Structural basis for syk tyrosine kinase ubiquity in signal transduction pathways revealed by the crystal structure of its regulatory SH2 domains bound to a dually phosphorylated ITAM peptide1

Article No. mb981964

J. Mol. Biol. (1998) 281, 523±537

Structural Basis for Syk Tyrosine Kinase Ubiquity in Signal Transduction Pathways Revealed by the Crystal Structure of its Regulatory SH2 Domains Bound to a Dually Phosphorylated ITAM Peptide Klaus FuÈtterer1, Jane Wong2, Richard A. Grucza1, Andrew C. Chan2,3 and Gabriel Waksman1* 1

Department of Biochemistry and Molecular Biophysics 2 Departments of Medicine and Pathology and 3Howard Hughes Medical Institute Washington University School of Medicine, Campus Box 8231 660 South Euclid Av. Saint Louis, MO 63110, USA

The Syk family of kinases, consisting of ZAP-70 and Syk, play essential roles in a variety of immune and non-immune cells. This family of kinases is characterized by the presence of two adjacent SH2 domains which mediate their localization to the membrane through receptor encoded tyrosine phosphorylated motifs. While these two kinases share many structural and functional features, the more ubiquitous nature of Syk has suggested that this kinase may accommodate a greater variety of motifs to mediate its function. We present the crystal structure of the tandem SH2 domain of Syk complexed with a dually phosphorylated ITAM peptide. The structure was solved by multiple isomorphous replacement Ê resolution. The asymmetric unit comprises six copies of the at 3.0 A liganded protein, revealing a surprising ¯exibility in the relative orientation of the two SH2 domains. The C-terminal phosphotyrosine-binding site is very different from the equivalent region of ZAP-70, suggesting that in contrast to ZAP-70, the two SH2 domains of Syk can function as independent units. The conformational ¯exibility and structural independence of the SH2 modules of Syk likely provides the molecular basis for the more ubiquitous involvement of Syk in a variety of signal transduction pathways. # 1998 Academic Press

*Corresponding author

Keywords: X-ray crystal structure; Syk kinase; SH2 domains; ITAM

Introduction ZAP-70 and Syk comprise a family of hematopoietic cell speci®c protein tyrosine kinases (PTKs) that are required for antigen and antibody receptor function (reviewed by Chan & Shaw, 1995; Wange & Samelson, 1996; Reth & Wienands, 1997; Kurosaki, 1997). While ZAP-70 is expressed in T and natural killer cells, Syk is expressed in B cells, mast cells, polymorphonuclear leukocytes, platelets, macrophages, and immature T cells. The absence of either ZAP-70 or Syk results in arrested Abbreviations used: ITAM, immunoreceptor tyrosinebased activation motif; PTK, protein tyrosine kinase; TCR, T cell antigen receptor; SH2, Src homology 2; BCR, B cell antigen receptor; r.m.s., root mean square; MIR, multiple isomorphous replacement; NCS, noncrystallographic symmetry. E-mail address of the corresponding author: [email protected] 0022±2836/98/330523±15 $30.00/0

T and B cell development and in functional defects of a variety of immune receptors including the T cell antigen receptor (TCR), the B cell antigen receptor (BCR), and the receptors for IgG and IgE (FcgRI and FceRI; Costello et al., 1996; Zhang et al., 1996; Crowley et al., 1997; and reviewed by Cheng & Chan, 1997 and by Kurosaki, 1997). In addition, this family of PTKs has been implicated in activating NK cells (Brumbaugh et al., 1997; Lanier et al., 1998) and signaling by non-immune receptors such as G-protein coupled and integrin receptors (Wan et al., 1996; Gao et al., 1997; Poole et al., 1997). The signaling components of the antigen and Ig receptors contain sequence motifs known as ITAMs (for Immunoreceptor Tyrosine-based Activation Motifs) which have the consensus sequence YxxL/I-x7/8YxxL/I (Reth, 1989). Phosphorylation of both tyrosine residues within the ITAM by the Src-family PTKs is required for ef®cient interaction of ZAP-70 and Syk with the receptor subunits and for receptor function (Iwashima et al., 1994; Isakov et al., # 1998 Academic Press

