Supplementary Materials for

Size: px

Start display at page:

Download "Supplementary Materials for"

Amy Norton
6 years ago
Views:

1 Supplementary Materials for Superbinder SH2 Domains Act as Antagonists of Cell Signaling Tomonori Kaneko, Haiming Huang, Xuan Cao, Xing Li, Chengjun Li, Courtney Voss, Sachdev S. Sidhu,* Shawn S. C. Li* *To whom correspondence should be addressed. (S.S.S.); (S.S.C.L.) Published 25 September 2012, Sci. Signal. 5, ra68 (2012) DOI: /scisignal The PDF file includes: Fig. S1. SH2 domain variants obtained by screening a phage-displayed library. Fig. S2. An alignment of the human SH2 domains showing the region for ptyr binding. Fig. S3. The specificity and affinity of the Fyn SH2 triple mutant in comparison to those of the wild-type domain. Fig. S4. Amino acid combinations of the ptyr-binding pocket in natural SH2 domains. Fig. S5. The dynamics of the BC loop and its stabilization in the Src SH2 triplemutant domain. Fig. S6. The structure of the ptyr-binding pocket of the Src SH2 domain triple mutant. Table S1. Minimal distances between pocket-forming residues in an SH2 domain and the ptyr residue of the ligand. Table S2. A list of biotinylated peptides used for screening the phage-displayed Fyn SH2 domain library. Table S3. A list of fluorescein-labeled peptides used for the in-solution binding assay. Table S4. Fitting error statistics for the K d values reported in Table 1. Table S5. Fitting error statistics for the K d values reported in Table 2. Table S6. Data collection and refinement statistics for x-ray crystallography.

(A) A list of SH2 variants, the corresponding ptyr-binding pocket residues

See table S2 for the sequences of the peptides. See the legend to Fig.

(B) Distribution of the number of substitutions in the ptyr-binding pocket

2 Fig. S1. SH2 domain variants obtained by screening a phage-displayed library. (A) A list of SH2 variants, the corresponding ptyr-binding pocket residues and the peptide probes used for their isolation. See table S2 for the sequences of the peptides. See the legend to Fig. 2A for the scheme in coloring of the pocket residues. (B) Distribution of the number of substitutions in the ptyr-binding pocket amongst the 63 SH2 variants. Variants with three substituted pocket residues were the most often observed. No clone identical to the wild-type (zero substitutions) was obtained from the phage selection.

3 Fig. S2. An alignment of the human SH2 domains showing the region for ptyr binding. The 120 human SH2 domain sequences were aligned as described in the Materials and Methods section. The C-terminal half of an SH2 domain, which is not involved in ptyr-binding, is omitted. The Arg at βb5 is highlighted.

4 Fig. S3. The specificity and affinity of the Fyn SH2 triple mutant in comparison to those of the wild-type domain. (A) Pull-down of phosphorylated proteins by the wildtype (Wt) and triple mutant (TrM) Fyn SH2 domains. GST or a GST-SH2 domain was used to precipitate phosphorylated proteins from the lysate of HeLa cells stimulated with sodium pervanadate. The complex was resolved on SDS-PAGE and immunoblotted with an antibody to ptyr (4G10). The bottom panel shows the loading control for the GST and GST-SH2 domains. Data shown is representative of three independent experiments. (B) Correlation of the binding free energies between the wild-type and triple mutant (TrM) Fyn SH2 domain for a group of ptyr-containing peptides (as labeled). The Gº values were derived from corresponding K d values shown in Table 1.

Fig. S4 Amino acid combinations of the ptyr-binding pocket in natural SH2

The 15 ptyr-binding pocket residues of 89 SH2 domains that have a

5 Fig. S4 Amino acid combinations of the ptyr-binding pocket in natural SH2 domains. The 15 ptyr-binding pocket residues of 89 SH2 domains that have a fiveresidue BC loop (including the Fyn, Src, and Grb2 SH2 domains) are listed. The following apolar residues are shaded in black: Ile, Val or Leu at position 8, Ala at position 10, Leu or Ile at position 15. The residues at these three positions in the Fyn, Src and Grb2 SH2 domains are boxed.

Fig. S5. The dynamics of the BC loop and its stabilization in the Src SH2 triple - mutant domain. (A) A stereo view of the ptyr-binding pocket as shown in Fig. 5A.

6 Fig. S5. The dynamics of the BC loop and its stabilization in the Src SH2 triple - mutant domain. (A) A stereo view of the ptyr-binding pocket as shown in Fig. 5A. (B) B-factor distribution of the triple mutant and the wild-type Src SH2 structures in apo form or in complex with the ptyr residue or a ptyr-containing peptide. The relative B- factor in the vertical axis was calculated as the individual B-factor divided by the average B-factor of the domain region. B-factor values of C α atoms were used. Loop residues generally produce peaks due to higher flexibility relative to the core residues in the domain. In the triple mutant structure, the BC loop gave a moderately lower peak compared to wild type structures. The peak corresponding to the BC loop is highlighted in red. The greater the B-factor value, the more flexible the region in general.

