PDBsum entry 3hw2

Signaling protein

PDB id: 3hw2

Contents

Protein chains

308 a.a.

105 a.a.

References listed in PDB file

Key reference

Title

Interaction between the sifa virulence factor and its host target skip is essential for salmonella pathogenesis.

Authors

L.Diacovich, A.Dumont, D.Lafitte, E.Soprano, A.A.Guilhon, C.Bignon, J.P.Gorvel, Y.Bourne, S.Méresse.

Ref.

J Biol Chem, 2009, 284, 33151-33160. [DOI no: 10.1074/jbc.M109.034975]

PubMed id

19801640

Abstract

SifA is a Salmonella effector that is translocated into infected cells by the pathogenicity island 2-encoded type 3 secretion system. SifA is a critical virulence factor. Previous studies demonstrated that, upon translocation, SifA binds the pleckstrin homology motif of the eukaryotic host protein SKIP. In turn, the SifA-SKIP complex regulates the mobilization of the molecular motor kinesin-1 on the bacterial vacuole. SifA exhibits multiple domains containing functional motifs. Here we performed a molecular dissection and a mutational study of SifA to evaluate the relative contribution of the different domains to SifA functions. Biochemical and crystallographic analysis confirmed that the N-terminal domain of SifA is sufficient to interact with the pleckstrin homology domain of SKIP, forming a 1:1 complex with a micromolar dissociation constant. Mutation of the tryptophan residue in the WXXXE motif, which has been proposed to mimic active form of GTPase, deeply affected the stability and the translocation of SifA while mutations of the glutamic residue had no functional impact. A SifA L130D mutant that does not bind SKIP showed a DeltasifA-like phenotype both in infected cells and in the mouse model of infection. We concluded that the WXXXE motif is essential for maintaining the tertiary structure of SifA, the functions of which require the interaction with the eukaryotic protein SKIP.

Figure 2.
Overall view of the SifA-SKIP(PH) complex. A, ribbon diagram of the SifA-SKIP(PH) complex, viewed in two orientations rotated by 90°. The SifA N- (residues 21–136) and C-terminal (residues 137–328) are shown in yellow and orange, respectively, and the SKIP(PH) (residues 772–876) is in green. The SifA conserved motifs WE(I/M)XXFF, which is important for translocation (13), and WXXXE, which has been proposed to mimic activated GTPase (16), are highlighted in pink and cyan, respectively. The Leu^130 position buried at the complex interface is displayed in red. B, molecular surface of the complex (left), color-coded, and oriented as in the left view of panel A, with the surface buried at the complex interface shown in green. Close-up view (right) of key residues involved in the binding interface. C, mapping sequence conservation in the SifB homolog onto the molecular surface of SifA (yellow and orange for the N- and C-terminal domain), oriented as in the left view of panel A, with non-conserved side chains from the N- and C-terminal domain shown in pink and magenta, respectively. Small patches of non-conserved surface regions (pink) are located in the N-terminal domain and within the binding interface (green) while large patches of non-conserved surface regions (magenta) are clustered within the C-terminal domain. D, surface electrostatic potential map of SifA, oriented as in the right view of panel A, showing a dominant electronegative potential except for a large patch of electropositive potential clustered near the translocation motif in the N-terminal domain. Electrostatic surface potentials are contoured at −3/+3 kT/e electrostatic units (k, Boltzmann constant; T, temperature in Kelvin; e, electronic charge), where red describes a negative and blue a positive potential. The figures were generated with PyMOL (DeLano Scientific (2004), San Carlos, CA), and panel D was generated with the APBS plug-in for PyMOL.

Figure 3.
Biochemical and functional analysis of point mutants of SifA: interaction with SKIP and RhoA, translocation, and formation of Sifs. A, pulldown analysis of the interaction between SKIP(PH) and SifA variants. GST::SKIP(PH) or GST were immobilized on beads and incubated with extracts of HeLa cells expressing SifA, SifA-(36–140), or SifA variants (AXXXE, WXXXA, AXXXA, and L130D) fused to the N terminus of GFP or GFP alone. Bound proteins were analyzed by Western blotting with an anti-GFP antibody. B and C, translocation analysis. HeLa cells were infected for 16 h with ΔsifA strains expressing GFP and 2HA-tagged version of wild-type or point-mutation variants of SifA. Cells were either fixed, immunolabeled for HA, and imaged by confocal microscopy for GFP (green) and HA (red) (scale bar, 10 μm) (B) or subjected to Triton X-100 extraction and differential centrifugation and analyzed by Western blotting for HA-tagged proteins in bacterial (BCT) and HeLa cell (HC) fractions (C). D, both SifA and SifA-(L130D) pull down GDP-bound RhoA. GST::SifA, GST::SifA-(L130D), or GST were immobilized on beads and incubated with extracts of HeLa cells expressing HA-tagged wild-type, GTP-bound (L63), or GDP-bound (N19) forms of RhoA. Pulled down proteins were analyzed by Western blotting with an anti-HA antibody. E, SifA-(L130D) does not support the formation of Sifs. HeLa cells were infected for 16 h, immunostained, and scored for the formation of HA-labeled Sifs.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2009, 284, 33151-33160) copyright 2009.