PDBsum entry 1h3h

Protein binding

PDB id: 1h3h

Contents

Protein chains

60 a.a. *

11 a.a. *

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Structural basis for specific binding of the gads sh3 domain to an rxxk motif-Containing slp-76 peptide: a novel mode of peptide recognition.

Authors

Q.Liu, D.Berry, P.Nash, T.Pawson, C.J.Mcglade, S.S.Li.

Ref.

Mol Cell, 2003, 11, 471-481. [DOI no: 10.1016/S1097-2765(03)00046-7]

PubMed id

12620234

Abstract

The SH3 domain, which normally recognizes proline-rich sequences, has the potential to bind motifs with an RxxK consensus. To explore this novel specificity, we have determined the solution structure of the Gads T cell adaptor C-terminal SH3 domain in complex with an RSTK-containing peptide, representing its physiological binding site on the SLP-76 docking protein. The SLP-76 peptide engages four distinct binding pockets on the surface of the Gads SH3 domain and upon binding adopts a unique structure characterized by a right-handed 3(10) helix at the RSTK locus, in contrast to the left-handed polyproline type II helix formed by canonical proline-rich SH3 ligands. The structure, and supporting mutagenesis and peptide binding data, reveal a novel mode of ligand recognition by SH3 domains.

Figure 1.
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 A^1PSIDRSTKPA^11, 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).

Figure 2.
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 (A^1PPLPPRN^8) 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.

The above figures are reprinted by permission from Cell Press: Mol Cell (2003, 11, 471-481) copyright 2003.

Secondary reference #1

Title

A high-Affinity arg-X-X-Lys sh3 binding motif confers specificity for the interaction between gads and slp-76 in t cell signaling.

Authors

D.M.Berry, P.Nash, S.K.Liu, T.Pawson, C.J.Mcglade.

Ref.

Curr Biol, 2002, 12, 1336-1341. [DOI no: 10.1016/S0960-9822(02)01038-2]

PubMed id

12176364

Figure 1.
Figure 1. Gads Binding Proteins Share an Amino Acid Motif Similar to SLP-76(A) Refinement of the minimal Gads SH3 domain binding motif in SLP-76. Peptides (15-mer), scanning through the binding region of SLP-76, were synthesized and spotted on a cellulose filter as previously described [9]. The filter was blocked with 5% skim milk powder in TBS-T and probed with full-length GST-Gads C-SH3 fusion protein (2 μg/ml for 3 hr). Bound fusion protein was detected by blotting with monoclonal anti-GST antibody.(B) Alignment of known and novel Gads SH3 domain binding proteins reveals a common binding motif. A 16-day mouse embryo protein library (Novagen) was screened with GST-Gads fusion protein (see Supplementary Material). Isolated cDNAs were partially sequenced and identified through the GenBank database. A search of the GenBank database for other proteins with the consensus PxxxR-X-X-KP identified both Gab2 and BLNK.(C) UBPY, Gab2, and BLNK can associate with Gads. Cos1 cells were transfected with 2 μg of pCMV-UBPY or pPuro-BLNK-Myc and harvested 24–48 hr later. Clarified cell lysate from transfected Cos1 cells or from K562 cells was incubated with 2 μg of GST or GST-Gads bound to glutathione-sepharose beads for 1–2 hr at 4°C. Beads were washed with NP-40 lysis buffer, and bound proteins were separated by SDS-PAGE and Western blotted with anti-UBPY (a gift from Paolo Di Fiore and Guillio Draetta), anti-myc (to detect myc-BLNK), or anti-Gab2 (Upstate Biotechnology).(D) Confirmation of binding sites in UBPY, AMSH, and BLNK. Peptides representing the predicted binding region of each protein were synthesized and spotted onto a cellulose filter as in (A). The filters were probed with Gads GST-C-SH3 fusion protein (2 μg/ml) and blotted with anti-GST monoclonal antibody (Upstate Biotechnology).

Figure 4.
Figure 4. The Gads SH3 Domain Confers SLP-76 Binding and TCR Signaling Properties to Grb2(A) Schematic of the Grb2-Gads chimera that was generated by PCR amplification and ligation of cDNA fragments encoding amino acids 1–154 of Grb2 (N-SH3 and SH2) and amino acids 257–322 of Gads (C-SH3). The chimeric cDNA was cloned into a pEF vector with a Flag epitope tag.(B) Jurkat T cells were electroporated with 40 μg of either empty pEF vector, or pEF containing full-length Gads, Grb2, or the chimera. Anti-Flag immunoprecipitations were performed on clarified lysates, and Western blots were probed with polyclonal anti-SLP-76 anti-sera (a gift from Gary Koretzky) (top). Total cell lysates from each condition were Western blotted with anti-Flag antibody (Sigma) to assess expression levels of the transfected proteins.(C) Grb2-Gads chimera synergizes with SLP-76 to activate NFAT downstream of the TCR. Jurkat cells (2 × 10^7) were electroporated with 20 μg NFAT-luciferase reporter construct, together with 40 μg of empty pEF vector or 40 μg of Flag epitope-tagged Gads, Grb2, or a Grb2 construct with the C-terminal SH3 of Gads. Luciferase assays were performed and data presented as described for Figure 2C.

The above figures are reproduced from the cited reference with permission from Cell Press