524 1995; Bu et al., 1995; Chen et al., 1996; Kurosaki et al., 1995). The Syk family of PTKs is characterized by a domain structure consisting of two N-terminal Srchomology 2 (SH2) domains and a C-terminal kinase domain separated from the SH2 domains by a linker or hinge region (reviewed by Chan & Shaw, 1995). The localization of ZAP-70 and Syk to the receptor is mediated through the high af®nity interaction between the SH2 domains of ZAP-70 and Syk with the ITAM phosphorylated on both tyrosine residues (Isakov et al., 1995; Bu et al., 1995; Chen et al., 1996). The inability of either tyrosine residue within the ITAM to be phosphorylated or mutation of either SH2 domain within ZAP-70 or Syk decreases the avidity of this interaction by >100-fold and results in a non-functional antigen receptor (Kong et al., 1995; Kurosaki et al., 1995; Isakov et al., 1995; Bu et al., 1995). The co-localization of ZAP-70 and Syk to the receptor complex, in part, permits the membrane-localized Src-PTKs to contribute to the phosphorylation and enzymatic activation of ZAP-70 and Syk (Chan et al., 1995; Kurosaki et al., 1995; El-Hillal et al., 1997). While ZAP-70 and Syk have similar structures and play overlapping roles in T cell biology, these two PTKs also exhibit many different characteristics. First, the Syk kinase domain is 100-fold more active than ZAP-70 (Latour et al., 1996). Second, the enzymatic activation of ZAP-70 is primarily dependent upon the trans-phosphorylation by Src-PTKs (reviewed by Chan & Shaw, 1995). In contrast, the enzymatic activity of Syk can be augmented both by Src-PTKs and by the binding of dually phosphorylated ITAM peptides (Rivera & Brugge, 1995; Rowley et al., 1995; Kurosaki et al., 1995; El-Hillal et al., 1997). Third, expression of Syk is more ubiquitous amongst hematopoietic cells and Syk has been shown to be activated by not only a large variety of immune response receptors, but also by cytokines, integrins, thrombin and G protein-coupled receptors (Minami et al., 1995; Corey et al., 1994; Wan et al., 1996; Taniguchi et al., 1993; Clark et al., 1994). Fourth, recent evidence has indicated that activation of Syk by nonimmune response receptors such as integrin and G protein-coupled receptors may require only one functional SH2 domain for activity and therefore may not involve a classical ITAM/Syk interaction (Gao et al., 1997; Wan et al., 1996). Although the distinct patterns of expression of ZAP-70 and Syk may explain their selective involvement in signal transduction pathways, differences in ITAM or phosphotyrosine-recognition and binding speci®city by the SH2 domains of these proteins may also account for their differences in function. Recently, the X-ray crystal structure of the liganded tandem SH2 domain of ZAP-70 was determined, providing the structural basis for the obligatory requirement of ZAP-70 for dually phosphorylated ITAMs and insights into ITAM recognition by this protein (Hatada et al., 1995). We present here the structure of the tandem SH2

Crystal Structure of the ITAM-bound SH2 Domains of Syk

domain of Syk bound to a dually phosphorylated ITAM. In contrast to ZAP-70, where one of the two phosphotyrosine binding pockets is shared by both SH2 domains, this binding pocket in Syk is selfcontained within the N-terminal SH2 domain, suggesting that the N and C-terminal SH2 domains can function independently. In addition, the two SH2 domains of Syk display a remarkable ¯exibility in their relative orientation, suggesting that Syk may accommodate a greater variety of spacing sequences between the ITAM phosphotyrosines as well as singly phosphorylated non-classical ITAM ligands. Hence, this structure provides a structural basis for the broader spectrum of recognition processes mediated by the Syk-PTK and addresses the issue of speci®city in the recruitment of Syk to immune and non-immune receptors.

Results and Discussion Structure determination The crystal structure of the Syk-tandem-SH2 domain complexed with a dually phosphorylated ITAM peptide, derived from the CD3e chain of the T cell receptor, was determined by multiple isomorphous replacement and anomalous scattering Ê (Figure 1 and Materials to a resolution of 3.0 A and Methods). The asymmetric unit contains six copies of the bound tandem-SH2 domain (Figure 2), which were built independently (details in Materials and Methods). Therefore, this structure reports six independent views of the protein bound to its ligand. Re®nement of the structure resulted in a ®nal R value of 22.6%, and a free R Ê resolution (Table 1). value of 31.7% at 3.0 A Conformational flexibility in the architecture of the tandem SH2 domain of Syk The overall topology of the Syk tandem-SH2 domain is similar to its counterpart in the ZAP-70 kinase (Figures 3 and 4; Hatada et al., 1995) and very different from that of the tyrosine phosphatase SHP-2, a protein which also contains tandem SH2 domains (Eck et al., 1996; Hof et al., 1998; and details in `The SH2-SH2 interface' section). It consists of two canonical SH2 domains, the N-terminal SH2 domain (Syk-N, residues 15 to 109), and the C-terminal SH2 domain (Syk-C, residues 168 to 261), connected by an intervening region, which together with the SH2 domains forms a Y-shaped molecule. Both SH2 domains are very similar in fold to other SH2 domains and consist of a large b-sheet ¯anked by two a-helices (Kuriyan & Cowburn, 1993). Both Syk-N and Syk-C superimpose with the Src-SH2 domain (Waksman et al., 1993) with a root-mean-square (r.m.s.) deviation in Ê , or with the N or C-terminal Ca atoms of 1.8 A SH2 domains of ZAP-70 (Hatada et al., 1995) with Ê , respectively. The r.m.s. deviations of 0.6 and 0.9 A inter-SH2 region in Syk is similar in topology and structure to that of ZAP-70 (r.m.s. deviation in Ca

