The phox domain of sorting nexin 5 lacks phosphatidylinositol 3-phosphate (PtdIns(3)P) specificity and preferentially binds to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)

Abstract

Subcellular retrograde transport of cargo receptors from endosomes to the trans-Golgi network is critically involved in a broad range of physiological and pathological processes and highly regulated by a genetically conserved heteropentameric complex, termed retromer. Among the retromer components identified in mammals, sorting nexin 5 and 1 (SNX5; SNX1) have recently been found to interact, possibly controlling the membrane binding specificity of the complex. To elucidate how the unique sequence features of the SNX5 phox domain (SNX5-PX) influence retrograde transport, we have determined the SNX5-PX structure by NMR and x-ray crystallography at 1.5 A resolution. Although the core fold of SNX5-PX resembles that of other known PX domains, we found novel structural features exclusive to SNX5-PX. It is most noteworthy that in SNX5-PX, a long helical hairpin is added to the core formed by a new alpha2'-helix and a much longer alpha3-helix. This results in a significantly altered overall shape of the protein. In addition, the unique double PXXP motif is tightly packed against the rest of the protein, rendering this part of the structure compact, occluding parts of the putative phosphatidylinositol (PtdIns) binding pocket. The PtdIns binding and specificity of SNX5-PX was evaluated by NMR titrations with eight different PtdIns and revealed that SNX5-PX preferentially and specifically binds to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). The distinct structural and PtdIns binding characteristics of SNX5-PX impart specific properties on SNX5, influencing retromer-mediated regulation of retrograde trafficking of transmembrane cargo receptors.

FIGURE 1.

Amino Acid sequence alignment of phox domains and domain architecture of the mammalian sorting nexin family. A, comparative sequence alignment of PX domains for residues equivalent to Gly⁴⁹–Leu¹¹⁹ of the p40-PX domain (adapted from Worby and Dixon (21)). Prolines in the Pro-X-X-Pro motif are highlighted in yellow, and residues involved in phospholipid binding in the p40-PX domain are boxed in magenta. Arg⁵⁸ and Arg¹⁰⁵ are marked with magenta triangles, and Tyr⁵⁹ and Lys⁹² are marked with black stars at the bottom of the sequences. The two conserved Arg residues and Lys⁹² of p40-PX in other PX domains are highlighted in dark blue boxes; those corresponding to Tyr⁵⁹ are boxed in green. The secondary structure elements of p40-PX are indicated by yellow arrows (β-sheets) and red ovals (α-helices). The three sequence stretches that are unique in SNX5-PX (or SNX6-PX) are enclosed in a bright blue box. B, domain architecture of SNX family members. The four classes within the SNX family are designated as PX SNXs, PX-BAR SNXs, SH3-PX-BAR, and PX-other domain SNXs. Each individual domain is depicted in a different color and/or shape. The following domains are depicted: PX (phox), BAR (Bin-Amphiphysin-Rvs), SH3 (Src homology 3), TM (transmembrane), PXA (PX domain-associated), RGS (regulator of G-protein signaling), MIT (microtubule interacting and trafficking), B41 (band 4.1 homology), TPR (tetratricopeptide repeat), PDZ (postsynaptic protein PSD-95/SAP90, the Drosophila melanogaster septate junction protein Discs-large, and the tight junction protein ZO-1), and RA (Ras association).

FIGURE 2.

¹H-¹⁵N two-dimensional HSQC spectrum of SNX5-PX. Backbone amide resonances are labeled by amino acid type and number. The amides of residues Arg⁴², Gln⁶⁸, His⁶⁹, and His⁸³ are marked by dashed circles. They exhibit faster solvent exchange than average and therefore are only observed at low contour levels (see insets). Assignments for the crowded region in the middle of the spectrum (gray contours) are provided in an expansion of this region in the lower right hand corner of the spectrum. Note that two sets of resonances are observed for residues Leu¹⁷², Ser¹⁷³, Val¹⁷⁴, and Arg¹⁷⁵ caused by C-terminal heterogeneity (blue dashed circles). The spectrum was recorded at 25 °C on a 0.1 mm protein sample in 20 mm Tris buffer, 100 mm NaCl, 0.02% NaN₃, pH 7.4.

FIGURE 3.

