PDBsum entry 3hk0

Signaling protein

PDB id: 3hk0

Contents

Protein chains

230 a.a.

216 a.a.

Ligands

SCN

Waters ×14

References listed in PDB file

Key reference

Title

Structural and functional studies of the ras-Associating and pleckstrin-Homology domains of grb10 and grb14.

Authors

R.S.Depetris, J.Wu, S.R.Hubbard.

Ref.

Nat Struct Biol, 2009, 16, 833-839. [DOI no: 10.1038/nsmb.1642]

PubMed id

19648926

Abstract

Growth factor receptor-binding proteins Grb7, Grb10 and Grb14 are adaptor proteins containing a Ras-associating (RA) domain, a pleckstrin-homology (PH) domain, a family-specific BPS (between PH and SH2) region and a C-terminal Src-homology-2 domain. Previous structural studies showed that the Grb14 BPS region binds as a pseudosubstrate inhibitor in the tyrosine kinase domain of the insulin receptor to suppress insulin signaling. Here we report the crystal structure of the RA and PH domains of Grb10 at 2.6-A resolution. The structure reveals that these two domains, along with the intervening linker, form an integrated, dimeric structural unit. Biochemical studies demonstrated that Grb14 binds to activated Ras, which may serve as a timing mechanism for downregulation of insulin signaling. Our results illuminate the membrane-recruitment mechanisms not only of Grb7, Grb10 and Grb14 but also of MIG-10, Rap1-interacting adaptor molecule, lamellipodin and Pico, proteins involved in actin-cytoskeleton rearrangement that share a structurally related RA-PH tandem unit.

Figure 1.
(a) Domain architecture of Grb7, Grb10 and Grb14 drawn to linear scale (human Grb10, isoform c, 536 residues). P, proline-rich region; RA, Ras-associating domain; PH, pleckstrin-homology domain; BPS, between PH and SH2 region; SH2, Src-homology-2 domain. (b) Ribbon diagram of the crystal structure of Grb10 RA-PH. One copy of RA-PH is colored violet (RA) and cyan (PH), and the second copy is colored orange (RA) and green (PH). For both copies, the RA-PH linker is gray. The binding sites for small GTPases on the RA domain and phosphoinositides on the PH domain (noncanonically) are indicated by the position of the labels 'RA' and 'PH'. An approximate twofold axis (vertical, in the plane of the figure) relates the two molecules in the asymmetric unit. Select secondary-structure elements are labeled, as are the N and C termini. Right, the structure has been rotated 90°, as indicated, with the molecular twofold axis perpendicular to the plane of the figure. (c) Stereo view of the dimerization interface. The view is the same as in the right panel of b. Side chains that mediate the interaction between the two RA-PH molecules are shown in stick representation. Hydrogen bonds and salt bridges are represented by black dashed lines, and the side chains of hydrophobic residues are shown with a van der Waals surface. (d) Stereo view of the interface between the RA and PH domains. Pictorial conventions as in c. Figures 1,3c,d and 6 were rendered with PyMol (http://pymol.sourceforge.net).

Figure 6.
The two insulin receptor kinase (IRK) domains are shown in surface representation (opaque), colored dark (N lobe) and light (C lobe) gray. The juxtamembrane regions linking the transmembrane helices to the kinase domains are colored black. Grb14 is shown in ribbon representation with a semitransparent surface. The RA domain is colored violet, the PH domain cyan, the BPS region orange and the SH2 domain green. The interdomain linker regions are gray. Two Ras molecules (blue) are shown in ribbon representation with semitransparent surfaces. The bound nucleotide (GMPPNP) is shown in sphere representation and colored black.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Struct Biol (2009, 16, 833-839) copyright 2009.