525

Crystal Structure of the ITAM-bound SH2 Domains of Syk

Figure 1. Representative regions of the averaged experimental electron density contoured at 1.2s. A, The region encompasses the C-terminal phosphotyrosinebinding site and shows continuous and unambiguous electron density for all elements of the phosphotyrosinebinding site of Syk-N. Helix aA, the peptide's C-terminal phosphotyrosine (pYc), as well as residues Arg-bB5, Arg-aA2, His-bD3, and His-bD4 in Syk-N are indicated. B, Same region as in A, rotated by 90 about the axis of the aA helix, illustrating the proximity of Syk-C. Residues in the C and N-terminal SH2 domains are distinguished by pre®xes C- and N-, respectively. Clear density is observed for the elements of the phosphotyrosine-binding site contributed by Syk-N, while Lys-bF1, the only residue of Syk-C participating in the coordination of the phosphotyrosine, is poorly de®ned. Unambiguous density for this side-chain was only observed in omit maps and in only three of the six molecules. The Figure was generated using the program O (Jones et al., 1991).

Ê ) and consists of three a-helices, two atoms of 1.4 A of them (aC and aD0 ) forming a helical coiled coil (Hatada et al., 1995). The presence of six molecules in the crystallographic asymmetric unit provided six different ``snapshots'' of the structure that revealed a substantial variability in the relative orientation of the

two SH2 domains. Figure 5 presents the superposition of molecules 1 through 6 with respect to SykN. In this view, it is evident that Syk-C can undergo major conformational re-arrangements in its position relative to Syk-N. The conformational change can be described by a translation of Syk-C along the direction indicated by the arrow in Figure 5, accompanied by a rotation about the axis of the Syk-C aB helix. The two extreme orientations of Syk-C (represented in green and blue in Figure 5) are related by a rotation of 18 and a Ê , with the other four orientranslation of 2.0 A tations representing intermediate states. The conformational change does not affect the total buried solvent-accessible area at the interface between Syk-N and Syk-C which remains constant (about Ê 2) across the six molecules. 910 A Changes in peptide conformation mirror the variability in the relative orientation of the two SH2 domains. These correspond to an adjustment of the dihedral angle de®ned by the Ca atoms of residues pYN ‡ 6 through pYN ‡ 9 from 111.9 to 136.5 (with pYN indicating the N-terminal phosphotyrosine in the peptide). The phosphorous atoms in the N-terminal phosphotyrosines of the Ê two extreme conformations of Figure 5 are 7.2 A apart, illustrating the magnitude of the re-arrangement undergone by the SH2 domains. Finally, the change in the relative orientation of the SH2 domains extends into the inter-SH2 domain region (Figure 5). Moreover, electron density for this region was poorly de®ned and interpretable only where crystal packing contacts were present, suggesting an inherent structural ¯exibility of this part of the structure. General features of ITAM binding The peptide ligand stretches across the C and Nterminal SH2 domains along the surface opposite to the inter-SH2 region, or, to use the Y-metaphor, across the branches of the fork (Figure 4). The protein's total solvent accessible surface area buried Ê 2 averaged across the six upon binding is 1035 A molecules. The peptide binds to the tandem SH2 domain head-to-tail, i.e. the N-terminal pYxxL/I-motif binds to the C-terminal SH2 domain and the C-terminal pYxxL/I motif binds to the N-terminal SH2 domain. The peptide conformation in the two motifs is extended while residues in the inter-motif region of the peptide form a nearly complete turn of an a-helix. These features are similar to those reported for ZAP-70 (Hatada et al., 1995) and therefore establish these as general properties for ITAM binding and recognition by tandem SH2 domains of the Syk family of kinases. Each SH2 domain of Syk contains two pockets which bind a pYxxL/I motif of the ITAM peptide. The mode of binding in each SH2 domain resembles closely that of the SH2 domains of the Src family of proteins: one pocket, positively charged, accommodates the phosphotyrosine,

526

Crystal Structure of the ITAM-bound SH2 Domains of Syk

Figure 2. Molecular content of the unit cell. The view is approximately along the 3-fold axis. Each of the six molecules are color-coded differently. For clarity, the peptide has not been included. This ®gure was generated using O (Jones & Thirup, 1986; Jones et al., 1991).

while the other, hydrophobic in nature, buries the non-polar Ile/Leu residue at the ‡3 position C-terminal to the phosphotyrosine (Waksman et al., 1992, 1993; Eck et al., 1993). However, while in ZAP-70, the C-terminal phosphotyrosine binding

site is formed by elements of both N-terminal SH2 domains, this site in contained within the N-terminal SH2 ZAP-70, the solvent accessible surface upon binding of the phosphotyrosine

the C and Syk is selfdomain. In area buried amounts to

Table 1. Data collection and re®nement statistics Data collection Data set

Radiation

Native K2PtCl4-1 K2PtCl4-2

Ê , BL7-1, 1.08 A SSRL CuKa, Raxis CuKa, Raxis

Refinement

Resolution 30-3.0 30-3.8 30-4.0

Ê A Ê A Ê A

Number of reflections Total Unique 202,021 77,547 31,079

40,670 18,192 13,659

Completenessa (%) Rsym (%)b 89.8 (74.1) 84.7 (66.1) 72.0 (48.0)

