A unique PDZ domain and arrestin-like fold interaction reveals mechanistic details of endocytic recycling by SNX27-retromer

Abstract

The sorting nexin 27 (SNX27)-retromer complex is a major regulator of endosome-to-plasma membrane recycling of transmembrane cargos that contain a PSD95, Dlg1, zo-1 (PDZ)-binding motif. Here we describe the core interaction in SNX27-retromer assembly and its functional relevance for cargo sorting. Crystal structures and NMR experiments reveal that an exposed β-hairpin in the SNX27 PDZ domain engages a groove in the arrestin-like structure of the vacuolar protein sorting 26A (VPS26A) retromer subunit. The structure establishes how the SNX27 PDZ domain simultaneously binds PDZ-binding motifs and retromer-associated VPS26. Importantly, VPS26A binding increases the affinity of the SNX27 PDZ domain for PDZ- binding motifs by an order of magnitude, revealing cooperativity in cargo selection. With disruption of SNX27 and retromer function linked to synaptic dysfunction and neurodegenerative disease, our work provides the first step, to our knowledge, in the molecular description of this important sorting complex, and more broadly describes a unique interaction between a PDZ domain and an arrestin-like fold.

Keywords: Alzheimer's disease; Down syndrome; Parkinson disease; endosomal recycling.

Fig. 1.

Identification of the VPS26-binding surface on SNX27 by NMR. (A) Alignment of the amino acid sequences of PDZ domains of human SNX27, C. elegans SNX27, and human NHERF1 and NHERF2. Perfectly conserved residues are highlighted in red. Alignment was generated with ESPript 2.2. (B) Structural comparison of the crystal structures of SNX27 PDZ domain (blue) and the first PDZ domain of NHERF1 (gray) and NHERF2 (purple) (PDB ID codes 3QDO, 1G9O, and 2OCS, respectively). The arrow indicates the conserved loop in SNX27 identified by the sequence alignment. (C) Residues showing significant line broadening and change of chemical shift on formation of the SNX27-VPS26A complex are shown in black and blue, respectively, and displayed as sticks on the SNX27 PDZ domain-Kir3.3 peptide complex crystal structure (PDB ID code 3QDO). Affected residues (see the HSQC spectra in Fig. S1) map to a region close to the Leu67-Pro77 loop, away from the binding site for cargos such as the Kir3.3 potassium channel (in yellow).

Fig. 2.

Crystal structure of the SNX27 PDZ domain-VPS26A complex. (A) Ribbon representation of the crystal structure of the SNX27 PDZ domain (blue) bound to VPS26A (spectrum red-yellow-green). The dashed black rectangle indicates a putative PDZbm-binding pocket. The dashed black circle indicates the VPS35 interaction surface. Loops 4, 8, and 10 of VPS26A and the β3-β4 hairpin of SNX27PDZ, which make direct contact, are highlighted. (B) Interaction surfaces of VPS26A and SNX27. VPS26A is gold, with its SNX27-binding surface in blue. SNX27 is blue, with its VPS26A-binding surface in gold. The PDZbm peptide of the Kir3.3 cargo protein (yellow) is modeled on the published structure with the SNX27 PDZ domain. (C) Detailed view of the interaction surface of the complex between the SNX27 PDZ domain β3-β4 loop and VPS26A loops (L4–L19). The SNX27 and VPS26A main chains are blue and spectrum red-yellow-green, respectively. Selected residues involved in the interaction are displayed as sticks. Electrostatic and hydrogen bonds, represented by dashed black lines, are measured in angstroms.

Fig. 3.

