CRYSTAL STRUCTURE OF THE BRUTON'S TYROSINE KINASE PH DOMAIN IN COMPLEX WITH Ins(1,3,4,5)P4: EXPLANATION OF THE BTK MUTATIONS IN XLA IMMUNODEFICIENCY
Elena Baraldi1, Marko Hyvoenen2, Matti Saraste1 and Kristina Djinovic2
1EMBL, Meyerhofsrasse 1-69117
Heidelberg (Germany)
2Department of Biochemistry , University of
Cambridge 80 Tennis Court Road CB2 1QW Cambridge (UK)
Keywords: E41K, PH domain, XLA mutations,
Ins(1,3,4,5)P4 binding.
X-linked immunodeficiency (XLA) is a hereditary immune disease which severely affects maturation and differentiation of B-cells. The cause of XLA are mutations in the gene coding for the Bruton's tyrosine kinase Btk. Btk is a member of Tec family tyrosine kinases. The structural composition of Btk and Tec kinases consists of Src Homology 3 (SH3), SH2 and catalytic domain SH1, like Src tyrosine kinases. Additionally the Tec kinases contain a Pleckstrin homology (PH) and Tec Homology (TH) domain at their N-terminus. These two domains occupy positions corresponding to the myristolation site in Src, responsible for Src membrane targeting.
The TH domain includes a Zn binding motif (the Btk motif) and a proline rich region. The Btk motif is only 26 amino acids long and is found always adjacent to PH domain in Tec kinases and in few other proteins. PH domains are 110 residues long signalling domains, found in many unrelated proteins. They associate by means of electrostatic interactions with negatively charged phosphatydil inositol (PtIns) phospholipids and serve to anchor proteins to membrane compartments. The spectrin PH domain and PLC-d PH domain structures have been solved in complex with the soluble head group of PtIns(4,5)P2, Ins(1,4,5,)P3. These structures point out the divergence in the binding sites of PH domains. Although the InsP3 binding site is located on the positively polarized surface in both cases, it involves different loops and different residues of the two domains.
The structure of Btk PH domain and its Btk motif has been solved recently in our laboratory (Hyvonen and Saraste, 1997). The Btk motif is essential for the PH domain structural stability. The PH domain shows structural features similar to other known PH domain. It is a seven stranded b-sheet with an C-terminal a-helix packing against it. Like other PH domains it is strongly electrostatically polarized. The Btk motif is a new fold of zinc binding motif. The Zn is coordinated by four conserved residues, three cysteins and a histidine. Unlike the majority of PH domains known to bind PtIns (4,5)P2, the Btk PH domain binds to PtIns (3,4,5)P3 or to the soluble counterpart Ins(1,3,4,5)P4.
Several mutations found in XLA patients map into the PH domain
of Btk. Based on the 3-dimensional structure of PH domain, these
mutations have been grouped in folding mutations, which
probably affect the structural stability of the PH domain, and in
functional mutations, which should affect its
PtIns(3,4,5)P3 binding function
(Hyvonen and Saraste, 1997). Contrary to these, the E41K mutation
of Btk PH was isolated as gain of function mutation (Li et
al., 1995). The Btk E41K protein appears constitutively
associated to membrane, hence constitutively active and able to
transform NIH3T3 cells (Li et al., 1997). We solved the crystal
structure of the E41K mutant of Btk PH domain in complex with
Ins(1,3,4,5)P4 ligand at 2.1 A resolution. The
Ins(1,3,4,5)P4 binding site
occupies a position similar to the InsP3
binding site of PLCd and involves many residues mutated in XLA
patient. Our results allow to explain at the atomic level the Btk
mutations provocking XLA.