5.10 8.00 6.40

Riso (%)c

23.8 20.8

Phasing Phasingd N Sites power

6 2.2 6 2.5 Êe FOM (SIRAS) 30-4 A 0.49

Resolution Number of Completeness R-Factor Rfree Ê) (A reflectionsf (%)f (%) (%)g 30.0-3.0 40,397 85.6 (72.2) 22.6 31.7 Number of non hydrogen atoms 12,225 Number of residues 1530 Number of waters 0 Ê )h 0.016 Bond length (A Bond angles ( ) 1.9 RMS deviations between NCS-related atoms Ê -0.38 A Ê protein main ch. 0.10 A Ê -0.47 A Ê peptide main ch. 0.19 A Ê 2) RMSD in B factor for bonded atoms (A main chain 1.9 side chain 2.7 Ê 2) Average B (A main chain 43 side chain 46 a Ê ) in parentheses. Completeness for I/s(I) > 2.0, high resolution shell (3.1-3.0 A b Rsym ˆ jI ÿ hIij/I, where I ˆ observed intensity, and hIi ˆ average intensity from multiple observations of symmetry-related re¯ections. c Riso ˆ jjFPHj ÿ jFPjj/jFPj, where FP ˆ protein structure factor amplitude, FPH ˆ heavy-atom derivative structure factor amplitude. d Phasing power ˆ RMS (jFHj/e), where FH ˆ heavy-atom structure factor amplitude, e ˆ residual lack of closure. e FOM ˆ Figure of Mert. f Ê ). Numbers re¯ect the ``Working Set'' of re¯ections at F/s(F) > 2.0, high resolution shell in parentheses (3.14 to 3.0 A g Rfree was calculated on the basis of 5% of the total number of re¯ections omitted from the re®nement. h Bond length and angle parameters from Engh & Huber (1991).

Crystal Structure of the ITAM-bound SH2 Domains of Syk

527

Figure 3. SH2 domain sequences and de®nition of the residue notation. A, Alignment of the sequences of the N and C-terminal SH2 domains of Syk, ZAP-70, and the SH2 domain of Src, based on the secondary structure de®nitions used by Eck et al. (1993), Waksman et al. (1993) and Hatada et al. (1995). B, Alignment of the sequences of the interSH2 domains of Syk and ZAP-70, based on the secondary structure de®nitions used by Hatada et al. (1995). Secondary structural elements are boxed and shown schematically as arrows (b-strands) and cylinders (a-helices) below the boxes. The notation for these elements is shown at the bottom. Functionally important residues are highlighted by thick vertical bars above the sequence and their relative position in the secondary structural elements is indicated. Residue sequence numbers for Syk are indicated above the sequences, with ®lled and open numbers referring to SykN and Syk-C, respectively.

Ê 2, of which 50 A Ê 2 (17.5%) is contributed by 285 A residues of the C-terminal SH2 domain (Hatada et al., 1995). In Syk, the corresponding solvent accessible surface area is on average smaller Ê 2). More remarkably, in three of the six mol(245 A ecules in the asymmetric unit, only 5% of the total buried surface area involved in phosphotyrosine binding is contributed by the C-terminal SH2 domain. In the other three molecules, this contribution is substantially increased (to 17.9%) by new contacts formed by a single side-chain, that of LysbF1. However, electron density for this side-chain was consistently missing in maps based on either experimental or model-combined phases (Figure 1B). Thus, building relied solely on simulated annealing omit maps (Hodel et al., 1992) of the corresponding regions, which displayed electron density for Lys-bF1 in only three of the six complexes. In contrast, all other elements of the phosphotyrosine binding site were clearly de®ned in all maps (experimental included) and in all mol-

ecules (Figure 1, A and B). These observations strongly suggest that Lys-bF1 is at least partially disordered and therefore may not contribute signi®cantly to binding. Thus, the molecular basis for ITAM recognition by Syk differs distinctly from ZAP-70, and suggests that the two SH2 domains of Syk may function as independent units. Binding of the N-terminal pYNEPI motif by the C-terminal SH2 domain All contacts with the amino-terminal portion the peptide (positions pYN-2 to pYN ‡ 3) are provided by Syk-C. Four arginine residues are in close proximity at the phosphotyrosine-binding site. Three of these residues, bB7, aA2 and bB5, contribute to the network of hydrogen bonds coordinating the phosphate group of the phosphotyrosyl residue (Figures 6C and 8A). Arg-aA2 also makes a weak amino-aromatic interaction with the phosphotyrosine ring, and is within hydrogen bonding distance

