Volume 11, Issue 2, Pages 471-481 (February 2003) Structural Basis for Specific Binding of the Gads SH3 Domain to an RxxK Motif- Containing SLP-76 Peptide  Qin Liu, Donna Berry, Piers Nash, Tony Pawson, C.Jane McGlade, Shawn Shun-Cheng Li  Molecular Cell  Volume 11, Issue 2, Pages 471-481 (February 2003) DOI: 10.1016/S1097-2765(03)00046-7

Figure 1 Solution Structure of a Gads SH3-C Domain – SLP-76 Peptide Complex (A) Stereo view of a superposition of 20 lowest-energy structures of the Gads SH3-C domain – SLP-76 peptide complex in backbone traces. The Gads SH3-C domain (residues 265–321) is shown in green and the SLP-76 peptide in violet. The peptide has the sequence A1PSIDRSTKPA11, of which the first ten residues were taken from the Gads SH3-C – binding site in SLP-76, and the last Ala was added to reduce end effects. Ala11 does not interact with the SH3 domain (see Figure 3B) and is hence omitted from all figures for clarity. (B) Ribbon representation of the same complex generated using coordinates of the lowest-energy structure. The β strands and the RT- and n-Src loops of the SH3 domain are labeled in black. The peptide backbone is depicted in violet with side chains shown in pale green or dark green (for residues located at the peptide-protein interface). Molecular Cell 2003 11, 471-481DOI: (10.1016/S1097-2765(03)00046-7)

Figure 2 A Comparison of Peptide Binding Surfaces between the Gads and c-Src SH3 Domains (A) Surface representation of the Gads SH3-C domain – SLP-76 peptide complex. Areas of positive and negative charges are shown in blue and red, respectively. Residues in the peptides that occupy the four binding pockets on the SH3 domain surface (identified in dotted circles) are labeled in black. Residue Glu275 of the protein, which encloses the second pocket, is labeled in red. (B) Surface representation of the c-Src SH3 domain – APP12 peptide complex (adapted from Feng et al., 1995). APP12 is a dodecapeptide that binds with high affinity to the c-Src SH3 domain (Kd = 1.2 μM). Since the last four residues of the peptide do not contribute significantly to SH3 binding (Feng et al., 1995), only the first eight residues of the peptide (A1PPLPPRN8) are shown for clarity. As in (A), key residues in peptide APP12 that engage the three binding pockets (identified as dotted circles) on the c-Src SH3 domain are labeled. Molecular Cell 2003 11, 471-481DOI: (10.1016/S1097-2765(03)00046-7)

Figure 3 Structure and Interactions of the RSTK Motif-Containing SLP-76 Peptide (A) Conformation and interactions mediated by the RSTK site of the SLP-76 peptide (in stereo view). Backbone and side chain heavy (except N and O) atoms of the peptide are shown in cyan, while those of the SH3 domain are shown in gray. Nitrogen and oxygen atoms are depicted in blue and red, respectively. Residues are identified in cyan while interacting residues in the SH3 domain in black. Potential electrostatic and hydrogen-bond interacting pairs are identified by arches. For simplicity, hydrogen atoms are not included in the figure. (B) An overlay of 15N-1H correlation spectra (HSQC) of peptide SLP-76 in free (colored in black) and bound (in red) states. The boxed peak is originated from the side chain of Arg6. Note that the intensity of the amide resonance of Ala11 is much greater than those of other residues and that its position moved very little from the free state, indicating that Ala11 is quite flexible in the bound state and hence unlikely involved in binding the SH3 domain. Molecular Cell 2003 11, 471-481DOI: (10.1016/S1097-2765(03)00046-7)

Figure 4 Specificity Determinants for the Interaction between the SLP-76 Peptide and the Gads SH3-C Domain (A) Peptide IC50 curves for the competition of binding to purified Gads SH3-C. Fluorescein-SLP-76 wt peptide (Fl-APSIDRSTKPA) bound to Gads SH3-C was competed away by SLP-76, SLP-76 mutant peptides, or a SOS peptide (VPPPVPPRRR) (Nash et al., 2002). Calculated IC50 values were obtained from the average of at least three independent experiments and are on the right of the competition curves. For purpose of comparison, relative IC50 values (with that of wt peptide set at 1.0) are given. (B) Equilibrium binding curves for Gads SH3-C mutants to fluorescein-SLP-76 peptide measured by fluorescence polarization. Binding data were analyzed using the Michaelis-Menton equation in order to obtain the dissociation constant (Kd) values. Average values from at least three independent experiments are reported. Molecular Cell 2003 11, 471-481DOI: (10.1016/S1097-2765(03)00046-7)

Figure 5 Mutations of Residues at the Interface of the Complex Affect Binding of Gads to SLP-76 in Cells (A) Binding of Flag-tagged SLP-76 mutants to endogenous Gads protein in the SLP-76-deficient Jurkat cell line, J14. Alanine substitutions at P233, P241, I235, or D236A led to a reduction in coimmunoprecipation with Gads. Substitution of I235 with a proline residue also led to decreased binding, while a glutamate residue at position 235 completely abrogated binding. (B) Binding of Gads SH3-C domain mutants to SLP-76 in Jurkat T cells. Flag-tagged wt Gads protein was compared with Flag-tagged Gads mutants for ability to precipitate endogenous SLP-76. Compared to wt Gads, single Ala substitutions at L277 and E279 had mild effects on binding, while single substitutions at E278, E281, or W300, or double mutations at positions 277, 278, 279, and 281, greatly reduced or completely abolished binding. Molecular Cell 2003 11, 471-481DOI: (10.1016/S1097-2765(03)00046-7)

Figure 6 Conformational Change in the RT Loop Alters SH3 Domain Specificity Superposition of the Gads SH3-C – SLP-76 peptide complex (peptide in violet, protein in red) with the c-Src SH3 – peptide complex (peptide in gold, protein in green). Key residues in the two peptides are labeled, as are the RT- and n-Src loops. Molecular Cell 2003 11, 471-481DOI: (10.1016/S1097-2765(03)00046-7)