Fig. 2. Sequence comparison and subclass definition of GYF domains. (A) Multiple sequence alignment of GYF domains available from the public database. The color coding of the Chroma program was used as follows: single residues with a conservation of >80% are printed as colored characters (glycine residues in red, hydrophobic residues in yellow) on a dark gray background, and the occurrence of an aromatic or a hydrophobic amino acid in >80% of the 26 GYF domains aligned leads to a yellow character on a light gray background. The red and brown color of the protein accession numbers suggest the subclasses as derived from analysis of the multiple sequence alignment. (B) Phylogenetic tree of the GYF domain as calculated with the program ClustalW (version 1.7) using the multiple sequence alignment obtained from the pileup option of the gcg program package. The individual GYF domains are labeled with the DDBJ/EMBL/GenBank accession numbers according to (A) with the species in which they occur in parentheses.

Fig. 4. Fyn SH3 domain binding to the CD2 sequence SHRPPPPGHRV. (A) Model of the complex of the Fyn SH3 domain with the SHRPPPPGHRV ligand of CD2. The ribbon diagram of the SH3 domain is color coded according to the chemical shift perturbations observed in the NMR shift mapping experiments (red, combined chemical shift index >1.0; pink, combined chemical shift index 0.5–1.0). The side chains of residues making direct contacts to the ligand are displayed in red and marked by residue type and number. The ligand is shown as a bonded structure in cyan. Functionally important elements (RT and nsrc loops, specificity pocket and the two proline-binding pockets) of the SH3 domain fold are highlighted. (B) Structural superposition of the Lck (light red) and Fyn SH3 domain (white). The model of the Fyn SH3 domain in complex with the SHRPPPPGHRV peptide from CD2 was overlapped with the Lck SH3 structure (1Lck; Eck et al., 1994). The side chains of residues in the conserved proline-binding pockets are shown to align extremely well for this part of the interaction site. The side chains of RT loop residues that contribute to the observed binding specificity by interacting with (in the case of Fyn) or sterically hindering (in the case of Lck) the insertion of Arg3 of the peptide into the specificity pocket are also displayed. The shaded area defines the exclusion volume of Arg3. As in (A), the ligand is shown as a bonded structure with its carbons colored cyan.

Fig. 6. In vitro competition of CD2BP2 GYF and Fyn SH3 domains for CD2-binding sites. The spectrum of the isolated ¹⁵N-labeled GYF domain (0.2 mM) is shown in black. Substoichiometric amounts of the CD2 tail (residues 245–351 of human CD2) were added and the corresponding spectrum is shown in blue. Then 0.4 mM unlabeled Fyn SH3 domain was added, a third spectrum obtained and displayed in red. Residues that are shifted upon binding of the CD2 tail are marked by residue type and number. The arrow for the NH peak of Gly32 clarifies the movement of this resonance.

Fig. 1. The GYF domain–ligand complex. (A) The combined chemical shift index (see Supplementary data) for the backbone NH groups of all GYF domain residues except proline is shown for the binding of an equimolar amount of the peptide SHRPPPPGHRV. Secondary structure elements are given below the sequence of the CD2BP2 GYF domain and are also present in the three-dimensional fold shown above the chemical shift indices. (B) Stereo view of the superposition of the best 15 NMR structures of the CD2BP2 GYF domain in complex with the peptide SHRPPPPGHRV. The side chains of aromatic residues of the binding site are colored red, and the prolines of the peptide are shown in green. All other side chains are omitted for clarity. (C) Lipophilic potential of the GYF domain. The surface was created by using the hydrophobicity scale of the tripos program package (Heiden et al., 1993). Hydrophobicity scaling is from yellow (most hydrophobic) to green (hydrophilic). The peptide ligand is shown as a bonded structure with labels for residues mentioned in the text. (D) Electrostatic potential of the GYF domain. The amber force field has been used to calculate the charge distribution (Case et al., 1997). Scaling is from acid (red) to basic (blue). Residues of the GYF domain that are important for the interaction with the two arginine side chains of the ligand are marked on the surface.

