Six X-linked agammaglobulinemia-causing missense mutations in the src homology 2 domain of Bruton's tyrosine kinase: Phosphotyrosine-binding and circular dichroism analysis

Src homology 2 (SH2) domains recognize phosphotyrosine (pY)-containing sequ ences and thereby mediate their association to ligands. Bruton's tyrosine k inase (Btk) is a cytoplasmic protein tyrosine kinase, in which mutations ca use a hereditary immunodeficiency disease, X-linked agammaglobulinemia (XLA ). Mutations have been found in all Btk domains, including SH2, We have ana lyzed the structural and functional effects of six disease-related amino ac id substitutions in the SH2 domain: G302E, R307G, Y334S, L358F, Y361C, and H362Q, Also, we present a novel Btk SH2 missense mutation, H362R, leading t o classical XLA, Based on circular dichroism analysis, the conformation of five of the XLA mutants studied differs from the native Btk SH2 domain, whi le mutant R307G is structurally identical, The binding of XLA mutation-cont aining SH2 domains to pY-Sepharose was reduced, varying between 1 and 13% o f that for the native SH2 domain. The solubility of all the mutated protein s was remarkably reduced. SH2 domain mutations were divided into three cate gories: 1) Functional mutations, which affect residues presumably participa ting directly in pY binding (R307G); 2) structural mutations that, via conf ormational change, not only impair pY binding, but severely derange the str ucture of the SH2 domain and possibly interfere with the overall conformati on of the Btk molecule (G302E, Y334S, L358F, and H362Q); and 3) structural- functional mutations, which contain features from both categories above (Y3 61C).