Fig. S6. The structure of the ptyr-binding pocket of the Src SH2 domain triple mutant. Side chains are shown as stick models and the main chain as ribbons.

7 Fig. S6. The structure of the ptyr-binding pocket of the Src SH2 domain triple mutant. Side chains are shown as stick models and the main chain as ribbons. Water molecules in the pocket region are shown as red balls. The three mutated residues are in green. Water or side chain atoms with hydrogen-bonding distance are connected by dotted lines. (A) The apo structure. (B) The structure of the SH2 mutant in complex with the phosphate ion. The soaked-in phosphate is shown as a stick model. The phosphate ion replaces water molecules identified in the apo structure, which results in the side chain of Glu6 (BC1) being flipped outward. (C) The ptyr complex structure. The soaked-in ptyr is shown as a yellow stick model. The side chain of Leu15 is rotated towards the phenyl ring of the ptyr for a closer fit. The side chain of Arg1 (αa2) is turned towards the phosphate group of ptyr to maximize electrostatic interactions. (D) The structure of the wild-type Src SH2 domain in complex with the phosphorylated tail (shown in yellow) of the kinase (PDB: 1FMK). The three residues found mutated in the triple mutant are colored green. The aliphatic side chain of Lys15 is aligned in parallel with the ptyr aromatic ring. In both the triple mutant and wild-type structures (panel C and D), the same set of five pocket residues, namely Arg1 (αa2), Arg4 (βb5), Ser5 (βb7), Glu6 (BC1), and Thr7 (BC2), is involved in polar contacts with the phosphate group.

8 Supplementary Tables Table S1. Minimal distances between pocket-forming residues in an SH2 domain and the ptyr residue of the ligand. See Materials and Methods for details of calculation. The average distance column denotes average distance values based on the Fyn, Src (wild type) and Lck domains. The substituted residues in the triple mutant Src SH2 domain are highlighted in bold and underscored. Fyn (PDB: 1AOT) Src (PDB: 1FMK) Lck (PDB: 1LCJ) Average Src triple mutant Position Amino Distance Amino Distance Amino Distance distance Amino Distance acid (Å) acid (Å) acid (Å) (Å) acid (Å) 1: αa2 ARG 3.0 ARG 2.8 ARG ARG 2.8 2: αa3 LYS 7.1 ARG 8.9 LYS ARG 9.4 3: αa5 ALA 7.5 SER 6.7 ALA SER 7.1 4: βb5 ARG 3.2 ARG 2.7 ARG ARG 2.8 5: βb7 SER 4.0 SER 3.2 SER SER 3.0 6: BC1 GLU 5.2 GLU 3.1 GLU GLU 3.5 7: BC2 THR 3.3 THR 2.7 SER THR 2.6 8: BC3 THR 7.6 THR 5.7 THR VAL 5.4 9: βc1 ALA 7.7 ALA 7.5 SER ALA : βc3 SER 4.0 CYS 4.0 SER ALA : βc4 LEU 7.5 LEU 8.5 LEU LEU : βc5 SER 7.1 SER 6.0 SER SER : βd3 LYS 7.4 LYS 8.6 LYS LYS : βd4 HIS 3.2 HIS 3.8 HIS HIS 3.9 βd5 TYR 5.5 TYR 6.2 TYR TYR : βd6 LYS 3.2 LYS 3.9 LYS LEU 3.8