528

Crystal Structure of the ITAM-bound SH2 Domains of Syk

Figure 4. Stereo ribbon diagram of the tandem SH2 domain of Syk in a complex with the CD3e-ITAM peptide. SykN is depicted in blue, Syk-C in green and the inter-SH2 region in brown. The ITAM peptide is shown in ball-andstick representation. Secondary structural elements are labeled according to the notation adopted in Figure 3. The Figure was generated using the programs MOLSCRIPT (Kraulis, 1991) and RASTER3D (Merritt & Murphy, 1994).

of Asp(pYN-1 ) of the peptide. A fourth arginine residue (bD6) closes up the phosphotyrosine binding site by forming a hydrophobic platform for the ring of the phosphotyrosine. Most of these structural features have been observed in other SH2 domains, including the C-terminal SH2 domain of ZAP-70 (Figure 6D; Waksman et al., 1993; Eck et al., 1993; Hatada et al., 1995). However, the C-terminal SH2 domain of Syk differs from that of ZAP-70 in two respects: (1) a larger number of positive charges is observed at the phosphotyrosine binding site of Syk; (2) Arg-bB7 in ZAP-70 does not form a salt bridge with the phosphate, but instead neutralizes Glu-BC2 (Figure 6D). As a result, the net positive charge in the phosphotyrosine binding site is larger in Syk than in ZAP-70. These structural features strongly suggest that the C-terminal SH2 domains of Syk and ZAP-70 may differ substantially in their binding properties. Such a suggestion is consistent with the reported observation that, in

contrast to ZAP-70, the C-terminal SH2 domain of Syk retains substantial binding af®nity for tyrosylphosphorylated targets (Shiue et al., 1995). The side-chain of Glu(pYN ‡ 1) in the peptide makes extensive hydrophobic contacts, observed between the aliphatic carbon atoms of its sidechain and the protein, while the terminal oxygen Ê of the positively charged atoms are within 5.3 A tip of Lys-bD1. Pro(pYN ‡ 2) is mostly solvent exposed. Finally, the side-chain of Ile(pYN ‡ 3) is almost completely buried in the hydrophobic binding pocket of the C-terminal SH2 domain and contacts are typical of those observed in other SH2 domain-peptide complexes (see details in Figure 8A; Waksman et al., 1993; Eck et al., 1993). Binding of the C-terminal pYCSGL motif to the N-terminal SH2 domain As mentioned above, a striking feature of the C-terminal phosphotyrosine (pYC) binding pocket

Figure 5. Conformational ¯exibility of the Syk tandem SH2 domain. Stereo ribbon diagram of molecules 1 to 6 of the Syk tandem SH2 domain superimposed with respect to the N-terminal SH2 domain. The view is approximately down the axis of the C-terminal SH2 aB helix. The arrow indicates the direction of the translational component (see the text). The Figure was generated using the programs MOLSCRIPT (Kraulis, 1991) and RASTER3D (Merritt & Murphy, 1994).

Crystal Structure of the ITAM-bound SH2 Domains of Syk

Figure 6 (legend on page 530)

529

530

Crystal Structure of the ITAM-bound SH2 Domains of Syk

Figure 7. Stereo diagram of the interface between the SH2 domains of Syk. Secondary structural elements in the N and C-terminal SH2 domain are shown in blue and green, respectively. Side-chains are shown in ball-and-stick representation. Broken lines indicate hydrogen bonds. The peptide is shown in transparent rendering for reference. The Figure was generated using the programs MOLSCRIPT (Kraulis, 1991) and RASTER3D (Merritt & Murphy, 1994).

of Syk is the absence of signi®cant interactions contributed by the C-terminal SH2 domain in three of the six complexes in the asymmetric unit. In the other three molecules, only one residue in this domain, Lys-bF1, seems to participate in the coordination of the phosphotyrosine. However, Lys-bF1 is partially disordered and, as discussed above, may not contribute signi®cantly to binding. The C-terminal phosphotyrosine forms hydrogen bonding contacts with arginine residues aA2 and bB5 through its phosphate group (Figures 6A and 8C). Arg-aA2 in addition makes a weak amino-aromatic interaction with the phenyl ring and forms a hydrogen bond with the carbonyl oxygen of Leu(pYC-1). Both features occur similarly at the N-terminal pYNEPI motif. Ser(pYC ‡ 1) interacts primarily with the side-chain of Tyr-bD5 and the main-chain atoms of His-bD4, while Gly(pYC ‡ 2) only makes sparse contacts. Leu(pYC ‡ 3) inserts deeply into the hydrophobic binding pocket of Syk-N. Finally, Asn(pYC ‡ 4) is the last ligand residue that appears to be stabilized by interactions with the protein (Figure 8C). It is interesting to note that the residue of Syk-N facing the ‡2 position of the peptide is Glu-bD0 1. The equivalent residue in Src kinases is Arg-bD0 1 which is responsible for a strong selectivity for Glu at the ‡2 position of the peptide (Waksman et al., 1993; Eck et al., 1993; Songyang et al., 1993). By analogy, one would predict a strong selectivity of

