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Rab1 guanine nucleotide exchange factor SidM is a major phosphatidylinositol 4-phosphate-binding effector protein of Legionella pneumophila.

Brombacher E, Urwyler S, Ragaz C, Weber SS, Kami K, Overduin M, Hilbi H - J. Biol. Chem. (2008)

Bottom Line: Purified SidM specifically and directly bound to PtdIns(4)P, whereas the SidM-interacting Icm/Dot substrate LidA preferentially bound PtdIns(3)P but also PtdIns(4)P, and the L. pneumophila Arf1 GEF RalF did not bind to any PIs.An L. pneumophila DeltasidM mutant strain displayed significantly higher amounts of SidC on LCVs, suggesting that SidM and SidC compete for limiting amounts of PtdIns(4)P on the vacuole.Thus, L. pneumophila exploits PtdIns(4)P produced by PtdIns 4-kinase IIIbeta to anchor the effectors SidC and SidM to LCVs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, ETH Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland.

ABSTRACT
The causative agent of Legionnaires disease, Legionella pneumophila, forms a replicative vacuole in phagocytes by means of the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system and translocated effector proteins, some of which subvert host GTP and phosphoinositide (PI) metabolism. The Icm/Dot substrate SidC anchors to the membrane of Legionella-containing vacuoles (LCVs) by specifically binding to phosphatidylinositol 4-phosphate (PtdIns(4)P). Using a nonbiased screen for novel L. pneumophila PI-binding proteins, we identified the Rab1 guanine nucleotide exchange factor (GEF) SidM/DrrA as the predominant PtdIns(4)P-binding protein. Purified SidM specifically and directly bound to PtdIns(4)P, whereas the SidM-interacting Icm/Dot substrate LidA preferentially bound PtdIns(3)P but also PtdIns(4)P, and the L. pneumophila Arf1 GEF RalF did not bind to any PIs. The PtdIns(4)P-binding domain of SidM was mapped to the 12-kDa C-terminal sequence, termed "P4M" (PtdIns4P binding of SidM/DrrA). The isolated P4M domain is largely helical and displayed higher PtdIns(4)P binding activity in the context of the alpha-helical, monomeric full-length protein. SidM constructs containing P4M were translocated by Icm/Dot-proficient L. pneumophila and localized to the LCV membrane, indicating that SidM anchors to PtdIns(4)P on LCVs via its P4M domain. An L. pneumophila DeltasidM mutant strain displayed significantly higher amounts of SidC on LCVs, suggesting that SidM and SidC compete for limiting amounts of PtdIns(4)P on the vacuole. Finally, RNA interference revealed that PtdIns(4)P on LCVs is specifically formed by host PtdIns 4-kinase IIIbeta. Thus, L. pneumophila exploits PtdIns(4)P produced by PtdIns 4-kinase IIIbeta to anchor the effectors SidC and SidM to LCVs.

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Structural analysis of SidM and fragments. A, sedimentation equilibrium analysis of full-length SidM revealed an ∼71-kDa species corresponding to a monomeric state. B, far-UV CD spectra of the full-length protein SidM (red) and the fragments M7 (blue), M9 (green), and M13 (black). The signal unit is converted into mean residue ellipticity (MRE). The helical structure is evidenced by strong negative ellipticities at around 220 and 208 nm. C, thermofluor assay for the full-length SidM protein and the fragments M7, M9, and M13. The estimated unfolding transition temperatures of full-length SidM and the fragment M7 were 62.3 and 71.4 °C, respectively, whereas the M9 and M13 fragments did not display cooperative unfolding transitions.
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fig4: Structural analysis of SidM and fragments. A, sedimentation equilibrium analysis of full-length SidM revealed an ∼71-kDa species corresponding to a monomeric state. B, far-UV CD spectra of the full-length protein SidM (red) and the fragments M7 (blue), M9 (green), and M13 (black). The signal unit is converted into mean residue ellipticity (MRE). The helical structure is evidenced by strong negative ellipticities at around 220 and 208 nm. C, thermofluor assay for the full-length SidM protein and the fragments M7, M9, and M13. The estimated unfolding transition temperatures of full-length SidM and the fragment M7 were 62.3 and 71.4 °C, respectively, whereas the M9 and M13 fragments did not display cooperative unfolding transitions.

Mentions: Structural Analysis of SidM and Fragments—AUC of purified full-length SidM revealed a single species of 71,282 ± 586 Da, indicating a homogeneous monomeric state (Fig. 4A). Further structural analysis of full-length SidM and the fragments M7, M9, and M13 by CD revealed that the α-helical content of the full-length protein and M7 fragment was similar and ∼67 or 71%, respectively (Fig. 4B and Table 1), matching the predicted secondary structure. In contrast, the M9 and M13 fragments were found by CD spectroscopy to adopt only ∼48 and 59% α-helical structure, compared with predictions of 69 or 73%, respectively. These results suggest that the M9 and M13 fragments are structurally less stable, a finding that is reinforced by the poorly resolved NMR spectra of 15N-labeled M13 protein (data not shown). As a corollary, we suggest that the entire PtdIns(4)P binding structural domain of SidM includes residues N-terminal to residue 444, which are present in the M7 construct. In agreement with this notion, the M9 and M13 fragments were found by Thermofluor assays to lack a thermal unfolding transition typical of a globular fold, whereas the longer constructs revealed an unfolding transition between 65 and 72 °C (Fig. 4C). This instability of the M9 and M13 SidM fragments likely accounts for their apparent 50-fold reduced affinity for PtdIns(4)P, compared with full-length SidM and the M7 fragment (Fig. 3C).


