Grb10 SH2 Domain

spinqualitylabelsall models Crystal Structure of the SH2 Domain of Grb10 (PDB entry 1NRV) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Introduction Grb10 (Growth factor Receptor-Binding protein) is a member of a family of adapter proteins (Grb7 and Grb14) that interacts with tyrosine kinases. Dimerization of the Grb10 SH2 Domain The crystal structure of Grb10 SH2 (Src Homology) domain (molecular mass = 12.4 kDa) was an important step to understanding how this protein interacts with IGF1 receptors, and although the SH2 domain functions as an independent segment, it forms a dimer in physiological environments. The dimer interface exists due to the middle hydrophobic Phe515 and uncharged Thr504 (labeled blue) residues packed into its equivalent counter parter on the other protomer, designated as Phe515' and Thr504' (labeled red); Gln511 (blue) forms a hydrogen bond to the backbone of Asp514' (red) while the side chain of Asn519 forms two hydrogen bonds to the backbone of Lys505'. The interface ends with Leu518 and Phe-496' via hydrophobic interactions. . The structure of Grb10 SH2 forms similar SH2 domains found in other proteins, which have an alpha helix on the outsides with anti-parallel beta sheets. To ensure the crystallographic structure of Grb10 SH2 is indeed a dimer in solution, Evan Stein and colleagues substituted Phe515 at the dimer interface with arginine (electrically charged side chain) and found, using gel filtration chromatography (Picture 1), that the Grb10 SH2 dimer had indeed become independent monomers. Picture 1 Picture 1 The gel used for filtration chromatography had 5 lanes with 5 different components: BPS-SH2_WT (Wild Type) BPS-SH2_F515R (mutant = Phe515 --> Arg) IRK_3P (tris-phosphorylated Insulin Receptor Kinase domain) BPS-SH2_WT + IRK_3P BPS-SH2_F515R + IRK_3P As seen in lanes 1 and 2, the BPS-SH2 proteins did not travel down the gel due to their high pI; to resolve this issue, the researchers added IRK_3P to the two BPS-SH2 proteins which then made a complex that was mobile. Lane 4 shows a band labeled 2:2 complex that shows the position of the SH2 dimer. The additional band found at the very top of lane 4 represents the BPS-SH2_WT protein that did not complex with high motility protein IRK_3P, i.e. it was not able to migrate through the gel due to its high pI. Lane 5 shows a band labeled 1:1 complex elucidating that the Arg substitution at Phe515 did indeed produce a monomer, which was able to travel farther down the gel. Why BPS-SH2? In order for the full-length Grb10 protein to interact with Insulin Receptor Kinase (IRK), the SH2 and BPS domain must be present.

Interaction Between Grb10 and E3 Ubiquitin Ligase NEDD4

spinqualitylabelsall models Crystal structure of the Nedd4 C2/Grb10 SH2 complex PDB entry 3M7F) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D

Grb10 has now been shown to not only inhibit insulin receptors and IGF1R kinase activity, but also interacts via its SH2 domain with the C2 domain of E3 ubiquitin ligase NEDD4 facilitating ubiquitation of IGF1R . It is hypothesized that the Grb10 SH2 interaction with the E3 domain of NEDD4 may allow NEDD4 to come in close proximity to IGF1R promoting degradation.

There are 3 interfaces at which Nedd4 C2 and Grb10 SH2 interact:

Interface I is the largest of the 3 interfaces between Grb10 SH2 and NEDD4 C2 domains. There are 8 hydrogen bonds formed at this interface, with the least important H bonds formed between ASN519 and LEU177, for disruption between these two residues with an induced mutation did not inhibit Grb10 SH2 and NEDD4 C2 to complex.

Interface II, the smallest of the 3, is composed of residues 429-434 of Grb10 SH2 interacting with residues 112-114 of NEDD4 C2; disruption of this interaction does not disrupt the SH2-C2 domain complex.

Interface III is made of NEDD4 C2's proline rich C terminus which complexes with Grb10 H2 N-terminal (residues 431-440)

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