Syk-N for a basic residue at the ‡2 position in the peptide. Binding of the inter-motif-(pYN‡4)RKGQRDL(pYCÿ1)Residues of the inter-motif account for only 22.1% (on average) of the total surface area of the peptide buried upon binding. Very few contacts are observed between the protein and peptide residues between Arg(pYN ‡ 4) and Arg(pYC ÿ 3) (see Figure 8B for peptide residue notation). In contrast, Asp(pYC ÿ 2) makes extensive contacts with the protein surface and is the only residue in the intermotif that interacts with residues in both SH2 domains, namely Arg-aA2 of Syk-N and Lys-aB12 of Syk-C. The side-chain of Leu(pYC ÿ 1) is completely solvent exposed. The SH2-SH2 interface The SH2-SH2 interface is polar in nature. There are two main regions of inter-domain contacts; in the ®rst region, the BC-loop in Syk-N faces the bF-strand, the FB-loop and the N-terminal tip of the aB helix of Syk-C; in the second region residues in the N-terminal end of the aA helix and in the AA loop of Syk-N make contact with residues in the C-terminal end of the aB helix of Syk-C (Figure 7).

Figure 6. Stereo diagrams of the interactions at the phosphotyrosine binding sites. The phosphotyrosine binding pocket of Syk-N (A), ZAP-N (B), Syk-C (C), and ZAP-C (D). Residues are shown in ball-and-stick representation with big spheres for peptide atoms and small spheres for protein atoms. Secondary structural elements are shown blue and green ribbons representing the N and C-terminal SH2 domains, respectively. Broken lines indicate hydrogen bonding interactions. In B and D, water molecules are represented by spheres in magenta. In A, Lys-bF1 is depicted in transparent rendering to indicate its disordered state in three of the six molecules (see text). The Figure was generated using the programs MOLSCRIPT (Kraulis, 1991) and RASTER3D (Merritt & Murphy, 1994).

531

Crystal Structure of the ITAM-bound SH2 Domains of Syk

In the ®rst region, Asp-FB1 of Syk-C appears to play a central role in the interface by forming hydrogen bonding interactions with the amide nitrogen of Leu-BC4 and with the carbonyl oxygen of Asn-BC2 of Syk-N. In addition, GlnaB4 in Syk-C is within hydrogen bonding distance of the carbonyl oxygen of Arg-BC1 of SykN, and Asn-BC2 of Syk-N makes a weak hydrogen bond with the carbonyl oxygen of Lys-bF2 of Syk-C. Thus, residues in Syk-C all contribute to the formation of a scaffold that maintains the phosphotyrosine binding loop (BC loop) of Syk-N in a favorable conformation for binding. In the

second region a network of polar interactions is formed between Glu-aA3 and Thr-aA1 in Syk-N, and Tyr-aB11 and Lys-aB12 in Syk-C. In fact, Lys-aB12 plays a dual role since it is also involved in contacts with Asp(pYC ÿ 2) of the peptide ligand. No main-chain/main-chain interaction is observed between the SH2 domains in this region. Finally, the SH2-SH2 interface of Syk is very different from that of SHP-2 with the latter formed by residues in the bA strand and AA loop of the N-terminal SH2 domain and in the BC and DE loops of the C-terminal SH2 domain.

Figure 8(a) (legend on page 533)

532

Crystal Structure of the ITAM-bound SH2 Domains of Syk

Figure 8(b) (legend opposite)

As a result, the two peptide-binding surfaces of SHP-2 run parallel to each other, as opposed to Syk where these binding sites are spatially colinear (Eck et al., 1996).

Conclusions From the crystal structures of the SH2 domains of the Syk and ZAP-70 kinases complexed with ITAM peptides, common structural themes emerge which can be seen as the general features of the mechanism of ITAM recognition and binding. Firstly, the tandem SH2 domain of the Syk family

of proteins bind tyrosyl-phosphorylated ITAM sequences with a de®ned polarity determined by the relative positioning of the two SH2 domains. Hence, the N-terminal pYxxL/I motif of the ITAM binds to the C-terminal SH2 domain and vice-versa. Secondly, both pYxxI/L motifs in the ITAM contribute signi®cantly to the total interaction and therefore both are required for selective and highaf®nity binding to the SH2 domains. However, the tandem SH2 domain of Syk differs from that of ZAP-70 in that the two SH2 domains of Syk do not form a common C-terminal phosphotyrosine-binding site and that the phosphotyrosine-binding pocket of its C-terminal

Crystal Structure of the ITAM-bound SH2 Domains of Syk

533

Ê and are averaged across the Figure 8. Schematic representation of peptide-protein contacts. Distances are given in A six molecules in the asymmetric unit, except when marked with an asterisk. Peptide residues are labeled according to their position relative to the phosphotyrosine residues, with pYN and pYC denoting the N and C-terminal phosphotyrosines, respectively. Protein residues are labeled according to the notation of Eck et al. (1993) and Waksman et al. (1993), preceded by an N: or C: where distinction between N and C-terminal SH2 domains is necessary. The peptide is depicted in three portions A, pYN ÿ 2 through pYN ‡ 3; B, pYN ‡ 4 through pYC ÿ 1; and C, pYC through pYC ‡ 4.

