www.sciencesignaling.org/cgi/content/full/3/120/ra34/DC1

Supplementary Materials for

Loops Govern SH2 Domain Specificity by Controlling Access to Binding Pockets

Tomonori Kaneko, Haiming Huang, Bing Zhao, Lei Li, Huadong Liu, Courtney K. Voss,

Chenggang Wu, Martin R. Schiller, Shawn Shun-Cheng Li*

*To whom correspondence should be addressed. E-mail: sli@uwo.ca

Published 4 May 2010, Sci. Signal. 3, ra34 (2010) DOI: 10.1126/scisignal.2000796

This PDF file includes:

Fig. S1. Representative SH2 domain structures alone or in complex with peptides. Fig. S2. Stereo representation of the crystal structure of the BRDG1 SH2 domain–NTAL complex. Fig. S3. Contact area per residue of the NTAL pTyr136 peptide with the BRDG1 SH2 domain. Fig. S4. The conserved hydrogen bonding network at P+1. Fig. S5. Structure-based sequence alignment of a nonredundant group of 63 SH2 domains. Fig. S6. Specificity switch by loop mutagenesis. Fig. S7. Schematic description of the binding pockets in the Fyn SH2 domain and the loop-deletion mutants. Table S1. Crystallographic statistics. Table S2. Dissociation constants of SH2-peptide interactions measured by fluorescence polarization. Table S3. Peptide binding assays using different BRDG1 SH2 domain constructs. References

Other Supplementary Material for this manuscript includes the following: (available at www.sciencesignaling.org/cgi/content/full/3/120/ra34/DC1)

Interactive figures. JMOL representations of the structures shown in Fig. 3A.

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Fig. S1. Representative SH2 domain structures alone or in complex with peptides. (A) The structure of the Lck SH2 domain determined by Eck and colleagues (1). Their secondary structure nomenclature shown here was employed in this entire paper. The SH2 domain consists of secondary structure elements βA-αA-βB-βC-βD-βE-βF-αB-βG. Two α helices are in orange and seven β strands are in green. The EF (between βE and βF strands) and BG (between αB helix and βG strand) loops are colored brown and magenta, respectively. The arginine βB5 (the fifth residue in the strand βB) and the tyrosine βD5 are shown in stick representation. (B) The v-Src SH2 domain in complex with the pTyr-Glu-Glu-Ile peptide (PDB: 1SPS) (2). In panel (B) and (C), ligand peptides are in stick representation and colored yellow, except for nitrogen atoms that are colored blue, oxygen atoms red and a phosphorus atom orange. Ribbon (left) and surface (right) representations are shown for each complex. Only pTyr through P+3 residues of peptides are shown. (C) The Grb2 SH2 domain in complex with the pTyr-Val-Asn-Val peptide (PDB: 1JYR) (3). The EF1 (the first residue of the EF loop) residue Trp121 is colored cyan. The dashed line represents an intramolecular H-bond in the -turn of the peptide backbone.

Fig. S2. Stereo representation of the crystal structure of the BRDG1 SH2 domain-NTAL complex. See also Fig. 2A. The cyan mesh is an Fo-Fc electron density map (the omit map), contoured at 2.5 , calculated without the coordinates for the NTAL peptide.

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Fig. S3. Contact area per residue of the NTAL pTyr136 peptide with the BRDG1 SH2 domain. The contact area was calculated from the complex structure. Residues pTyr and Leu at P+4 exhibited the largest contact areas and, therefore, contribute the most to binding. The calculation was conducted with the program AREAIMOL in the CCP4 suite (4).

Fig. S4. The conserved hydrogen bonding network at P+1. A hydrogen bond between the ligand’s P+1 amide nitrogen and the SH2 domain’s D4 carbonyl oxygen is commonly observed in SH2 domains, as well as a water molecule connecting P+1 and D6 (5). Both elements are conserved in the BRDG1 SH2-peptide complex structure (right). The water molecule is replaced by the amide oxygen of Asn at P+2 of the peptide in the Grb2-peptide complex (left).

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Fig. S5. Structure-based sequence alignment of a nonredundant group of 63 SH2 domains. Each SH2 domain is identified by the corresponding protein name, species, N- or C-terminal location within the protein (in case of two SH2 domains from a single protein), and the PDB code. The pentagon residues (brown), the universally conserved arginine (blue), the five most conserved residues (besides the universal arginine and the leucine anchor) (black), BG loop regions (magenta), pentagon plug residues (green) are shaded with colors. The secondary structure assignment of the Src SH2 domain is according to convention (see fig. S1) (1). The lower case letters in sequences indicate disordered residues in crystal structures, which do not have assigned coordinates.

