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. 2018 Feb 13;115(7):E1446-E1454.
doi: 10.1073/pnas.1717383115. Epub 2018 Jan 31.

Mechanism of inhibition of retromer transport by the bacterial effector RidL

Jialin Yao  1 , Fan Yang  1 , Xiaodong Sun  2 , Shen Wang  3 , Ninghai Gan  4 , Qi Liu  1 , Dingdong Liu  1 , Xia Zhang  1 , Dawen Niu  1 , Yuquan Wei  1 , Cong Ma  3 , Zhao-Qing Luo  4 , Qingxiang Sun  5 , Da Jia  6
Affiliations

Mechanism of inhibition of retromer transport by the bacterial effector RidL

Jialin Yao et al. Proc Natl Acad Sci U S A. .

Abstract

Retrograde vesicle trafficking pathways are responsible for returning membrane-associated components from endosomes to the Golgi apparatus and the endoplasmic reticulum (ER), and they are critical for maintaining organelle identity, lipid homeostasis, and many other cellular functions. The retrograde transport pathway has emerged as an important target for intravacuolar bacterial pathogens. The opportunistic pathogen Legionella pneumophila exploits both the secretory and recycling branches of the vesicle transport pathway for intracellular bacterial proliferation. Its Dot/Icm effector RidL inhibits the activity of the retromer by directly engaging retromer components. However, the mechanism underlying such inhibition remains unknown. Here we present the crystal structure of RidL in complex with VPS29, a subunit of the retromer. Our results demonstrate that RidL binds to a highly conserved hydrophobic pocket of VPS29. This interaction is critical for endosomal recruitment of RidL and for its inhibitory effects. RidL inhibits retromer activity by direct competition, in which it occupies the VPS29-binding site of the essential retromer regulator TBC1d5. The mechanism of retromer inhibition by RidL reveals a hotspot on VPS29 critical for recognition by its regulators that is also exploited by pathogens, and provides a structural basis for the development of small molecule inhibitors against the retromer.

