PDBsum entry 2otx

Signaling protein

PDB id: 2otx

Contents

Protein chains

170 a.a. *

Waters ×23

* Residue conservation analysis

PDB id:

2otx

Name:

Signaling protein

Title:

Crystal structure of a n-terminal fragment of skap-hom containing both the helical dimerization domain and the ph domain

Structure:

Src kinase-associated phosphoprotein 2. Chain: a, b. Fragment: n-terminal fragment (residues 12-222). Synonym: src family-associated phosphoprotein 2, src kinase- associated phosphoprotein 55-related protein, skap55 homolog, skap- hom, src-associated adapter protein with ph and sh3 domains, pyk2/raftk-associated protein. Engineered: yes. Mutation: yes

Source:

Mus musculus. House mouse. Organism_taxid: 10090. Gene: skap2, prap, ra70, saps, scap2, skap55r. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

2.60Å R-factor: 0.181 R-free: 0.216

Authors:

Y.Tang,K.D.Swanson,B.G.Neel,M.J.Eck

Key ref:

K.D.Swanson et al. (2008). The Skap-hom dimerization and PH domains comprise a 3'-phosphoinositide-gated molecular switch. Mol Cell, 32, 564-575. PubMed id: 19026786 DOI: 10.1016/j.molcel.2008.09.022

Date:

09-Feb-07 Release date: 27-Mar-07

Protein chains

Q3UND0  (SKAP2_MOUSE) -  Src kinase-associated phosphoprotein 2 from Mus musculus

Seq: 358 a.a.

Struc: 170 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1016/j.molcel.2008.09.022

Mol Cell 32:564-575 (2008)

PubMed id: 19026786

The Skap-hom dimerization and PH domains comprise a 3'-phosphoinositide-gated molecular switch.

K.D.Swanson, Y.Tang, D.F.Ceccarelli, F.Poy, J.P.Sliwa, B.G.Neel, M.J.Eck.

ABSTRACT

PH domains, by binding to phosphoinositides, often serve as membrane-targeting modules. Using crystallographic, biochemical, and cell biological approaches, we have uncovered a mechanism that the integrin-signaling adaptor Skap-hom uses to mediate cytoskeletal interactions. Skap-hom is a homodimer containing an N-terminal four-helix bundle dimerization domain, against which its two PH domains pack in a conformation incompatible with phosphoinositide binding. The isolated PH domains bind PI[3,4,5]P(3), and mutations targeting the dimerization domain or the PH domain's PI[3,4,5]P(3)-binding pocket prevent Skap-hom localization to ruffles. Targeting is retained when the PH domain is deleted or by combined mutation of the PI[3,4,5]P(3)-binding pocket and the PH/dimerization domain interface. Thus, the dimerization and PH domain form a PI[3,4,5]P(3)-responsive molecular switch that controls Skap-hom function.

Selected figure(s)

Figure 2.
Figure 2. Interface between the Skap-hom PH and DM Domains
(A) The PH domain is shown in red and the DM domain in blue and green as in Figure 1C. Salt bridges are indicated by dashed lines. Note the helical conformation of the β1-β2 loop (residues Arg127–Phe135) and the insertion of PH domain residues Phe132 and Leu133 into a hydrophobic pocket on the DM domain.
(B) Stereo representation of the DM domain showing the side chains of all residues, with salt bridges highlighted. Representative charged residues are labeled.
(C) View of one subunit of the DM domain showing the hydrophobic and charged residues in the dimerization interface.
(D) Superposition of the Skap-hom DM (green and blue) and the dimerization domain of nuclear export protein Nep/Ns2 of influenza A (magenta) (Akarsu et al., 2003), which shares a similar overall topology but no clear sequence relationship.

Figure 6.
Figure 6. Localization of GFP-Tagged Skap-hom Variants in Skap-hom^−/− BMMs
Skap-hom^−/− BMMs were reconstituted with (A) GFP-tagged WT Skap-hom, (B) GFP alone, or (C–G) the indicated GFP-tagged variants of Skap-hom. (H–N) Cells were fixed and counterstained with rhodamine-labeled phalloidin to visualize the actin cytoskeleton. (O–U) Merged GFP and Rhodamine channels for each infection. WT Skap-hom (A, H, and O) colocalizes with actin-rich ruffles. The DM^* dimerization domain triple mutant (C, J, and Q) and the R140M PH domain mutant (D, K, and R) are diffusely localized. Deletion of the PH domain (ΔPH; [E], [L], and [S]) results in a protein that localizes to actin ruffles. Skap-hom bearing both the R140M and either the D129K (D129K:R140M, [F], [M], and [T]) or the K56A (K56A:R140M, [G], [N], and [U]) mutations colocalize with actin ruffles.

The above figures are reprinted from an Open Access publication published by Cell Press: Mol Cell (2008, 32, 564-575) copyright 2008.

Figures were selected by an automated process.