Fig. 5.

Fourth-order substrate detection by Btk. (A) Using the kinetic rate constants derived from the sequential kinetic model, the dimer fraction was calculated for the hypothetical case in which the PH-TH module may dimerize with only one PIP₃ (second order) and for the actual case where two are required (fourth order). (B) An alternative method to achieve fourth-order PIP₃ detection, single lipid binding followed by tetramerization to activation (Lower), was considered and compared with the observed mechanism (Upper). (C) The proposed activation mechanism for Btk. (D) Calcium flux for Btk-deficient chicken B cells transfected with variants of Btk and activated with BCR antibody (Left) and relative integrated calcium response for WT, gain-of-function (E41K), kinase dead (D521N), dimer interface mutant, and peripheral site mutant (Right).