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-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.


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. 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.
Baraldi
E
E
European Molecular Biology Laboratory, Meyerhofstrasse 1, Postfach 102209, D-69012 Heidelberg, Germany.
Djinovic Carugo
K
K
Hyvönen
M
M
Surdo
P L
PL
Riley
A M
AM
Potter
B V
BV
O'Brien
R
R
Ladbury
J E
JE
Saraste
M
M
D000077329:Agammaglobulinaemia Tyrosine Kinase; D000361:Agammaglobulinemia / Q000201:enzymology / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002151:Calorimetry; D018360:Crystallography, X-Ray; D019281:Dimerization; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism*; D008563:Membrane Lipids / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014960:X Chromosome / Q000235:genetics
D007295:Inositol Phosphates; D008563:Membrane Lipids; D010716:Phosphatidylinositols; D011993:Recombinant Fusion Proteins; C081655:inositol-1,3,4,5-tetrakisphosphate; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human