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 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...

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Bibliographic Details
Published in:	The Journal of immunology (1950) 2000-04, Vol.164 (8), p.4170-4177
Main Authors:	Mattsson, Pekka T, Lappalainen, Ilkka, Backesjo, Carl-Magnus, Brockmann, Eeva, Lauren, Susanna, Vihinen, Mauno, Smith, C. I. Edvard
Format:	Article
Language:	eng
Subjects:	Agammaglobulinaemia Tyrosine Kinase agammaglobulinemia Agammaglobulinemia - enzymology Agammaglobulinemia - genetics Amino Acid Substitution - genetics Arginine - genetics Btk protein Circular Dichroism Genetic Linkage Glycine - genetics Histidine - genetics Humans Medicin och hälsovetenskap Mutation, Missense Peptide Fragments - genetics Peptide Fragments - metabolism Phosphotyrosine - metabolism Protein Binding - genetics Protein Conformation Protein-Tyrosine Kinases - biosynthesis Protein-Tyrosine Kinases - chemistry Protein-Tyrosine Kinases - genetics Protein-Tyrosine Kinases - metabolism SH2 domain Solubility src Homology Domains - genetics Structure-Activity Relationship X Chromosome - genetics X-linked agammaglobulinemia
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Summary:	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).
ISSN:	0022-1767 1550-6606