9 Table S2. A list of biotinylated peptides used for screening the phage-displayed Fyn SH2 domain library ptyr peptide Sequence (a) Notes ptyr+2 Asn group peptide VEGFR1-pY 1213 biotin-a-a-d-v-r-py-v-n-a-a-k-f-amide wt b : DVRpYVNAFKF ShcA-pY 239 biotin-a-a-d-h-q-py-y-n-d-a-p-g-amide wt b : DHQpYYNDFPG β2-adrenoreceptor-py 350 c biotin-a-a-s-k-a-py-g-n-g-a-s-s-amide wt b : SKApYGNGYSS PDGFRβ-pY 716 biotin-a-a-a-e-l-py-s-n-a-a-p-v-amide wt b : AELpYSNALPV ErbB2-pY 1139 biotin-a-a-q-p-e-py-v-n-q-a-d-v-amide wt b : QPEpYVNQPDV TIE2-pY 1102 biotin-a-a-r-k-t-py-v-n-t-t-l-y-amide ptyr+3 Leu/Ile group peptide MidT-pY324mod d biotin-a-e-p-q-py-e-e-i-e-e-amide wt b : EPQpYEEIPI FCERB-pY 219 biotin-a-a-d-r-v-py-e-e-l-n-i-y-s-amide SIG11-pY 668 biotin-a-a-t-t-e-py-s-e-i-k-i-h-t-amide CD79A-pY 188 biotin-a-a-e-n-l-py-e-g-l-n-l-d-d-amide CEA20-pY 578 biotin-a-a-e-s-i-py-e-v-l-g-m-q-q-amide ptyr+4 Leu/Ile group peptide TRAF7-pY 275 biotin-a-a-q-d-t-py-e-t-h-l-e-t-amide (See footnote e) MALT1-pY 470 biotin-a-a-r-n-d-py-d-d-t-i-p-i-amide (See footnote e) RSKL-pY 423 biotin-a-a-y-q-h-py-d-l-d-l-k-d-amide B-raf-pY 85 biotin-a-a-y-e-e-py-t-s-k-l-d-a-amide (See footnote e) EGFR ptyr sites f 869 (845) EGFR-pY biotin-a-a-e-k-e-py-h-a-e-g-g-k-amide 915 (891) EGFR-pY biotin-a-a-s-k-p-py-d-g-i-p-a-s-amide EGFR-pY 944 (920) biotin-a-a-i-d-v-py-m-i-m-v-k-a-amide wt g : IDVpYMIMVKC 978 (954) EGFR-pY biotin-a-a-p-q-r-py-l-v-i-q-g-d-amide 998 (974) EGFR-pY biotin-a-a-s-n-f-py-r-a-l-m-d-e-amide 1016 (992) EGFR-pY biotin-a-a-a-d-e-py-l-i-p-q-q-g-amide 1069 (1045) EGFR-pY biotin-a-a-l-q-r-py-s-s-d-p-t-g-amide 1092 (1068) EGFR-pY biotin-a-a-v-p-e-py-i-n-q-s-v-p-amide 1110 (1086) EGFR-pY biotin-a-a-n-p-v-py-h-n-q-p-l-n-amide 1125 (1101) EGFR-pY biotin-a-a-d-p-h-py-q-d-p-h-s-t-amide 1138 (1114) EGFR-pY biotin-a-a-n-p-e-py-l-n-t-v-q-p-amide 1172 (1148) EGFR-pY biotin-a-a-n-p-d-py-q-q-d-f-f-p-amide 1197 (1173) EGFR-pY biotin-a-a-n-a-e-py-l-r-v-a-p-q-amide Selected ErbB4 ptyr sites

10 ErbB4-pY 1035 ErbB4-pY 1066 ErbB4-pY 1208 ErbB4-pY 1221 ErbB4-pY 1301 biotin-a-a-p-p-i-py-t-s-r-a-r-i-amide biotin-a-a-q-f-v-py-r-d-g-g-f-a-amide biotin-a-a-e-p-l-py-l-n-t-f-a-n-amide biotin-a-a-k-a-e-py-l-k-n-n-i-l-amide biotin-a-a-p-p-p-py-r-h-r-n-t-v-amide (a) The letter a in a peptide denotes a 6-aminohexanoic acid. (b) These synthetic peptides contain a modification from corresponding wild-type (wt) sequences. The wt sequence is shown in the notes column, and substituted residues are underlined. Modifications were introduced into these peptides so that each peptide can be unambiguously categorized in one of three groups: Asn at the ptyr + 2 position, a hydrophobic residue at the ptyr + 3 position, or a hydrophobic residue at the ptyr + 4 position. (c) Derived from the hamster sequence. (d) Derived from hamster polyomavirus middle-t antigen. (e) These are not known physiological phosphorylation sites. (f) For EGFR, two series of residue numbering systems are commonly used. In this paper, we followed the UniProt entry EGFR_HUMAN. The numbering system in parenthesis refers to the alternative convention (Foley, J. et al. EGFR signaling in breast cancer: bad to the bone. Semin Cell Dev Biol 21, , (2010)). (g) Cys in this peptide is replaced with Ala, to avoid disulfide bond formation.

