PDBsum entry: 2k42

Signaling protein

PDB id: 2k42

Contents

Protein chains

72 a.a. *

36 a.a. *

* Residue conservation analysis

PDB id:

2k42

Name:

Signaling protein

Title:

Solution structure of the gtpase binding domain of wasp in complex with espfu, an ehec effector

Structure:

Wiskott-aldrich syndrome protein. Chain: a. Fragment: crib domain, unp residues 242-310. Synonym: wasp. Engineered: yes. Espfu. Chain: b. Fragment: unp residues 268-300. Synonym: tir-cytoskeleton coupling protein.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: was, imd2. Expressed in: escherichia coli. Escherichia coli o157:h7. Organism_taxid: 83334. Gene: tccp, ecs2715.

NMR struc:

20 models

Authors:

H.-C.Cheng,B.M.Skehan,K.G.Campellone,J.M.Leong,M.K.Rosen

Key ref:

H.C.Cheng et al. (2008). Structural mechanism of WASP activation by the enterohaemorrhagic E. coli effector EspF(U). Nature, 454, 1009-1013. PubMed id: 18650809 DOI: 10.1038/nature07160

Date:

27-May-08 Release date: 22-Jul-08

Protein chain

P42768  (WASP_HUMAN) -  Wiskott-Aldrich syndrome protein

Seq: 502 a.a.

Struc: 72 a.a.*

Protein chain

P0DJ88  (ESFU2_ECO57) -  Secreted effector protein EspF(U)

Seq: 337 a.a.

Struc: 36 a.a.*

* PDB and UniProt seqs differ at 5 residue positions (black crosses)

 Gene Ontology (GO) functional annotation 

• Cellular component: actin cytoskeleton (1 term)
• Biological process: protein complex assembly (2 terms)
• Biochemical function: protein binding (2 terms)

DOI no: 10.1038/nature07160

Nature 454:1009-1013 (2008)

PubMed id: 18650809

Structural mechanism of WASP activation by the enterohaemorrhagic E. coli effector EspF(U).

H.C.Cheng, B.M.Skehan, K.G.Campellone, J.M.Leong, M.K.Rosen.

ABSTRACT

During infection, enterohaemorrhagic Escherichia coli (EHEC) takes over the actin cytoskeleton of eukaryotic cells by injecting the EspF(U) protein into the host cytoplasm. EspF(U) controls actin by activating members of the Wiskott-Aldrich syndrome protein (WASP) family. Here we show that EspF(U) binds to the autoinhibitory GTPase binding domain (GBD) in WASP proteins and displaces it from the activity-bearing VCA domain (for verprolin homology, central hydrophobic and acidic regions). This interaction potently activates WASP and neural (N)-WASP in vitro and induces localized actin assembly in cells. In the solution structure of the GBD-EspF(U) complex, EspF(U) forms an amphipathic helix that binds the GBD, mimicking interactions of the VCA domain in autoinhibited WASP. Thus, EspF(U) activates WASP by competing directly for the VCA binding site on the GBD. This mechanism is distinct from that used by the eukaryotic activators Cdc42 and SH2 domains, which globally destabilize the GBD fold to release the VCA. Such diversity of mechanism in WASP proteins is distinct from other multimodular systems, and may result from the intrinsically unstructured nature of the isolated GBD and VCA elements. The structural incompatibility of the GBD complexes with EspF(U) and Cdc42/SH2, plus high-affinity EspF(U) binding, enable EHEC to hijack the eukaryotic cytoskeletal machinery effectively.

Selected figure(s)

Figure 1.
Figure 1: A single repeat of EspF[U] activates WASP/N-WASP with high potency. a, Sequence alignment of WASP and N-WASP VCA C regions and the fifth repeat element of EspF[U]. Helix residues in the autoinhibited GBD–C structure and GBD–R33 complex are blue; residues in the extended EspF[U] arm are red; aligned hydrophobic residues are boxed in yellow. EspF[U] residues that contact the GBD are indicated by black circles. Asterisks indicate sites of EspF[U] mutations used in b and d. The proline-rich motif is boxed in grey. The C termini of EspF[U] single repeat constructs used throughout this work are indicated below the sequence. b, Pyrene-actin fluorescence measured during assembly of 4 M actin (5% pyrene-labelled) plus 10 nM Arp2/3 complex (black) and 25 nM N-WASP[C] (blue) and 500 nM of: R47 (green), R33 (red), R18 (orange), R14 (cyan), R33**^L (pink) or R33*** (V4A/L8A/L12A, grey). c, Concentration of filament barbed ends produced during assembly of 4 M actin by 10 nM Arp2/3 complex, 25 nM N-WASP[C] and increasing concentrations of R47 (black squares; red curve shows fit to single-site binding isotherm) or 500 nM Cdc42–GMPPNP (green triangle). The blue circle shows barbed ends produced by actin plus Arp2/3 plus 25 nM N-WASP VCA. d, For the EspF[U] proteins listed, the table shows the free energy of unfolding of GBD–EspF[U] fusion (G[u]), the dissociation constant for binding to N-WASP[C] and actin filament barbed ends (B.E.) produced by assays in b. In the 'none' row, G[u] represents melting of the isolated GBD and B.E. represents assays performed with only N-WASP[C] and Arp2/3 complex.

Figure 2.
Figure 2: Structures of the WASP GBD in complex with different ligands. a, b, The complex of WASP GBD and EspF[U] R33 is shown. Layer 1 (WASP 250–276) is shown in yellow, layer 2 (WASP 277–310) in blue and layer 3 (EspF[U] 2–20) in green. In b, GBD is shown as a surface representation; R33 is shown as a ribbon with sticks for side chains that contact the GBD. Views are related by a 180° rotation about a vertical axis. c, d, Autoinhibited WASP (GBD–C^7). GBD is coloured as in a; the C region of WASP VCA is coloured red. Representation and views are as in a and b.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2008, 454, 1009-1013) copyright 2008.

Figures were selected by the author.

Author's comment

See also Padrick et al., Mol Cell. 2008 Nov 7;32(3):426-38 for the distinct mechanism by which multiple EspFu repeats provide additional activation of WASP/N-WASP.