Figure 4.

Determination of the cross-linking site on BTK protein and the proposed ligand-directed cross-linking mechanism. (a) Sequence of the full-length His₆-(TEV)-G-hBTK_387–659. The MS/MS spectrum for probe 1a-modified tripeptide fragment, EMR, is shown with the fragment ions annotated on the structure. (b) A docking model of probe 1a bound to BTK (PDB code: 3K54) showing a proximal Glu-488 residue located on a loop 6.9 Å away from the C⁵ of the tetrazole ring. (c) Proposed mechanism of the ligand-dependent nucleophilic addition to the carboxy-nitrile imine followed by the O → N acyl shift to generate the specific photoadduct.