11 Table S3. A list of fluorescein-labeled peptides used for the in-solution binding assay ptyr (or Tyr) peptide ptyr+2 Asn group peptide 1213 (a) VEGFR1-pY fluorescein-g-g-d-v-r-py-v-n-a-a-k-f-amide ptyr+3 Leu/Ile group peptide 324 (b) MidT-pY fluorescein-g-g-e-p-q-py-e-e-i-p-i-y-l-amide ptyr+4 Leu/Ile group peptide RSKL-pY 423 EGFR peptide EGFR-pY 978 EGFR-pY 1110 ShcA peptide ShcA-pY 239 ShcA-pY 317 Sequence fluorescein-g-g-y-q-h-py-d-l-d-l-k-d-amide fluorescein-g-g-p-q-r-py-l-v-i-q-g-d-amide fluorescein-g-g-n-p-v-py-h-n-q-p-l-n-amide fluorescein-g-g-d-h-q-py-y-n-d-f-p-g-amide fluorescein-g-g-d-p-s-py-v-n-v-q-n-l-amide ptyr or Tyr peptide (c) GGpYGG fluorescein-g-g-py-g-g-amide GGYGG fluorescein-g-g- Y-G-G-amide (a) Modified from the wild-type sequence: DVRpYVNAFKF. See also the table S2 footnote. (b) Derived from hamster polyomavirus middle-t antigen. (c) Designed peptides.

12 Table S4. Fitting error statistics for the dissociation constant (K d ) values reported in Table 1. Fyn wild-type Fyn T8V Fyn S10V Peptide K d ± SEM K d ± SEM K d ± SEM VEGFR1-pY ± ± ± 0.91 EGFR-pY ± ± ± 0.31 EGFR-pY ± ± ± 1.1 MidT-pY ± ± ± RSKL-pY ± ± ± 0.23 ShcA-pY ± ± ± ShcA-pY ± ± ± 0.13 GGpYGG 72 ± ± ± 3.6 Fyn S10A Fyn K15L FynS10A/K15L Peptide K d ± SEM K d ± SEM K d ± SEM VEGFR1-pY ± ± ± EGFR-pY ± ± ± EGFR-pY ± ± ± MidT-pY ± ± ± RSKL-pY ± ± ± ShcA-pY ± ± ± ShcA-pY ± ± ± GGpYGG 9.0 ± ± ± 1.0 FynT8V/K15L FynT8I/S10A/K15L Fyn T8V/S10A/K15L Peptide K d ± SEM K d ± SEM K d ± SEM VEGFR1-pY ± ± ± EGFR-pY ± ± ± EGFR-pY ± ± ± MidT-pY ± ± ± RSKL-pY ± ± ± ShcA-pY ± ± ± ShcA-pY ± ± ± GGpYGG 3.1 ± ± ± Footnote: The K d and standard error of the mean (SEM) values derived from binding curve fitting are listed, in the micromolar unit. Data with K d > 30 µm are out of the titration range and exact values are not reliable.

13 Table S5. Fitting error statistics for the dissociation constant (K d ) values reported in Table 2. Src wild-type Src T8V/C10A Peptide K d ± SEM K d ± SEM VEGFR1-pY ± ± 0.21 EGFR-pY ± ± EGFR-pY ± ± 0.39 MidT-pY ± ± RSKL-pY ± ± ShcA-pY ± ± ShcA-pY ± ± GGpYGG 46 ± ± 5.0 Src K15L Src T8V/C10A/K15L Peptide K d ± SEM K d ± SEM VEGFR1-pY ± ± EGFR-pY ± ± EGFR-pY ± ± MidT-pY ± ± RSKL-pY ± ± ShcA-pY ± ± ShcA-pY ± ± GGpYGG 25 ± ± Footnote: The K d and standard error of the mean (SEM) values derived from binding curve fitting are listed, in the micromolar unit. Data with K d > 30 µm are out of the titration range and exact values are not reliable.

14 Table S6. Data collection and refinement statistics for X-ray crystallography. Apo form Phosphate ion complex Phosphotyrosine complex Data collection Space group P6 1 P6 1 P6 1 Cell dimensions a, b, c (Å) 67.1, 67.1, , 67.0, , 67.6, 46.8 Resolution (Å) ( )* ( )* R merge (0.264) (0.267) (0.266) I/σI 7.9 (2.8) 10.0 (2.9) 7.9 (2.9) Completeness (%) 97.0 (85.9) 99.6 (100.0) 90.2 (100.0) Redundancy 5.8 (5.4) 5.9 (5.8) 8.2 (8.0) ( )* Refinement Resolution (Å) No. reflections R work/ R free / / / No. atoms Protein Phosphate or ptyr 5 17 Water B-factors Protein Phosphate or ptyr Water R.m.s. deviations Bond lengths (Å) Bond angles (º) *The highest resolution shell is shown in parenthesis.

Supplementary Figure 3 a. Structural comparison between the two determined structures for the IL 23:MA12 complex. The overall RMSD between the two

Supplementary Figure 3 a. Structural comparison between the two determined structures for the IL 23:MA12 complex. The overall RMSD between the two Supplementary Figure 1. Biopanningg and clone enrichment of Alphabody binders against human IL 23. Positive clones in i phage ELISA with optical density (OD) 3 times higher than background are shown for