Fig. 3

Impact of the R392W substitution and other SH2B3 abnormalities. a 104 amino acid sequence comprising 92% of the SH2 domain of human SH2B3 aligned to the mouse SH2B1 sequence. Black arrows indicate three arginine residues required to coordinate pTyr813 of JAK2 within the mouse SH2B1 SH2 binding pocket. Red arrow shows the position of the R392W arginine to tryptophan substitution identified in patient 25. b Ribbon diagram based on the published co-crystalized structure of the mouse SH2B1 SH2 domain and an eleven residue phosphopeptide surrounding tyrosine pTyr813 within the activation loop of JAK2 [40]. The structure has a canonical SH2 domain architecture with a central β-sheet (magenta) flanked by two α-helices (cyan) that form two binding pockets for the JAK2 phosphopeptide (shown as a stick representation with carbon atoms in green, oxygen atoms in red, nitrogen atoms in dark blue and phosphate atoms in orange). c A similar canonical SH2 domain structure is seen in a homology model of 104 residues of the SH2 domain of human SH2B3 based on the mouse SH2B1 crystal structure. Overall sequence identity between the two SH2 domains was 71%, with a structural homology of 1 Å RMSD when superposed. d Detailed view of superposed models of the phosphotyrosine binding pocket of SH2 domains of mouse SH2B1 (grey ribbons) and human SH2B3 (magenta ribbons) demonstrating close structural homology for this region. The JAK2 phosphopeptide and three arginine residues (R534, R555 and R560) that coordinate pTyr813 within the binding pocket through salt bridges (broken black lines) are indicated by stick representations, with carbon atoms of arginine in grey and other atoms coloured as in 3B. The R392W substitution in human SH2B3 is also shown as a stick representation with carbon atoms coloured in magenta. Tryptophan in this position is unable to form salt bridges with pTyr813, the R392W substitution therefore results in loss of two of four critical interactions with phosphotyrosine and is predicted to destabilise binding of SH2B3 to activated JAK. e Summary of abnormalities of SH2B3 identified in this study. At the top the SH2B3 linear protein structure is represented with major functional domains marked; DIME (dimerization), PH (Pleckstrin homology), SH2 (Src homology 2). For each patient, genomic abnormalities of the SH2B3 coding region, as indicated in the key, are aligned with the domain representation. Numbers on the left are patient ids, genomic position of CN-LOH and deletion are indicated on the right. The codon position and consequence of somatically acquired mutations are as labelled, with arrows marking their positions on the SH2B3 protein. Through combinations of mutation, deletion and CN-LOH, SH2B3 abnormalities were all homozygous and predicted to result in absence of a coding transcript (patients 3, 12, 28, 72 and 78), production of truncated and likely unstable transcripts / proteins (patients 44, 61 and 88) or production of a protein with functionally impaired SH2 domain (patient 25)