Defining SH2 domain and PTP specificity by screening combinatorial peptide libraries

Abstract

Src homology 2 (SH2) domains mediate protein-protein interactions by recognizing short phosphotyrosyl (pY) peptide motifs in their partner proteins. Protein tyrosine phosphatases (PTPs) catalyze the dephosphorylation of pY proteins, counteracting the protein tyrosine kinases. Both types of proteins exhibit primary sequence specificity, which plays at least a partial role in dictating their physiological interacting partners or substrates. A combinatorial peptide library method has been developed to systematically assess the sequence specificity of SH2 domains and PTPs. A "one-bead-one-compound" pY peptide library is synthesized on 90-microm TentaGel beads and screened against an SH2 domain or PTP of interest for binding or catalysis. The beads that carry the tightest binding sequences against the SH2 domain or the most efficient substrates of the PTP are selected by an enzyme-linked assay and individually sequenced by a partial Edman degradation/mass spectrometry technique. The combinatorial method has been applied to determine the sequence specificity of 8 SH2 domains from Src and Csk kinases, adaptor protein Grb2, and phosphatases SHP-1, SHP-2, and SHIP1 and a prototypical PTP, PTP1B.

Fig. 1

Apparatus for peptide library synthesis. It consists of a dry-ice cold trap connected to house vacuum (part A), a vacuum manifold with a solvent reservoir (part B), and a rotary shaker (part C).

Fig. 2

Specific labeling of a ybbR-tagged protein by Sfp-mediated transfer of an S-alkylated phosphopantetheinyl group from CoA-SR to the ybbR tag. CoA-SR, S-alkylated coenzyme A where R is biotin or a fluorescent group.

Fig. 3

Screening of the pY peptide library against an SH2 domain. (a) Scheme showing the steps involved in colorimetric screening of the pY peptide library against a biotinylated MBP-SH2 domain. Key: MBP, maltose binding protein; SA, streptavidin; AP, alkaline phosphatase. (b) A photograph of a portion of the screened library (~50x magnification). A positive bead (turquoise colored) is shown.

Fig. 4

Peptide sequencing by PED/MS. (a) Scheme showing the reactions involved in partial Edman degradation. Reagents and conditions: (a) 10–30:1 PITC:Fmoc-OSU; (b) TFA; (c) Repeat steps a and b 7 times; (d) 20% piperidine in DMF; and (e) CNBr in 70% TFA. (b) The MALDI mass spectrum of peptide TAFIpYDNVLNBBRM and its degradation products (from a single 90 μm bead). Key: M*, homoserine lactone.

Fig. 5

Sequence specificity of c-Src, Grb2 and Csk SH2 domains. Displayed are the amino acids identified at each position from −2 to +3 relative to pY (position 0). Number of occurrence on the y axis represents the number of selected sequences that contain a particular amino acid at a certain position. Key: M, norleucine; C, α-aminobutyric acid. The pY library screened against c-Src and Grb2 SH2 domains did not contain α-aminobutyric acid.

Fig. 6

Reactions involved in pY library screening against a PTP.