SH2 domain is characterized by a more positive electrostatic surface potential than its ZAP-70 counterpart. These observations may relate to the fact that at least in two signal transduction pathways, those mediated by integrins and G proteincoupled receptors, recruitment of Syk into the pathways may not necessitate the involvement of both SH2 domains (Gao et al., 1997; Wan et al., 1996). Whereas the C-terminal SH2 domain is not required for Syk activation by the aIIbb3 integrin

receptor (Gao et al., 1997), Syk-N is not required for activation of the MAP kinase pathway by the Gq-coupled muscarinic acetylcholine receptor (Wan et al., 1996). These observations are consistent with the interpretation that each individual SH2 domain can function as an independent unit, thus supporting the structural information provided by the present study. The six different conformations of the tandem SH2 domain of Syk demonstrate a remarkable

534 ¯exibility in domain structure, which may enable Syk to tolerate changes in the size of the ITAM's inter-motif sequence. Consistent with this hypothesis, Syk binds ITAMs derived from the FcgRII and colony-stimulating factor receptors which both have inter-motif regions signi®cantly longer (by four amino acids) than the canonical ITAM (Corey et al., 1994; Chacko et al., 1996). Interestingly, Syk binds with higher af®nity to the FcgRII ITAM than does ZAP-70, while both Syk and ZAP-70 have comparable af®nity for the CD3e ITAM (Bu et al., 1995; J.W. & A.C.C., unpublished), suggesting that the tandem SH2 domain of ZAP-70 may not be as ¯exible as that of Syk. Due to the presence of only one molecule in the asymmetric unit, the crystal structure of the ITAM-bound SH2 domains of ZAP-70 could not provide evidence for possible variations in the relative orientation of the SH2 domains (Hatada et al., 1995). Together, these studies indicate that discrimination in the function of Syk and ZAP-70 may be achieved independently by two mechanisms. First, tissue selective expression undoubtedly dictates the potential function of these two kinases. Second, the selective involvement of Syk or ZAP-70 in signal transduction pathways may reside in differences in binding speci®city and mode determined by their SH2 domains. Src-homology 2 domains, as single binding units, have evolved binding selectivity, which when linked into a two-unit protein provides for a uniquely selective macromolecular template. Together with selectivity in expression, recruitment of SH2-containing proteins can then be modulated in an exquisitely precise way resulting in their selective involvement in signal transduction pathways.

Materials and Methods Purification, crystallization and data collection The tandem SH2 domain fragment of Syk (residues 7 to 269) was expressed and puri®ed as described by Bu et al. (1995). The pure protein was complexed with the CD3e-chain ITAM peptide (sequence PDpYEPIRKGQRDLpYSGLNQR, from Quality Controlled Biochemicals) at a ratio of 1:1.1 protein to ligand, and concentrated to typically 35 mg/ml. Crystals were grown at room temperature using the hanging drop vapor diffusion method (Mc Pherson, 1990). Drops of 2 ml protein were mixed with 2 ml of 9 to 12% (w/v) PEG 8000, 10% (v/v) PEG 200, 0.1 M Tris-HCl pH 8.5, (Sigma) and placed over a reservoir of the same composition. Crystals (0.4 mm  0.3 mm  0.1 mm) appeared within one week. Flash-frozen crystals, protected in 15% Ê in a laboratory setting (Rglycerol, diffracted to 2.9 A axis IIc image plate detector mounted on an RU-200 Ê at the rotating anode generator, Rigaku) and to 2.5 A synchrotron (beamline 7.1, Stanford Synchrotron Radiation Laboratory). However, scattering was strongly anisotropic, thus limiting the effective maximum resolution Ê at a I/s(I) ratio greater than 2.0 (see Table 1). to 3.0 A The crystals belonged to space group P21 with Ê , b ˆ 146.9 A Ê , c ˆ 91.5 A Ê , and b ˆ 97.6 , and a ˆ 85.5 A