X-ray structure of SNX5-PX domain. A, stereo view of the overall structure of SNX5-PX. β-strands (β1, β2, and β3) and α-helices (α1, α2′, α3′, α3, α_3–10, and α4) are colored in yellow and red, respectively. Residues are numbered at every 10th position. The polyproline (PXXP) motif is located in the irregular strand connecting helices α1 and α2′. B, intermolecular interaction between the N-terminal residues (Val²³–Asp²⁸) of one molecule and the β1-strand of the neighboring one. Backbone hydrogen bonds are formed between the amide of Asn²⁶ and the carbonyl oxygen of Ala³⁸, the carbonyl oxygen of Asn²⁶ and amide of Ser⁴⁰, the amide of Asp²⁸ and the carbonyl oxygen of Ser⁴⁰, and the carbonyl oxygen of Asp²⁸ and the amide of Arg⁴².

FIGURE 4.

Comparison between the structures of SNX5-PX and selected PX domains. A, architecture of selected structures of PX domains. PX domains used for comparison are as follows: PDB numbers 1XTE (crystal structure of mouse CISK-PX domain), 1OCU (crystal structure of yeast SNX3 (Grd19p), 2AR5 (crystal structure of human C-2 α-phosphatidylinositol 3-kinase), 1O7K (crystal structure of human p47-PX), and 1H6H (crystal structure of human p40-PX). The color coding in the ribbon representations is as follows: all β-strands are shown in yellow, helix α1 is in bright green, helices α2′ and α3′ in SNX5-PX are in blue and cyan, and helices α3 and α4 in SNX5-PX and their counterparts (α2 and α3) in other PX domains are in red and dark green, respectively. B, best fit superposition of SNX5-PX (blue) and p40-PX (gray) structures. The most striking differences are the additional helical insertion at the bottom (cyan oval), the altered conformation of the irregular strand that harbors the PXXP motif (cyan arrow), causing tighter packing of this strand against the body of the protein, and a tighter turn connecting β-strands β1 and β2 (cyan arrow). C, detailed view of the area around the double PXXP motif in SNX5-PX. The compactness is highlighted by indicating the ∼7.5 Å Cα–Cα distance between Val⁴⁵ and Pro⁹⁷. D, detailed view of the corresponding region in p40-PX. In this case, the equivalent Cα–Cα distance between Phe³⁹ and Val⁹³ is ∼13.5 Å, almost twice that in SNX5-PX.

FIGURE 5.

NMR titration and chemical shift mapping of PtdIns binding to SNX5-PX. A, superposition of the ¹H-¹⁵N HSQC spectra of free (black) and PtdIns(0)P-saturated (green) SNX5-PX. B, superposition of the ¹H-¹⁵N HSQC spectra of PtdIns(0)P-saturated (green) and PtdIns(3)P-saturated (blue) SNX5-PX. C, superposition of the ¹H-¹⁵N HSQC spectra of free (black) and PtdIns(4,5)P₂-saturated (magenta) SNX5-PX. D, superposition of the ¹H-¹⁵N HSQC spectra of PtdIns(3)P-saturated (blue) and PtdIns(4,5)P₂-saturated (magenta) SNX5-PX. Titration curves for selected resonances (Asp⁴³, Lys⁴⁴, Arg¹⁰³, and Lys¹⁰⁵) are shown in the inset. E, structural mapping of residues affected non-specifically by PtdIns(3)P binding. The protein is displayed in gray surface representation, and residues whose resonances are perturbed in the titration are colored in blue. F, structural mapping of residues affected specifically by PtdIns(4,5)P₂. The protein is displayed in gray surface representation, and residues whose resonances are perturbed in the titration are colored in magenta. G, detailed depiction of the PtdIns(4,5)P₂ binding site in SNX5-PX. Selected residues are labeled by residue name and number. Pro⁹⁷ is shown in green.

FIGURE 6.

Side-by-side view of the PtdIns binding sites in the p40-PX/PtdIns(3)P complex and in SNX5-PX. The proteins are show in space-filling representation, and the ligand and selected side chains are show in in ball-and-stick representation. A, contacts between the positively charged Arg⁵⁸, Arg⁶⁰, Lys⁹², and Arg¹⁰⁵ side chains and the negatively charged phosphate or hydroxyl groups on the PtdIns(3)P ligand in the p40-PX/PtdIns(3)P complex structure are shown by dashed lines. Tyr⁵⁹ and Tyr⁹⁴ side chains involved in stabilizing the inositol ring and the acyl chain, respectively, are also highlighted. B, PtdIns binding site in free SNX5-PX into which a PtdIns(3)P ligand was positioned in the same orientation as in the p40-PX/PtdIns(3)P structure. Note the presence of severe steric clash between Glu⁷⁰ and the phosphate group at the C-3 position and between Lys⁹⁶ and the inositol ring.