Mutation of the VPS26-binding site of SNX27 prevents endosome-to-plasma membrane recycling of GLUT1. (A) GFP-SNX27 mutant constructs expressed in HEK293T cells were analyzed for binding to the retromer VPS subcomplex by GFP-trap immunoprecipitation and immunoblotting with the indicated antibodies. (B) GST-SNX27 mutant constructs purified from bacteria were tested for binding to purified VPS26A-myc in a direct binding assay. (C) Effective suppression of SNX27 expression using siRNA in HeLa cells. (D) HeLa cells in which SNX27 expression was suppressed using siRNA were transiently transfected with GFP-SNX27 constructs, stained for lysosomal membrane-associated protein 1 (LAMP1) and GLUT1, and imaged on a confocal microscope. (Scale bar: 10 µm.) (E) Pearson correlation coefficient (PCC) and overlap coefficient (Ky) for LAMP1 and GLUT1 in SNX27-suppressed cells expressing the indicated GFP-SNX27 construct. The graph shows the mean of three independent experiments, in each of which at least 30 cells were analyzed. Error bars represent SD. *P < 0.05, two-tailed t test compared with scrambled.

Fig. 4.

VPS26A and VPS26B function redundantly in SNX27-retromer mediated endosome-to-plasma membrane recycling. (A) GFP-VPS26A mutant constructs expressed in HEK293T cells were analyzed for binding to SNX27 by GFP-trap immunoprecipitation and immunoblotting with the indicated antibodies. (B) GST-VPS26A-myc constructs purified from bacteria were tested for binding to the purified SNX27 PDZ domain in a direct binding assay. (C) GFP-VPS26B mutant constructs expressed in HEK293T cells were analyzed for binding to SNX27 by GFP-trap immunoprecipitation and immunoblotting with the indicated antibodies. (D) HeLa cells, in which VPS26A and VPS26B expression was suppressed using siRNA (knockdown efficiency shown in Fig. S5), were transiently transfected with GFP-VPS26 constructs, stained for LAMP1 and GLUT1, and imaged on a confocal microscope. (Scale bar: 10 µm.) (E) PCC and overlap coefficient (Ky) for LAMP1 and GLUT1 in VPS26-suppressed cells expressing the indicated GFP-VPS26 construct. The graph shows the mean of three independent experiments, in each of which at least 30 cells were analyzed. Error bars represent SD. *P < 0.05, two-tailed t test compared with scrambled.

Fig. 5.

SNX27 binding to cargo motifs is allosterically enhanced by VPS26. (A) (Upper) The GLUT1 PDZbm peptide binds the SNX27 PDZ domain in vitro and has enhanced affinity for the SNX27 PDZ domain-VPS26A complex. Mutation in the SNX27 PDZbm pocket (H114A) abolishes the peptide interaction. Mutations in the VPS26A- and SNX27-binding interface reverse the allosteric effect. (Lower) This effect was observed for the Kir3.3 potassium channel peptide as well. Raw data are at the top; integrated normalized data, at the bottom. Colors indicate the proteins used in the ITC cell. (B) (Upper) Overlay of free (green) and complexed (orange) forms of VPS26A, aligned based on their N-terminal subdomains to highlight the conformational flexibility between the N- and C-terminal subdomains in the free and bound structures. Asp171 Cα, represented as a sphere, is used as an arbitrary reference point to quantify C-terminal domain flexibility. (Lower) Overlay of the SNX27 PDZ domain bound to the Kir3.3 PDZbm (gray) or VPS26A (blue) (PDB ID code 3QGL and this study, respectively). The SNX27 PDZ domain protein superposition highlights the rigidity of the β3-β4 β-hairpin and the canonical PDZ-binding pocket with the Kir3.3 peptide in yellow.

Fig. 6.

Model of the SNX27-retromer complex. Low-resolution retromer trimeric structure (VPS26-VPS35-VPS29) and full length SNX27 structure were obtained from previous SAXS experiments. The overall complex was derived from overlaying these complexes with the VPS26A-SNX27 PDZ domain crystal structure. The PDZ domain (blue) and the FERM-like domain (green) of SNX27 bind PDZbm- and NPxY/NxxY-containing transmembrane cargo, respectively, whereas the PX domain of SNX27 (red) associates with the endosomal lipid phosphatidylinositol 3-phosphate (PtdIns3P). VPS26 (orange) binds the PDZ domain of SNX27, VPS35 (yellow) binds VPS26, and VPS29 (purple) in turn binds VPS35.