Keywords: endosomal sorting; host–pathogen interaction; retromer; vesicular trafficking.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The RidL–retromer interaction is mediated by the N terminus of RidL and VPS29 subunit of retromer. (A) GST–RidL or GST pull-down of purified retromer VPS35/VPS26/VPS29, VPS35/VPS26, and VPS29. Shown are Coomassie blue stained SDS/PAGE gels of purified proteins used (Left) and bound samples (Right). (B) Isothermal titration calorimetry (ITC) of RidL titrated into VPS35/VPS26/VPS29 in a buffer containing 20 mM Tris⋅HCl, pH 8.0, 200 mM NaCl, 5 mM βME at 20 °C. Top and Bottom show raw and integrated heat from injections, respectively. The black curve at Bottom represents a fit of the integrated data to a single-site binding model. (C) Affinity between retromer proteins and full-length RidL, RidL-N, or TBC1d5, determined by ITC. Association constant (Ka) are shown together with errors from data fitting. (D) GST–RidL, RidL-N, RidL-C, or GST pull-down of purified VPS29. Shown are Coomassie blue stained SDS/PAGE gels of purified proteins used (Left) and bound samples (Right).
Fig. 2.
Fig. 2.
Structural mechanism of VPS29 recognition by RidL-N. (A) Ribbon diagram of the VPS29/RidL complex (VPS29, gray; RidL, cyan). N and C terminus of proteins are labeled. The secondary structure elements of RidL are labeled. (B) VPS29–RidL interactions in detail. Critical VPS29 and RidL residues are shown in stick modes. A partially transparent electrostatic surface potential map is presented for VPS29. Blue dash represents hydrogen bond between RidL and VPS29. (C and D) Coomassie blue stained SDS/PAGE gels of bound proteins are shown. Results are representative of three independent experiments. Amount of VPS29 retained was expressed relative to the amount of GST–RidL in the bound sample and then normalized to the amount of wild-type protein. All values are presented as mean ± SD, derived from three independent experiments. In C, immobilized GST–RidL-N or its mutants (Y166A, P168A, I170A, I170E, I70L, I170W, P171A, and P172A) was used to pull down VPS29. In D, immobilized GST–RidL-N was used to pull down VPS29 WT or mutants (L2A, L25A, K30A, Y163A, Y165A, and R176A).
Fig. 3.
Fig. 3.
The sequence and conformation of the RidL loop are essential for the binding to VPS29. (A) GST–RidL from related Legionella species pull-down of purified VPS29. Shown are Coomassie blue stained SDS/PAGE gels of purified proteins used (Left) and bound samples (Right). (B) Detailed VPS29–RidL interactions highlight RidL residues that are distant from the binding sites of VPS29, but critical for the conformation of the binding loop. Residues L162, N176, K177, and S178 are shown in stick representation. Hydrogen bonds are denoted with blue dash lines. (C) GST–RidL-N, RidL-N-NKS, loopLP, RidLLS-loopLP pull-down of purified VPS29. Shown are Coomassie blue stained SDS/PAGE gels of purified proteins used (Left) and bound samples (Right). (D) GST–RidL, RidLLP-loopLS, and GST pull-down of purified VPS29. Shown are Coomassie blue stained SDS/PAGE gels of purified VPS29 protein (Left) and bound samples (Right).
Fig. 4.
Fig. 4.
The RidL localizes to endosomes and inhibits Shiga toxin transport through its interaction with retromer. (A) HeLa cells were transfected with GFP, or GFP–RidL-N WT, Y166A, I170A, P172A (green), and then fixed and labeled with anti-VPS35 (red) antibody. (B) Quantitation of GFP colocalization with VPS35 in cells in A. Each dot represents Pearson’s correlation coefficients from one cell. P values shown are the result of one-way ANOVA, post hoc Tukey’s test. (C) RidL inhibits retrograde transport of STxB through its interaction with the retromer. Cells were first transformed for 24 h with GFP, GFP–RidL-N WT, or mutants (green), and then fed with purified STxB protein. The trafficking of STxB was analyzed by staining STxB with antibody (red) and determining the colocalization with the trans-Golgi marker, TGN46 (white). (D) Quantitation of STxB colocalization with TGN46 in cells in C. Each dot represents Pearson’s correlation coefficients from one cell. P values shown are the result of one-way ANOVA, post hoc Tukey’s test.
Fig. 5.
Fig. 5.
RidL and TBC1d5 compete with each other for binding to retromer. (A) Structural comparison of VPS29/RidL and VPS29/TBC1d5-Ins1 overlaid by VPS29 (Left). (VPS29, gray; RidL, cyan; and TBC1d5, magenta). (Right) Zoomed-in view of the VPS29-interacting regions of TBC1d5 and RidL. Key interacting residues from TBC1d5 and RidL are shown in stick representation. (B and C) Coomassie blue stained SDS/PAGE gels of bound proteins are shown. Results are representative of three independent experiments. Amount of VPS29 (B) or VPS35 (C) retained was expressed relative to the amount of immobilized GST-fusion proteins in the bound sample and then normalized to control sample (no competing protein). All values are presented as mean ± SD, derived from three independent experiments. The molar ratio of immobilized protein and competing protein is indicated at the Bottom of the table. (B) GST–RidL or TBC1d5 pull-down of purified VPS29 in the absence or presence of competing TBC1d5 or RidL. (C) GST–RidL or TBC1d5 pull-down of purified VPS35/VPS29 in the absence or presence of competing TBC1d5 or RidL. (D and E) RidL inhibits endosomal localization of TBC1d5 through its interaction with the retromer. Cells were first transformed with GFP, GFP–RidL-N WT, or mutants (green), then fixed and stained with anti-TBC1d5 (white) and EEA1 (red) antibodies. Each dot represents Pearson’s correlation coefficients from one cell.
Fig. 6.
Fig. 6.
Model showing how RidL inhibits retromer-dependent transport. (Left) Retromer requires TBC1d5 for endosomal transport. TBC1d5 forms a tight complex with retromer through interacting both VPS35 and VPS29 and may function to regulate retromer assembly and turnover on endosomal membranes. Retromer cargoes, Sortin Nexins, VARP, and other known regulators are omitted for simplicity. (Right) During L. pneumophila infection or ectopic expression of RidL, RidL replaces TBC1d5. Upon ectopic expression of RidL, RidL is recruited to endosomes through interaction with VPS29 and endosomal lipid PtdIns(3)P (green dot). RidL replaces TBC1d5, and likely VARP, to block retromer-mediated trafficking. During L. pneumophila infection, retromer subunits are recruited to Legionella-containing vacuoles (LCVs) through their interaction with RidL and potentially other bacterial effector proteins.
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References

    1. Burd C, Cullen PJ. Retromer: A master conductor of endosome sorting. Cold Spring Harb Perspect Biol. 2014;6:a016774. - PMC - PubMed
    1. Bonifacino JS, Hurley JH. Retromer. Curr Opin Cell Biol. 2008;20:427–436. - PMC - PubMed
    1. McMillan KJ, Korswagen HC, Cullen PJ. The emerging role of retromer in neuroprotection. Curr Opin Cell Biol. 2017;47:72–82. - PMC - PubMed
    1. Lucas M, Hierro A. Retromer. Curr Biol. 2017;27:R687–R689. - PubMed
    1. Seaman MN, McCaffery JM, Emr SD. A membrane coat complex essential for endosome-to-Golgi retrograde transport in yeast. J Cell Biol. 1998;142:665–681. - PMC - PubMed

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