Sleuthing biochemical evidence to elucidate unassigned electron density in a CBL/SLAP2 crystal complex

Abstract

The Src-like adaptor proteins (SLAP/SLAP2) bind to CBL E3 ubiquitin ligase to downregulate antigen, cytokine, and tyrosine kinase receptor signaling. In contrast to phospho-tyrosine dependent binding of CBL substrates through its tyrosine kinase binding domain (TKBD), CBL TKBD associates with the C-terminal tail of SLAP2 in a phospho-independent manner. To understand the distinct nature of this interaction, a purification protocol for SLAP2 in complex with CBL TKBD was established and the complex crystallized. However, determination of the complex crystal structure was hindered by apparent SLAP2 degradation during the crystallization process, such that only CBL TKBD residues could be modeled initially. Close examination of the CBL TKBD structure revealed a unique dimer interface that included two short segments of electron density of unknown origin. To elucidate which residues of SLAP2 to model in this unassigned density, a co-expression system was generated to test SLAP2 deletion mutants and define the minimal SLAP2 binding region. As well, SLAP2 degradation products were analyzed by mass spectrometry. Model building and map generation features of the Phenix software package were employed, leading to successful modeling of the C-terminal tail of SLAP2 in the unassigned electron density segments.