Surface Loops in a Single SH2 Domain Are Capable of Encoding the Spectrum of Specificity of the SH2 Family

Abstract

Src homology 2 (SH2) domains play an essential role in cellular signal transduction by binding to proteins phosphorylated on Tyr residue. Although Tyr phosphorylation (pY) is a prerequisite for binding for essentially all SH2 domains characterized to date, different SH2 domains prefer specific sequence motifs C-terminal to the pY residue. Because all SH2 domains adopt the same structural fold, it is not well understood how different SH2 domains have acquired the ability to recognize distinct sequence motifs. We have shown previously that the EF and BG loops that connect the secondary structure elements on an SH2 domain dictate its specificity. In this study, we investigated if these surface loops could be engineered to encode diverse specificities. By characterizing a group of SH2 variants selected by different pY peptides from phage-displayed libraries, we show that the EF and BG loops of the Fyn SH2 domain can encode a wide spectrum of specificities, including all three major specificity classes (p + 2, p + 3 and p + 4) of the SH2 domain family. Furthermore, we found that the specificity of a given variant correlates with the sequence feature of the bait peptide used for its isolation, suggesting that an SH2 domain may acquire specificity by co-evolving with its ligand. Intriguingly, we found that the SH2 variants can employ a variety of different mechanisms to confer the same specificity, suggesting the EF and BG loops are highly flexible and adaptable. Our work provides a plausible mechanism for the SH2 domain to acquire the wide spectrum of specificity observed in nature through loop variation with minimal disturbance to the SH2 fold. It is likely that similar mechanisms may have been employed by other modular interaction domains to generate diversity in specificity.

Keywords: Antibiotics; Autophagy; Breast Cancer; Cancer Biology; Cancer Stem Cells; Chemoresistance; Clinical Proteomics; Mitochondria; Mitochondria Function or Biology; NMR; NMR-metabolomic; Stem Cells.

Graphical abstract

Fig. 1.

Directed evolution of the Fyn SH2 domain via systematic changes in the EF and BG loops. A, Structure of the FYN-SH2 domains in complex with a tyrosine-phosphorylated peptide (PDB ID 4U1P). The bound peptide is colored magenta, with the side chains of pTyr and p + 3 Ile residues shown as sticks. The p + 3 Ile is located between the EF and BG loops. Residues targeted for evolution via combinatorial mutagenesis are identified with blue balls for the EF loop (EF1, EF2 and EF3) and red balls for the BG loop (BG2, BG3 and BG4). B, The phage display library design. The three resides from the EF and BG loops targeted for mutagenesis were underscored in the sequence of the human Fyn-SH2 domain. The length of both loops was unchanged in the 3 + 3 library, whereas the length of BG loop was varied in the 3+x library, from two-residue shorter to three-residue longer, but not 3.

Fig. 2.

Loop sequences of the variants identified by different bait peptides. Residues in the bait peptide and EF/BG loop sequences are colored according to their chemical nature. The 29 variants selected for subcloning and further characterization are annotated with the variant numbers. A, Variants identified by bait peptides containing the pY+2N motif. B, Variants identified by bait peptides that contain a hydrophobic residue at either the pY+3 or pY+4 position.

Fig. 3.

Specificity of SH2 loop variants revealed by OPAL. A, A representative OPAL binding profile for the variant V6. Each sublibrary was printed in quadruplicate (marked by a square). Neutravidin was included as the negative control (marked by red rectangle). GST was employed as positive control (for GST fusion proteins used to probe the OPAL) and identified by a green rectangle. The Fyn-SH2 variant V6 showed p + 2N specificity. B, A heat map to show the preference of the 29 variants for residues at the p + 2 position. The Fyn, BRDG1 and PI3K-p85α SH2 domains were included as controls. The heat map was generated using the corresponding Z-scores on the OPAL.

Fig. 4.

Bait peptides affect specificity of the isolated loop variants. A, Pie charts showing the number of variants belonging to the specificity group p + 2N or p + 3[I/L/V] based on the corresponding OPAL binding profiles. B, The variants in (A) are divided into four groups based on their Z scores.

Fig. 5.

Characterization of variant specificity by peptide ligand array. A heat map depicting the binding specificity of the loop variants (top) for the different peptides (right) included in the ligand array. Rectangle “a” identifies variants with strong binding for pY+2N peptides, in agreement with the OPAL data. Similarly, variants showing p + 3 [ILV] preference on the OPAL exhibited strong binding to peptides containing these residues at pY+3 position (rectangle “b”).

Fig. 6.

The specificity defining residues in the loop variants. Ranking of variants for p + 2N (A), p + 3[I/V/L] (B) and p + 4[L/F] (C) specificity based on the corresponding Z scores on OPAL.

Fig. 7.

A cartoon model depicting the mechanism of p + 4 recognition by V17 (A) and p + 4 and p + 2N recognition by V29 (B, C). The peptide ligand is shown in orange with specificity residues shown. Specificity-determining residues in the EF and BG loops are shown.