Both proline-rich sequences in the TH region of Bruton's tyrosine kinase stabilize intermolecular interactions with the SH3 domain.

The Tec homology (TH) region located N-terminal to the Src homology 3 (SH3) domain of Bruton's tyrosine kinase (Btk) contains two proline-rich SH3-binding sequences (PRRs). We have previously demonstrated that the TH region acts to stabilize intermolecular interactions in N-terminally extended SH3 (PRR-SH3) fragments. Here, we analyze six PRR-SH3 fragments with different proline-to-alanine substitutions in the two PRRs. Gel permeation chromatography and nuclear magnetic resonance spectroscopy show that both PRRs can stabilize self-association. This observation provides an explanation to why the TH region of Btk makes intermolecular interactions, whereas the corresponding interaction in the related Itk kinase with only one PRR, is intramolecular.