Abstract
The Src-like adaptor proteins (SLAP/SLAP2) bind to CBL E3 ubiquitin ligase to downregulate antigen, cytokine and tyrosine kinase receptor signalling. In contrast to the phosphotyrosine-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 was crystallized. However, determination of the complex crystal structure was hindered by the apparent degradation of SLAP2 during the crystallization process, such that only the CBL TKBD residues could initially be modelled. 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 into this unassigned density, a co-expression system was generated to test SLAP2 deletion mutants and define the minimal SLAP2 binding region. SLAP2 degradation products were also analysed by mass spectrometry. The model-building and map-generation features of the Phenix software package were employed, leading to successful modelling of the C-terminal tail of SLAP2 into the unassigned electron-density segments.