Solution structure and backbone dynamics of the AF-6 PDZ domain/Bcr peptide complex

Abstract

The human AF-6, a scaffold protein between cell membrane-associated proteins and the actin cytoskeleton, plays an important role in special cell-cell junctions and signal transduction. It can be phosphorylated by the protein kinase Bcr, which allows efficient binding of the C terminus of Bcr to the PDZ domain of AF-6 and consequently enhances the binding affinity of AF-6 to Ras. Formation of the AF-6, Bcr, and Ras ternary complex results in down-regulation of the Ras-mediated signal transduction pathway. To better understand the molecular basis for the recognition of the AF-6 PDZ domain and Bcr, we solve the solution structure of the AF-6 PDZ domain complexed with the C-terminal peptide of Bcr and explore the interactions between them in detail. Compared with previously reported structures, the complex exhibits a noncanonical binding mode of PDZ/peptide. Owing to the distinct residues involved in the AF-6 PDZ domain and Bcr peptide interaction, the interaction mode does not adapt to the existing classification rules that have been put forward, based on the ligand or the PDZ domain specificity. Furthermore, the PDZ domain of AF-6 can bind to the C terminus of Bcr efficiently after phosphorylation of AF-6 by the Bcr kinase. The phosphorylation may induce a conformational change of AF-6, which makes the binding surface on the PDZ domain accessible to Bcr for efficient binding. This study not only characterizes the structural details of the AF-6 PDZ/Bcr peptide complex, but also provides a potential target for future drug design and disease therapy.

Figure 1.

Structure of the AF-6 PDZ/Bcr complex. (A) Backbone overlay stereoview of the 20 lowest-energy NMR structures of the PDZ domain from human AF-6 complexed with the C-terminal peptide from Bcr, superimposed using backbone atoms (N, C_α, C′). (Blue) The PDZ domain; (purple) the Bcr peptide. This figure was prepared using MOLMOL (Koradi et al. 1996). (B) Ribbon diagram of a representative NMR structure of the complex generated with MOLSCRIPT (Kraulis 1991) and Raster3D (Merritt and Murphy 1994). The β-strands of the PDZ domain are labeled βA–βF, and the α-helices are labeled αA and αB. The ligand peptide (β0) inserts between the βB-strand and the αB-helix of the PDZ domain, forming an antiparallel β-sheet with βB.

Figure 2.

Detailed interaction between the AF-6 PDZ and Bcr peptide. (A) Structural comparison between (left) AF-6 PDZ/Bcr and (right) the canonical class I complex (PSD-95 PDZ3/peptide, PDB code 1BE9). Hydrogen bonds (dotted pink lines) between residues of the PDZ domain (blue) and the Bcr peptide (yellow) were deduced from the geometry of the structure. (Red) Oxygen atoms; (green) nitrogen atoms. The AF-6 PDZ/Bcr interaction differs significantly from that of the canonical class I PDZ domains for the absence of a hydrogen bond between the −2 position residue of the peptide and the αB:1-position residue His of the PDZ. For clarity, side chains of only selected residues are shown. The programs MOLSCRIPT and Raster3D were used to generate this figure. (B) Structural comparison between (left) AF-6 PDZ/Bcr and (right) the canonical class II complex (Grip1 PDZ6/peptide, PDB code 1N7F). Surface representation of the packing interface is generated with PyMOL (available at www.pymol.org). (Yellow) The hydrophobic residues (Ala, Ile, Leu, Met, Pro, Phe, Tyr, and Val); (red) negatively charged residues (Asp and Glu); (blue) positively charged residues (Arg, His, and Lys); and (white) polar residues (Asn, Gln, Gly, Ser, and Thr). The AF-6 PDZ/Bcr interaction differs obviously from that of the canonical class II PDZ domains for the absence of the second hydrophobic pocket at the −2 position residue of the ligand peptide.

Figure 3.

Comparison of the AF-6 PDZ in the (blue) ligand-free and (yellow) ligand-bound states; the peptide of Bcr is not shown (stereoview). Both figures were prepared using the program PyMol. (A) The overall structure of the PDZ domain is only slightly changed upon Bcr binding. (B) Although the overall structure of the PDZ domain is very similar in the free and complex forms, backbone deviations are observed in αB, βB region.

Figure 4.

Backbone dynamics of the AF-6 PDZ/Bcr peptide complex. (A) The backbone ¹⁵N relaxation parameters for (open triangles) free and (filled triangles) peptide-bound states of the AF-6 PDZ domain are plotted versus the residue number. The error bars represent standard deviations. (B) The amides of the AF-6 PDZ domain in the complex form showing enhanced mobility on a sub-nanosecond timescale, as evident by reduced {¹H}–¹⁵N NOE values, are highlighted in color on the representation of the ensemble of NMR-derived structures. (Red) Residues with {¹H}–¹⁵N NOE values of <0.2 (highest mobility); (orange) between 0.2 and 0.4; (yellow) between 0.4 and 0.6; and (gray) >0.6. (Blue) Proline residues or residues for which data are not available (e.g., because of spectral overlap). (Pink) The Bcr peptide in ball-and-stick models. This figure was prepared using PyMOL.