Structural basis for SH3 domain-mediated high-affinity binding between Mona/Gads and SLP-76

Abstract

SH3 domains are protein recognition modules within many adaptors and enzymes. With more than 500 SH3 domains in the human genome, binding selectivity is a key issue in understanding the molecular basis of SH3 domain interactions. The Grb2-like adaptor protein Mona/Gads associates stably with the T-cell receptor signal transducer SLP-76. The crystal structure of a complex between the C-terminal SH3 domain (SH3C) of Mona/Gads and a SLP-76 peptide has now been solved to 1.7 A Ê . The peptide lacks the canonical SH3 domain binding motif P±x±x±P and does not form a frequently observed poly-proline type II helix. Instead, it adopts a clamp-like shape around the circumfence of the SH3C b-barrel. The central R±x±x±K motif of the peptide forms a 3 10 helix and inserts into a negatively charged double pocket on the SH3C while several other residues complement binding through hydrophobic interactions, creating a short linear SH3C binding epitope of uniquely high af®nity. Interestingly, the SH3C displays ion-dependent dimerization in the crystal and in solution, suggesting a novel mechanism for the regulation of SH3 domain functions.

Intracellular protein interaction mapping with FRET hybrids

“…Importantly, although the P2 peptide possesses an exceptionally high affinity for Mona (22), most SH3 domain ligands exhibit K D values that range from 100 nM to 200 M (31), in agreement with the K D values reported in Table 1. Because K D values determined by using FRET in cell lysates were lower (i.e., higher apparent affinity) than those measured by using either isothermal titration calorimetry or fluorescence polarization, we also determined the K D for selected FRET hybrids after purification from cell lysates.…”

“…3), enabling calculation of an apparent dissociation constant (Table 1), despite varying maximum FRET signals. With this ranking method, the K D of the Mona-P2 interaction was determined to be Ϸ10 nM, Ϸ10-to 20-fold lower than that determined by using isothermal titration calorimetry (22). Similarly, a known PDZ binding peptide, derived from the C terminus of the NMDA receptor NR2 subunit (29,30), exhibited a K D 3-fold lower than that obtained by using fluorescence polarization (30) ( Table 1).…”

“…To validate this approach, a model interaction pair was chosen consisting of the C-terminal SH3 domain from monocytic adaptor protein (21) (hereafter referred to as Mona) and a high-affinity peptide ligand known as P2 (K D ϭ 100-200 nM) (22). The structure of the Mona-P2 complex has been determined by crystallography to 1.7-Å resolution, and the ligand preferences of Mona have been characterized extensively (22)(23)(24). Mona was genetically fused to the C terminus of YPet (YPet-Mona), and P2 was similarly tethered to CyPet (CyPet-P2) to create fluorescent fusion proteins capable of FRET (FRET hybrids) upon complex formation.…”

“…FRET-Based Screening for Ligands Binding to Protein-Interaction Domains. We then investigated whether FRET hybrids would enable identification of optimal peptide ligands for two types of widely occurring protein-interaction modules: the C-terminal SH3 domain from Mona (22) and the second PDZ domain from postsynaptic density protein PSD-95 (28) (PDZ2). Combinatorial 15-mer peptide libraries, composed of 2 ϫ 10 6 members, were constructed as C-terminal fusions to CyPet (CyPetpeptide) in bicistronic expression vectors that contained a gene encoding the target-domain fused to YPet.…”

The LAT Story: A Tale of Cooperativity, Coordination, and Choreography

“…The proline-rich domain of SLP-76 binds SH3 domains of Grb2 and Gads; however SLP-76 binds Gads with a 40 -50-fold stronger affinity than Grb2 (Berry et al 2002;Harkiolaki et al 2003). Gads recruits SLP-76 to LAT by way of the interaction of its SH2 domain with phospho-LAT (Liu et al 1999).…”