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Fig. S6. Specificity switch by loop mutagenesis. Representative binding curves of N-terminally fluorescein-labeled peptides to the wild-type BRDG1 SH2 or the L240A mutant. A full list of peptides and Kd values can be found in Table 2. The vertical axis is fluorescence polarization in an arbitrary unit.

Fig. S7. Schematic description of the binding pockets in the Fyn SH2 domain and the loop-deletion mutants. Corresponding binding data are shown in Table 3. Three residues (Leu238-Cys-Cys) including the BG4-plug Leu238 of the BG loop in the Fyn SH2 domain were deleted in Fyn BG. Subsequently, the EF1 residue Thr216 was mutated to methionine to create the mutant Fyn BG/T216M.

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Table S1. Crystallographic statistics. Data collection Space group P6222 Cell dimensions (Å) a=b=72.64, c= 97.84 Resolution (Å) 29.2-1.90 (1.97-1.90)* Number of reflections (total / unique) 127844 / 12619 Rmerge (%) 5.0 (35.7) I/I 7.0 (2.1) Completeness (%) 100.0 (100.0) Redundancy 10.1 (10.0) Refinement Resolution (Å) 29.2-1.9 Number of reflections 12583 Rwork / Rfree (%) 21.2 / 23.6 Number of atoms BRDG1 SH2 domain 801 NTAL peptide 85 Water molecules 83 malonate ion 3.5 (at special position) B-factors (Å2) BRDG1 SH2 domain 31.4 NTAL peptide 34.6 Water molecules 40.1 malonate ion 45.7 Root-mean-square deviations Bond lengths (Å) 0.012 Bond angles (°) 1.5 *Values in parentheses are for the highest-resolution shell.

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Table S2. Dissociation constants of SH2-peptide measured by fluorescence polarization.

A. Data for GST-BRDG1 SH2-peptide binding Source protein Peptide sequence Kd ± standard error (µM)

STS-1 pY546 S-E-S-pY-D-T-Y-I-S-R-NH2 0.19 ± 0.01 CD22 pY807 V-G-D-pY-E-N-V-I-P-D-F-NH2 0.19 ± 0.01 CD22 pY822 G-I-H-pY-S-E-L-I-Q-F-G-NH2 0.61 ± 0.03 NTAL pY136 (C142A) A-N-S-pY-E-N-V-L-I-A-K-NH2 0.28 ± 0.01 1 tublin pY418 R-H-H-pY-S-L-Y-V-H-NH2 0.50 ± 0.03 Titin pY23044 G-E-E-pY-S-F-R-V-S-NH2 0.91 ± 0.05 PTPMT1 pY75 N-E-E-pY-E-T-R-F-L-NH2 0.57 ± 0.04 STAT1 pY22 H-Q-L-pY-D-D-S-F-P-M-NH2 1.42 ± 0.05 CD84 pY262 K-T-I-pY-T-Y-I-M-A-S-NH2 1.36 ± 0.12 c-RET pY1096 D-S-V-pY-A-N-W-M-L-S-NH2 1.89 ± 0.10 FcRIIB pY292 T-I-T-pY-S-L-L-M-H-P-NH2 2.48 ± 0.24 ZNF598 pY163 I-F-T-pY-E-R-K-W-Y-NH2 1.69 ± 0.17 RAO pY465 V-G-N-pY-D-Y-I-W-D-NH2 2.42 ± 0.14 KIAA0256 pY874 E-K-E-pY-E-T-N-W-R-NH2 4.29 ± 0.30 COL11A2 pY344 E-G-P-pY-D-Y-T-Y-G-NH2 11.13 ± 0.83 CD46 pY147 N-G-T-pY-E-F-G-Y-Q-NH2 24.00 ± 1.99 Nephrin pY1217 Y-E-D-P-R-G-I-pY-D-Q-V-A-A-D-M-NH2 18.31 ± 0.75 Syk pY342 G-G-T-E-V-pY-E-S-P-pY-A-D-NH2 49.62 ± 5.21

B. Data for His-tagged BKS SH2-peptide binding

Source protein Peptide sequence Kd ± standard error (µM)

