Abstract LB-103: Structural characterization of SPRY2-Cbl interactions

Abstract

Abstract Sprouty 2 (SPRY2) is a feedback modulator of receptor tyrosine kinase (RTK)-ERK signaling, and a potential tumor suppressor. Cbl, an E3 ubiquitin ligase and scaffold protein, binds SPRY2 with high affinity, and sequesters or targets it for degradation. Cbl also binds several growth factor receptors. Hence SPRY2, Cbl and receptors are in a dynamic equilibrium. Truncation and site-mutagenesis studies previously mapped SPRY2-Cbl interactions to two discrete domains, TKB and RING. A pY binding pocket in the TKB SH2 region is critical for binding, but the role(s) of other TKB regions and/or RING are undefined. Interactions of three Cbl recombinant proteins (P47-G351 (TKBD), and two phosphomimetic RING active-conformation constructs, P47-D435 Y371D/E) with three SPRY2 peptides (P1: Q36-N53, a putative RING binder; P2: Q36-T60 (pY55), TKB binder; and P3: 54-60(pY55), a truncated TKB binder) were characterized by computational simulation, surface plasmon resonance spectroscopy (SPR), fluorescence polarization (FP), and X-ray crystallography. In silico modeling suggested possible low-affinity binding of P1 in the groove between TKBD SH2 and 4-helix (4H) regions and near the RING, but this was not confirmed by FP, SPR, or crystallography. Apo and P2 ligand-bound structures agreed with prior published data; three previously unknown 4H interaction sites were also identified. Data on the effects of site-specific 4H mutants on P2 binding affinities are pending. Binding affinities for P2 with the TKBD, Y371E, and Y371D constructs were 160±5, 92±9, and 45±3 nM, respectively, by SPR; FP results were similar. Direct interactions with the RING were not confirmed, but the higher P2 binding affinities observed with RING constructs suggest that this domain indirectly facilitates SPRY2 binding. Binding of P2 induced 16.9° rotation of the SH2 region and formation of new H-bonds to 4H, compressing the TKBD. Importantly, P4 induced identical conformational changes as P2, but had no direct contact with 4H. These observations are consistent with a refined model of SPRY2-Cbl interaction, where ligand docking at the pY SH2 site is sufficient to globally change TKB architecture, facilitating SPRY2 binding at previously unrecognized sites in the 4H region (Support: KU COBRE-PSF (NIH/RR017708, NIH/GM103420), KU Cancer Center, Kansas Bioscience Authority, and the George & Floriene Lieberman Endowment). Citation Format: Na Zhang, Xuan Zhang, Laurie Washington, Asokan Anbanandam, Kevin Battaile, Philip Gao, Aaron Smalter Hall, Scott Lovell, Anuradha Roy, Robert Hanzlik, Raymond Perez. Structural characterization of SPRY2-Cbl interactions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-103. doi:10.1158/1538-7445.AM2015-LB-103