present study will lay the foundation for future studies on the interaction between Fer and diverse signaling molecules.

4. MATERIALS AND METHODS

4.1. Expression and purification of Fer SH2 domain

N‐terminally glutathione S‐transferase (GST)‐tagged Fer (human, residues 453–552; UniProt code, P16591) was expressed in the Escherichia coli host strain BL21‐CodonPlus(DE3)RIL (Stratagene) at 20°C from pGEX‐TEV.41 The cells were harvested, frozen in liquid nitrogen, and stored at −25°C. The cell pellets were resuspended in buffer A (10 mM Tris–HCl pH 7.5, 0.3 M NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride [PMSF], and 2 mM 2‐mercaptoethanol) and lysed by sonication on ice. All subsequent steps were performed at 4°C. Tween20 was added to 0.1%, and clarified lysates were incubated with glutathione‐Sepharose 4B (GE Healthcare) for 4 h. After washing the beads with buffer B (10 mM Tris–HCl pH 7.5, 0.3 M NaCl, 0.05% Tween20, and 2 mM 2‐mercaptoethanol), the GST‐tag was removed with His‐TEV protease (80 μg/mL) overnight. Fer SH2 domain released from the resin was finally purified over Superdex200 (GE Healthcare) in buffer C (10 mM Tris–HCl pH 7.5, 0.3 M NaCl, and 2 mM 2‐mercaptoethanol). Fer SH2 domain was concentrated to 4 mM using a 3 kDa molecular weight cutoff Amicon Ultra centrifugal filter (Millipore).

4.2. Crystallization, data collection, and structure determination

A phosphopeptide (D‐E‐pY‐E‐N‐V‐D; referred to as DEpYENVD peptide) was synthesized by GenScript. Crystals of human Fer SH2 domain bound to the DEpYENVD peptide were grown at 20°C from 1.2 mM Fer SH2 domain and 1.4 mM DEpYENVD peptide by hanging drop vapor diffusion against 0.1 M HEPES (pH 6.8), 0.4 M NaCl, and 34% PEG3350. Crystals were harvested directly from the drop and flash‐cooled in liquid nitrogen. X‐ray diffraction datasets were collected at 95 K at Photon Factory beamline BL‐17A using an EIGER X 16M detector at a wavelength of 0.98 å. Diffraction data were processed using MOSFLM and CCP4 programs.42 The structure was solved by molecular replacement using MOLREP43 using the solution structure of human Fer SH2 domain (PDB code, 2KK6) as a search model. The structure was refined by iterative cycles of model building using COOT44 and refinement using PHENIX.45 MolProbity46 was used to validate the final model. Structural figures were produced using CCP4MG.47

4.3. Accession number

The coordinates and structure factors of human Fer SH2 domain bound to the DEpYENVD peptide have been deposited in the PDB with accession code 6KC4.

CONFLICT OF INTEREST

The author declares no conflict of interest in publishing the results of this study.

Supporting information

Figure S1 Two orthogonal views of the six Fer‐peptide complexes in the asymmetric unit of the crystal. Fer is shown in ribbon representation, whereas the DEpYENVD peptide is shown in stick representation.

Figure S2. The Fer SH2 domain structure in ribbon representation with the helices, strands, and loops labeled.

Figure S3. Stereo diagrams of the Fer SH2 domain (surface representation) bound to the DEpYENVD peptide (stick representation) in three distinct binding modes (A, binding mode 1; B, binding mode 2; C, binding mode 3). The molecular surface of Fer is colored according to electrostatic potential, shaded from blue (potential +500 mV) through white (potential 0 mV) to red (potential −500 mV).

Figure S4. (A, B) Stereo diagrams of the structure of Grb2 SH2 domain (ribbon representation in (A) and surface representation in (B)) bound to a phosphopeptide (stick representation) containing pY‐V‐N‐V sequence (PDB code, 1TZE). Dashed lines represent hydrogen bonds or salt bridges. In (B), the molecular surface of Grb2 is colored according to electrostatic potential, shaded from blue (potential +500 mV) through white (potential 0 mV) to red (potential −500 mV). (C) Stereo diagram of overlay of the Grb2 SH2‐phosphopeptide complex (PDB code, 1TZE) with Fer SH2‐DEpYENVD peptide complex (binding mode 1). Grb2 in light blue, Fer in orange. The main‐chain traces of the bound peptides are represented in the corresponding colors with the side chains of the pY and pY + 2 residues shown in stick representation.

Figure S5. (A, B) Stereo diagrams of the structure of Src SH2 domain (ribbon representation in (A) and surface representation in (B)) bound to a phosphopeptide (stick representation) containing pY‐E–E‐I sequence (PDB code, 1SPS). Dashed lines represent hydrogen bonds or salt bridges. In (B), the molecular surface of Src is colored according to electrostatic potential, shaded from blue (potential +500 mV) through white (potential 0 mV) to red (potential −500 mV). (C) Stereo diagram of overlay of the Src SH2‐phosphopeptide complex (PDB code, 1SPS) with Fer SH2‐DEpYENVD peptide complex (binding mode 3). Src in light blue, Fer in orange. The main‐chain traces of the bound peptides are represented in the corresponding colors with the side chains of the pY and pY + 3 residues shown in stick representation.

Figure S6. The microenvironment surrounding the DEpYENVD peptide (stick representation with yellow carbons) in the binding mode 2 bound to chain G of Fer SH2 domain (ribbon representation in gray) in the crystal. Adjacent molecules of Fer SH2 domain are shown in cyan and green (ribbon representation and transparent molecular surface). Superposed DEpYENVD peptide in the binding mode 1 (stick representation with orange carbons) suggests that the C‐terminal residue of this peptide would clash with the Fer molecule in cyan at the position marked by a black dashed circle if the peptide adopted the conformation of the binding mode 1. Likewise, superposed DEpYENVD peptide in the binding mode 3 (stick representation with magenta carbons) suggests that the C‐terminal residue of this peptide would clash with the Fer molecule in green at the position marked by a red dashed circle if the peptide adopted the conformation of the binding mode 3.

Figure S7. The microenvironment surrounding the DEpYENVD peptide (stick representation with yellow carbons) in the binding mode 3 bound to chain C of Fer SH2 domain (ribbon representation in gray) in the crystal. An adjacent molecule of Fer SH2 domain is shown in purple (ribbon representation and transparent molecular surface). Superposed DEpYENVD peptide in the binding mode 1 (stick representation with orange carbons) suggests that the C‐terminal residue of this peptide would clash with the Fer molecule in purple at the position marked by a dashed circle if the peptide adopted the conformation of the binding mode 1.

Figure S8. The microenvironment surrounding the DEpYENVD peptide (stick representation with yellow carbons) in the binding mode 3 bound to chain C of Fer SH2 domain (ribbon representation in gray) in the crystal. Two adjacent molecules of Fer located close the C‐terminus of the peptide in the crystal are shown in ribbon representation in green and light blue. Water molecules are depicted as cyan spheres. Black dashed lines represent hydrogen bonds, whereas the dashed line in magenta represent long‐range electrostatic interaction. These interactions may help stabilize the extended conformation of the peptide in the binding mode 3.

Matsuura Y. High‐resolution structural analysis shows how different crystallographic environments can induce alternative modes of binding of a phosphotyrosine peptide to the SH2 domain of Fer tyrosine kinase. Protein Science. 2019;28:2011–2019. 10.1002/pro.3713