Rab1 guanine nucleotide exchange factor SidM is a major phosphatidylinositol 4-phosphate-binding effector protein of Legionella pneumophila.

Brombacher E, Urwyler S, Ragaz C, Weber SS, Kami K, Overduin M, Hilbi H - J. Biol. Chem. (2008)

Structural analysis of SidM and fragments. A, sedimentation equilibrium analysis of full-length SidM revealed an ∼71-kDa species corresponding to a monomeric state. B, far-UV CD spectra of the full-length protein SidM (red) and the fragments M7 (blue), M9 (green), and M13 (black). The signal unit is converted into mean residue ellipticity (MRE). The helical structure is evidenced by strong negative ellipticities at around 220 and 208 nm. C, thermofluor assay for the full-length SidM protein and the fragments M7, M9, and M13. The estimated unfolding transition temperatures of full-length SidM and the fragment M7 were 62.3 and 71.4 °C, respectively, whereas the M9 and M13 fragments did not display cooperative unfolding transitions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2643517&req=5

fig4: Structural analysis of SidM and fragments. A, sedimentation equilibrium analysis of full-length SidM revealed an ∼71-kDa species corresponding to a monomeric state. B, far-UV CD spectra of the full-length protein SidM (red) and the fragments M7 (blue), M9 (green), and M13 (black). The signal unit is converted into mean residue ellipticity (MRE). The helical structure is evidenced by strong negative ellipticities at around 220 and 208 nm. C, thermofluor assay for the full-length SidM protein and the fragments M7, M9, and M13. The estimated unfolding transition temperatures of full-length SidM and the fragment M7 were 62.3 and 71.4 °C, respectively, whereas the M9 and M13 fragments did not display cooperative unfolding transitions.
Mentions: Structural Analysis of SidM and Fragments—AUC of purified full-length SidM revealed a single species of 71,282 ± 586 Da, indicating a homogeneous monomeric state (Fig. 4A). Further structural analysis of full-length SidM and the fragments M7, M9, and M13 by CD revealed that the α-helical content of the full-length protein and M7 fragment was similar and ∼67 or 71%, respectively (Fig. 4B and Table 1), matching the predicted secondary structure. In contrast, the M9 and M13 fragments were found by CD spectroscopy to adopt only ∼48 and 59% α-helical structure, compared with predictions of 69 or 73%, respectively. These results suggest that the M9 and M13 fragments are structurally less stable, a finding that is reinforced by the poorly resolved NMR spectra of 15N-labeled M13 protein (data not shown). As a corollary, we suggest that the entire PtdIns(4)P binding structural domain of SidM includes residues N-terminal to residue 444, which are present in the M7 construct. In agreement with this notion, the M9 and M13 fragments were found by Thermofluor assays to lack a thermal unfolding transition typical of a globular fold, whereas the longer constructs revealed an unfolding transition between 65 and 72 °C (Fig. 4C). This instability of the M9 and M13 SidM fragments likely accounts for their apparent 50-fold reduced affinity for PtdIns(4)P, compared with full-length SidM and the M7 fragment (Fig. 3C).

Bottom Line: Purified SidM specifically and directly bound to PtdIns(4)P, whereas the SidM-interacting Icm/Dot substrate LidA preferentially bound PtdIns(3)P but also PtdIns(4)P, and the L. pneumophila Arf1 GEF RalF did not bind to any PIs.An L. pneumophila DeltasidM mutant strain displayed significantly higher amounts of SidC on LCVs, suggesting that SidM and SidC compete for limiting amounts of PtdIns(4)P on the vacuole.Thus, L. pneumophila exploits PtdIns(4)P produced by PtdIns 4-kinase IIIbeta to anchor the effectors SidC and SidM to LCVs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, ETH Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland.

ABSTRACT
The causative agent of Legionnaires disease, Legionella pneumophila, forms a replicative vacuole in phagocytes by means of the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system and translocated effector proteins, some of which subvert host GTP and phosphoinositide (PI) metabolism. The Icm/Dot substrate SidC anchors to the membrane of Legionella-containing vacuoles (LCVs) by specifically binding to phosphatidylinositol 4-phosphate (PtdIns(4)P). Using a nonbiased screen for novel L. pneumophila PI-binding proteins, we identified the Rab1 guanine nucleotide exchange factor (GEF) SidM/DrrA as the predominant PtdIns(4)P-binding protein. Purified SidM specifically and directly bound to PtdIns(4)P, whereas the SidM-interacting Icm/Dot substrate LidA preferentially bound PtdIns(3)P but also PtdIns(4)P, and the L. pneumophila Arf1 GEF RalF did not bind to any PIs. The PtdIns(4)P-binding domain of SidM was mapped to the 12-kDa C-terminal sequence, termed "P4M" (PtdIns4P binding of SidM/DrrA). The isolated P4M domain is largely helical and displayed higher PtdIns(4)P binding activity in the context of the alpha-helical, monomeric full-length protein. SidM constructs containing P4M were translocated by Icm/Dot-proficient L. pneumophila and localized to the LCV membrane, indicating that SidM anchors to PtdIns(4)P on LCVs via its P4M domain. An L. pneumophila DeltasidM mutant strain displayed significantly higher amounts of SidC on LCVs, suggesting that SidM and SidC compete for limiting amounts of PtdIns(4)P on the vacuole. Finally, RNA interference revealed that PtdIns(4)P on LCVs is specifically formed by host PtdIns 4-kinase IIIbeta. Thus, L. pneumophila exploits PtdIns(4)P produced by PtdIns 4-kinase IIIbeta to anchor the effectors SidC and SidM to LCVs.

Show MeSH
Related in: MedlinePlus