Baraldi E, Djinovic Carugo K, Hyvönen M, Surdo P L, Riley A M, Potter B V, O'Brien R, Ladbury J E, Saraste M
European Molecular Biology Laboratory, Meyerhofstrasse 1, Postfach 102209, D-69012 Heidelberg, Germany.
Structure. 1999 Apr 15;7(4):449-60. doi: 10.1016/s0969-2126(99)80057-4.
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.
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-tetra-kisphosphate (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-delta. 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 (Kd = 40 nM), and several XLA-causing mutants have been measured using isothermal titration calorimetry.
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. Mis-sense 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 Galpha protein.
布鲁顿酪氨酸激酶（Btk）的活性对B细胞的成熟至关重要。该酶的多种点突变会导致一种严重的人类免疫缺陷，即X连锁无丙种球蛋白血症（XLA）。Btk含有一个普列克底物蛋白同源（PH）结构域，该结构域特异性结合磷脂酰肌醇3,4,5-三磷酸，因此可响应通过磷脂酰肌醇3-激酶的信号传导。PH结构域中的点突变可能会消除膜结合，从而阻止通过Btk的信号传导。
我们确定了野生型PH结构域和功能获得性突变体E41K与D-肌醇1,3,4,5-四磷酸（Ins(1,3,4,5)P4）形成复合物的晶体结构。肌醇Ins(1,3,4,5)P4结合到一个与磷脂酶C-δ的PH结构域中的肌醇1,4,5-三磷酸结合位点相似的位点。第二个Ins(1,3,4,5)P4分子与E41K突变体的结构域相关联，这表明了其与膜组成型相互作用的机制。已使用等温滴定量热法测量了Ins(1,3,4,5)P4与野生型（Kd = 40 nM）以及几种导致XLA的突变体的亲和力。
我们的数据为与磷脂酰肌醇3,4,5-三磷酸相互作用的特异性和高亲和力提供了解释，并导致了对位于Btk PH结构域中的XLA突变的分类。并非简单地破坏PH折叠的错义突变要么直接影响与脂质头部基团磷酸的相互作用，要么改变脂质结合位点的静电性质。一个点突变（Q127H）无法用这些事实来解释，这表明Btk的PH结构域具有额外的功能，例如与Gα蛋白相互作用。