CD22 pY807 V-G-D-pY-E-N-V-I-P-D-F-NH2 0.61 ± 0.03 STS-1 pY546 S-E-S-pY-D-T-Y-I-S-R-NH2 0.66 ± 0.06 NTAL pY136 (C142A) A-N-S-pY-E-N-V-L-I-A-K-NH2 1.78 ± 0.13 ALEX3 pY237 T-N-E-pY-Q-H-M-L-A-NH2 2.09 ± 0.22 CD22 pY822 G-I-H-pY-S-E-L-I-Q-F-G-NH2 4.14 ± 0.28 RasGAP pY723 E-E-E-pY-S-E-F-K-E-L-NH2 4.28 ± 0.38 STAT1 pY22 H-Q-L-pY-D-D-S-F-P-M-NH2 4.66 ± 0.37 c-RET pY1096 D-S-V-pY-A-N-W-M-L-S-NH2 4.77 ± 0.51 Akt1 pY229 V-M-E-pY-A-N-G-G-E-L-NH2 12.25 ± 1.44 Nephrin pY1217 Y-E-D-P-R-G-I-pY-D-Q-V-A-A-D-M-NH2 30.96 ± 3.38

IL2R0 pY384 Y-F-T-pY-D-P-Y-S-E-E-NH2 39.89 ± 4.02

interactions

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Table S3. Peptide binding assays using different BRDG1 SH2 domain constructs. GST-WT (containing BRDG1 Glu161-Ser270), His-WT (containing BRDG1 Asp167-Ser285) and His-C269A (a mutation introduced for crystallization) were examined for binding to the listed fluorescein-labeled peptides by fluorescence polarization.

Peptide sequence Kd ± standard error (µM) GST-WT His-C269A His-WT

A-N-S-pY-E-N-V-L-I-A-K-NH2(*) 0.28 ± 0.01 0.21 ± 0.01 0.47 ± 0.03 H-Q-L-pY-D-D-S-F-P-M-NH2 1.42 ± 0.05 0.77 ± 0.03 1.76 ± 0.09 E-E-E-pY-S-E-F-K-E-L-NH2 11.85 ± 0.81 3.52 ± 0.11 4.87 ± 0.26 S-E-S-pY-D-T-Y-I-S-R-NH2 0.19 ± 0.01 0.13 ± 0.01 — V-G-D-pY-E-N-V-I-P-D-F-NH2 0.19 ± 0.01 0.15 ± 0.01 — G-I-H-pY-S-E-L-I-Q-F-G-NH2 0.61 ± 0.03 0.42 ± 0.02 — I-F-T-pY-E-R-K-W-Y-NH2 1.69 ± 0.17 1.33 ± 0.05 — D-S-V-pY-A-N-W-M-L-S-NH2 1.89 ± 0.10 1.35 ± 0.05 — V-M-E-pY-A-N-G-G-E-L-NH2 11.94 ± 0.72 10.21 ± 0.42 — Y-F-T-pY-D-P-Y-S-E-E-NH2 15.65 ± 0.89 30.85 ± 2.67 — Y-E-D-P-R-G-I-pY-D-Q-V-A-A-D-M-NH2 18.31 ± 0.75 22.28 ± 1.12 —

Supplementary References 1. M. J. Eck, S. E. Shoelson, S. C. Harrison, Recognition of a high-affinity phosphotyrosyl

peptide by the Src homology-2 domain of p56lck. Nature 362, 87-91 (1993).

2. G. Waksman, S. E. Shoelson, N. Pant, D. Cowburn, J. Kuriyan, Binding of a high affinity phosphotyrosyl peptide to the Src SH2 domain: crystal structures of the complexed and peptide-free forms. Cell 72, 779-790 (1993).

3. P. Nioche, W. Q. Liu, I. Broutin, F. Charbonnier, M. T. Latreille, M. Vidal, B. Roques, C. Garbay, A. Ducruix, Crystal structures of the SH2 domain of Grb2: highlight on the binding of a new high-affinity inhibitor. Journal of Molecular Biology 315, 1167-1177 (2002).

4. M. D. Winn, An overview of the CCP4 project in protein crystallography: an example of a collaborative project. Journal of synchrotron radiation 10, 23-25 (2003).

5. R. T. Nolte, M. J. Eck, J. Schlessinger, S. E. Shoelson, S. C. Harrison, Crystal structure of the PI 3-kinase p85 amino-terminal SH2 domain and its phosphopeptide complexes. Nat Struct Biol 3, 364-374 (1996).