Fig. 3. Binding of proline-rich CD2 peptides to the GYF domain. (A) Overlap of the ¹⁵N–¹H correlation spectra of the isolated GYF domain (green), the GYF domain upon addition of 0.73 mM of the short CD2 peptide segment (red) and the GYF domain after addition of 0.2 mM of the long CD2 peptide sequence (blue). The eight NH resonances with the largest chemical shift difference between the bound and unbound state are labeled according to residue type and number. The inset shows the change of the ¹⁵N chemical shift for Tyr33 of the GYF domain as a function of the ligand concentration of either the short (SHRPPPPGHRV) or the long peptide (HPPPPGHRSQAPSHRPPPPGHRVQHQPQK). The short peptide corresponds to the CD2 sequence 294–304 and the long peptide to the sequence 281–310. (B) ¹⁵N–¹H correlation spectrum of the isotopically labeled long peptide before (blue) and after (red) addition of a 3-fold excess of unlabeled GYF domain. The resonances of the glycine and histidine residues of the two PPPPGHR motifs are overlapping and highlighted within the spectra. Inset: SDS–PAGE of EGS cross-linking reaction products. The GYF domain (∼26 µM) and long CD2 tail peptide (∼140 µM) were cross-linked in 50 µl of reaction buffer with EGS. The molecular weights of migration markers are shown on the right of the gel. The positions of various cross-linked complexes are indicated with arrows, where G indicates the GYF domain and P indicates the CD2 tail peptide. The identity of cross-linked bands was confirmed by MALDI-TOF mass spectrometry using the molecular weights of the monomers. The GGP band (∼20 kDa) shows the presence of a complex with two GYF domains and one CD2 tail peptide. Lane 1, GYF and CD2 tail peptide mixture without cross-linker; lane 2, GYF and CD2 tail peptide mixture with cross-linker.

Fig. 5. Comparison of GYF and SH3 domain–ligand interactions. (A) The CD2BP2 GYF domain in complex with the PPPPGHR sequence from CD2 and the Fyn SH3 domain in complex with the peptide YPPPPVP are shown side by side. The surface of the GYF and the SH3 domain is shown as a grid with the residues Trp8, Glu15 and Tyr17 of the GYF domain displayed in red. Conserved residues important for binding are marked by residue type and sequence number. The peptides are shown as a bonded structure with residues 9 and 10 of the Fyn ligand colored red to highlight the extension of the proline helix as compared with the GYF domain ligand. (B) Schematic model showing the two proline-binding sites for the SH3 (right) and the single pocket in the GYF domain (left). Residues of the protein domains are displayed as rectangular structures, with labels for protein residues as either black (conserved domain residues) or white (non-conserved residues). Peptide residues are shown as circles labeled inside by residue type. The two prolines of the PxxP SH3 recognition motif are shaded gray.

Fig. 7. Distinct cellular compartmentation of CD2BP2 and Fyn proteins in vivo. Flag-tagged CD2BP2 Jurkat T-cell transfectants (Nishizawa et al., 1998) were used for lipid raft preparation according to a previously published protocol (Yang and Reinherz, 2001). (A) The distribution of CD2BP2 as well as Fyn proteins in sucrose density gradients. Each of the 12 fractions are shown from least to most dense (1–12, respectively). CD2BP2 western blotting was performed using the anti-Flag M2 antibody. (B) The distribution of molecules in the lipid raft and detergent-soluble fraction in the presence (+) or absence (–) of prior CD2 cross-linking. Sucrose gradient fraction 10 was used as a representative detergent-soluble fraction, and fraction 4 as the raft fraction. Identical results were obtained with each of two independent Jurkat CD2BP2 stable transfectants. The distribution of CD2, LAT and actin in the sucrose gradient system is consistent with results described previously (Yang and Reinherz, 2001). (C) An equal volume of raft or detergent-soluble fractions from CD2-stimulated (+) or non-stimulated (–) samples was diluted in octyl-glucoside-containing binding buffer, immunoprecipitated (IP) with either anti-Fyn (top row) or anti-T11₁ as designated (bottom row) and immunoblotted (WB) with anti-CD2 hetero-antisera (both rows).