Six X-Linked Agammaglobulinemia-Causing Missense Mutations in the Src Homology 2 Domain of Bruton’s Tyrosine Kinase: Phosphotyrosine-Binding and Circular Dichroism Analysis

Abstract

Src homology 2 (SH2) domains recognize phosphotyrosine (pY)-containing sequences and thereby mediate their association to ligands. Bruton’s tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase, in which mutations cause a hereditary immunodeficiency disease, X-linked agammaglobulinemia (XLA). Mutations have been found in all Btk domains, including SH2. We have analyzed the structural and functional effects of six disease-related amino acid substitutions in the SH2 domain: G302E, R307G, Y334S, L358F, Y361C, and H362Q. Also, we present a novel Btk SH2 missense mutation, H362R, leading to classical XLA. Based on circular dichroism analysis, the conformation of five of the XLA mutants studied differs from the native Btk SH2 domain, while mutant R307G is structurally identical. The binding of XLA mutation-containing SH2 domains to pY-Sepharose was reduced, varying between 1 and 13% of that for the native SH2 domain. The solubility of all the mutated proteins was remarkably reduced. SH2 domain mutations were divided into three categories: 1) Functional mutations, which affect residues presumably participating directly in pY binding (R307G); 2) structural mutations that, via conformational change, not only impair pY binding, but severely derange the structure of the SH2 domain and possibly interfere with the overall conformation of the Btk molecule (G302E, Y334S, L358F, and H362Q); and 3) structural-functional mutations, which contain features from both categories above (Y361C).

Functional Interaction of Caveolin-1 with Bruton's Tyrosine Kinase and Bmx

“…Plasmid pcDNA3.1/GS encoding human caveolin-1 cDNA was obtained from Invitrogen. The cDNA of native Btk as well as that of the E41K mutant were cloned into the expression vector pSGT using NotI and XhoI restriction sites, as described previously (39). The recombinant proteins for Btk (wild type) and its truncated form Btk ⌬SH1, lacking the catalytic domain, were purified from Sf 9 and Escherichia coli cells, respectively, as previously described (39).…”

BTKbase: the mutation database for X-linked agammaglobulinemia

“…We have predicted the consequences of all the mutations on protein structure and function based on sequence and structure information [Holinski-Feder et al, 1998;Jin et al, 1995;Korpi et al, 2000;Maniar et al, 1995;Mattsson et al, 2000;Okoh and Vihinen, 1999;Saha et al, 1997;Shen and Vihinen, 2004;Speletas et al, 2001;Vihinen et al, 1995aVihinen et al, ,b, 1994aVoøchovský et al, 1995;Zhu et al, 1994]. In Figure 6, all the disease causing missense mutations are indicated within PH, SH2, and the kinase domains.…”

Phylogeny of Tec Family Kinases: Identification of a Premetazoan Origin of Btk, Bmx, Itk, Tec, Txk, and the Btk Regulator SH3BP5

“…BTKbase has constantly grown from 188 cases to the current number of 1096 patients (Lindvall et al, 2005;Väliaho et al, 2006;Vihinen et al, 1995aVihinen et al, ,b, 1996Vihinen et al, , 1997bVihinen et al, , 1998Vihinen et al, , 1999Vihinen et al, , 2001). Experimental and modeled structures for BTK domains have extensively been used to explain protein structure-function relationships and consequences of mutations (Holinski-Feder et al, 1998;Jin et al, 1995;Korpi et al, 2000;Lindvall et al, 2005;Maniar et al, 1995;Mao et al, 2001;Mattsson et al, 2000;Speletas et al, 2001;Väliaho et al, 2006;Vihinen et al, 1994bVihinen et al, ,c, 1995bVorechovsky et al, 1995Vorechovsky et al, , 1997Zhu et al, 1994). These studies have allowed us to provide putative functional and/or structural explanation for all XLA-causing mutations.…”