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. 2017 Jun 30:2:17030.
doi: 10.1038/sigtrans.2017.30. eCollection 2017.

Structural and functional insights into sorting nexin 5/6 interaction with bacterial effector IncE

Qingxiang Sun  1 , Xin Yong  1   2 , Xiaodong Sun  3 , Fan Yang  1   2 , Zhonghua Dai  4 , Yanqiu Gong  1 , Liming Zhou  3 , Xia Zhang  1 , Dawen Niu  1 , Lunzhi Dai  1 , Jia-Jia Liu  4 , Da Jia  1   2
Affiliations

Structural and functional insights into sorting nexin 5/6 interaction with bacterial effector IncE

Qingxiang Sun et al. Signal Transduct Target Ther. .

Abstract

The endosomal trafficking pathways are essential for many cellular activities. They are also important targets by many intracellular pathogens. Key regulators of the endosomal trafficking include the retromer complex and sorting nexins (SNXs). Chlamydia trachomatis effector protein IncE directly targets the retromer components SNX5 and SNX6 and suppresses retromer-mediated transport, but the exact mechanism has remained unclear. We present the crystal structure of the PX domain of SNX5 in complex with IncE, showing that IncE binds to a highly conserved hydrophobic groove of SNX5. The unique helical hairpin of SNX5/6 is essential for binding, explaining the specificity of SNX5/6 for IncE. The SNX5/6-IncE interaction is required for cellular localization of IncE and its inhibitory function. Mechanistically, IncE inhibits the association of CI-MPR cargo with retromer-containing endosomal subdomains. Our study provides new insights into the regulation of retromer-mediated transport and illustrates the intricate competition between host and pathogens in controlling cellular trafficking.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Crystal structure of IncE109–132:PX5 complex. (a) SPR experiment flowing IncE109–132peptide over cell immobilized with PX5 protein. Upper panel shows the reference-subtracted concentration-dependent-binding response and 1:1 fitting model. Bottom panel shows the residual plots for the quality of fitting. (b) Crystal structure of PX5 (green) in complex with IncE109–132 (cyan). (c) IncE (ribbon and line representation) is docked into a hydrophobic groove on PX5 (electrostatic surface representation). The viewing angle in b is slightly rotated here to better illustrate the groove. (d) Overlay of PX5:IncE109–132with structure of apo PX5(PDB:3HPC). The dash line shows the movement of the helical hairpin tip. The view is set by rotating b around the horizontal line by 90° clockwise.
Figure 2
Figure 2
The IncE:PX interface. (a) Zoom-in view of interactions between PX5 and IncE from the crystal structure. For clarity, IncE is shown in ribbon representation. Possible interacting (except hydrogen bonds) residues are shown in stick representation. (b) Glutathione S-transferase (GST) pull down of GST-IncE109–132 WT and mutants with purified PX5 protein. (c) SPR steady state analysis of GST-IncE109–132 WT and two mutants binding to immobilized PX5. (d) MBP pull down of MBP-IncE109–132 with GST-PX5 WT and mutants. Before running SDS-PAGE, the samples are digested with excess of tobacco etch virus to allow visualization of bound PX5 protein. (e) Immunoprecipitation of green fluorescent protein (GFP)-SNX6 using GFP antibody with purified GST-IncE WT and two mutants.
Figure 3
Figure 3
SNX5’s unique protruding helical hairpin is essential for IncE binding. (a) Structural comparison of PX5 with PX domains. IncE is colored in cyan. Note that PX5 has a unique long helical hairpin. (b) GST pull down of PX5 WT and Δ102–131 mutant with immobilized GST-IncE109–132. (c) SNX6 WT and Δ124–148 co-immunoprecipitated with SNX1. HEK293T cells were transfected with empty vector, or Flag-YFP-SNX6 constructs, p100 fractions were prepared, and immunopurified with anti-Flag antibody. Lysates were immunoblotted with anti-SNX1 or anti-Flag antibodies. (d) GST pull down of purified SNX1/SNX6 protein complex with immobilized GST, GST-IncE109–132 WT or V114A.
Figure 4
Figure 4
Interaction between IncE and SNX5/6 is conserved, and IncE inhibits CI-MPR loading to retromer vesicles and transport to TGN. (a) Sequence alignment of the C terminus of IncE from Chlamydia species, Chlamydia trachomatis (ct), Chlamydia suis (cs) or Chlamydia muridarum (cm). Multiple PX sequences are aligned using ClustalW (* for invariant, for conserved, for less conserved changes). Residues at the interface are highlighted in machaccino. Residues whose mutation disrupt or weaken the binding in Figure 2 are labeled with black triangles on top. (b) GST pull down with GST or GST-IncE with purified 5PX. (c) HeLa cells were transfected with GFP, or GFP-IncEcm WT, and F125A (green), and then fixed and labeled with anti-CI-MPR (red) and TGN46 (white) antibodies. (d) Quantitation of CI-MPR co-localization with TGN46 in cells expressing GFP, or various GFP-IncEcm. 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. (e) Subcellular localization CI-MPR, VPS35 and IncEcm. HeLa cells were transfected with GFP, or various GFP-IncEcm WT, V123A and F125A (green), and then fixed and labeled with anti-CI-MPR (red) and VPS35 (white) antibodies. (f) Quantitation of CI-MPR co-localization with VPS35 in cells expressing GFP, or various GFP-IncEcm. 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. (g) A model of controlling SNX5/6-mediated transport by effector protein IncE. IncE binds to SNX5/6 and inhibits the loading of CI-MPR to SNX5/6-retromer-coated endosomal structures.
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