Crystal Structure of the ITAM-bound SH2 Domains of Syk contained six tandem SH2/ITAM complexes per asymmetric unit. All data were processed with the DENZO and SCALEPACK software packages (Otwinowski, 1993). Structure determination The structure was determined by multiple isomorphous replacement with anomalous scattering. Platinum derivatized crystals were obtained by soaking native crystals in 2 mM K2PtCl4 for 12 and 24 hours, respectively. Complete diffraction data on derivatized frozen Ê resolution in the labcrystals could be collected to 4.0 A oratory. The isomorphous difference Patterson analysis revealed distinct peaks in the Harker section (v ˆ 1/2) and ®ve platinum positions could be readily determined using the program HASSP (Terwilliger et al., 1987). A sixth position could be found by difference Fourier methods. The Pt-derivative data showed an anomalous Ê in consignal that could be exploited for phasing to 4 A junction with the isomorphous differences using the program MLPHARE (CCP4 package (CCP4, 1994); see Table I for relevant statistics). The electron density map that resulted from subsequent solvent ¯attening using DM (CCP4, 1994) showed weak secondary structural features together with a well de®ned molecular envelope. Non-crystallographic symmetry (NCS) averaging (program DM) was used to further improve the initial phases. The NCS-matrices were derived from the coordinates of the platinum atoms and found in agreement with the peaks in the self-rotation function (program POLARRFN, CCP4, 1994). In an iterative procedure of building recognizable secondary structural elements, rede®ning the molecular envelope and re®ning the NCSmatrices, the density could be improved to a level that allowed us to manually dock SH2 domains into the electron density, thus unequivocally identifying the location of the six molecules of the asymmetric unit. NCS averaging was also exploited to extend experimental phases Ê (Table 1). to 3.0 A At a later stage of this project the program SHARP (De La Fortelle & Bricogne, 1997) became available and was used to recalculate the experimental phases. NCSaveraging of these phases led to a much improved electron density map that was very bene®cial for model building. Model building and refinement A poly(Ala) model for the SH2 domains of the molecule best de®ned in the averaged electron density was built using the program O (Jones & Thirup, 1986; Jones et al., 1991), and subsequently re®ned, subjected to strict NCS constraints (program XPLOR, BruÈnger, 1992a). However, as the variable orientation between the two SH2 domains became apparent, all six pairs of SH2 domains were built independently and re®ned by applying NCS restraints to Syk-N and Syk-C separately. The inter-SH2 regions (residues 118 to 163) were only poorly de®ned and the density deteriorated upon NCS-averaging. Interpretable density for these regions was obtained, but only in three of the six molecules, by combining the phases calculated from the partial model with the unaveraged experimental phases (program SIGMAA, CCP4 package, 1994). The peptide could be built in all six molecules based on both experimental and phasecombined maps.

Crystal Structure of the ITAM-bound SH2 Domains of Syk

Progress in the re®nement that included conjugate gradient minimization as well as simulated annealing in both cartesian and torsional angle spaces (BruÈnger et al., 1990; Rice & BruÈnger, 1994) was assessed by monitoring the free R-factor (BruÈnger, 1992b; see Table 1). NCSmatrix sets for nine different structural groups were de®ned, i.e. residues 9 to 117 (Syk-N), 167 to 262 (SykC), 118 to 136 (helix aC in the inter-SH2 region), 137 to 140 (the loop connecting the aC and aD helices), 141 to 151 (helix aD in the inter-SH2 region), 152 to 166 (helix aD0 in the inter-SH2 region), residues pYN ÿ 2 to pYN ‡ 3, residues pYC ÿ 2 to pYC ‡ 4, and residues pYN ‡ 4 to pYC ÿ 3 in the peptide ligand (see notation in Figure 8). The NCS restraints were such that the r.m.s. deviations between main-chain atoms in equivalent Ê in the restrained regions were between 0.10 and 0.38 A Ê in the peptide protein and between 0.19 and 0.47 A ligand. Individual B-factor re®nement was used, resultÊ 2 for maining in average temperature factors of 43 A 2 Ê chain atoms and 46 A for side-chain atoms, and r.m.s. deviations for bonded main-chain or side-chain atoms of Ê 2, respectively. After bulk solvent correc1.9 and 2.7 A tion, the re®nement converged to a ®nal R-factor of 22.6 Ê resolution range; % with an R-free of 31.7 % (30 to 3.0 A jFj/s(jFj) > 2.0) with good stereochemistry (Table 1). The model includes residues 9 to 262 in all six molecules with an interruption in the inter-SH2 region in three molecules between residues 118 and 151. Electron density was not clearly interpretable for a few residues (Ser9, Lys124, Lys133, Lys222 and Lys261) which were built as alanine. The model does not contain water molecules. All residues are in the allowed region of the Ramachandran plot (Ramachandran & Sasisekharan, 1968). Relevant statistics for the re®nement are summarized in Table 1. The coordinates of the structure of the tandem SH2 domain of Syk bound to the dually phosphorylated CD3e-ITAM peptide have been deposited (PDB entry code 1A81).

Acknowledgments We thank Ian Wilson and Jack Johnson (Scripps Research Institute) for advice on NCS averaging, C.S. Ricard for advice on protein puri®cation, S. Korolev and A.B. Herr for help in data collection, M. Hatada (ARIAD Pharmaceuticals) for providing the ZAP-70 tandem SH2 domain coordinates, the staff of Beamline 7.1 at SSRL for assistance during data collection, and F.S. Mathews for comments on the manuscript. This work was supported by P®zer Inc. (G.W.), by funds from the Washington University School of Medicine (G.W.), and by National Institute of Health grant CA71516 (C.C.A.). J.W. was supported by National Institute of Health training grant 5T32DK07126.

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Edited by D. Rees (Received 6 April 1998; received in revised form 22 May 1998; accepted 22 May 1998)