Structure of the PH domain from Bruton's tyrosine kinase in complex with inositol 1,3,4,5-tetrakisphosphate

Background: The activity of Bruton's tyrosine kinase (Btk) is important for the maturation of B cells. A variety of point mutations in this enzyme result in a severe human immunodeficiency known as X-linked agammaglobulinemia (XLA). Btk contains a pleckstrin-homology (PH) domain that specifically binds phosphatidylinositol 3,4,5-trisphosphate and, hence, responds to signalling via phosphatidylinositol 3-kinase. Point mutations in the PH domain might abolish membrane binding, preventing signalling via Btk. Results: We have determined the crystal structures of the wild-type PH domain and a gain-of-function mutant E41K in complex with D-myo-inositol 1,3,4,5- tetrakisphosphate (Ins (1,3,4,5)P₄). The inositol Ins (1,3,4,5)P₄ binds to a site that is similar to the inositol 1,4,5-trisphosphate binding site in the PH domain of phospholipase C-δ. A second Ins (1,3,4,5)P₄ molecule is associated with the domain of the E41K mutant, suggesting a mechanism for its constitutive interaction with membrane. The affinities of Ins (1,3,4,5)P₄ to the wild type (K(d) = 40 nM), and several XLA-causing mutants have been measured using isothermal titration calorimetry. Conclusions: Our data provide an explanation for the specificity and high affinity of the interaction with phosphatidylinositol 3,4,5-trisphosphate and lead to a classification of the XLA mutations that reside in the Btk PH domain. Missense mutations that do not simply destabilize the PH fold either directly affect the interaction with the phosphates of the lipid head group or change electrostatic properties of the lipid-binding site. One point mutation (Q127H) cannot be explained by these facts, suggesting that the PH domain of Btk carries an additional function such as interaction with a Gα protein.