Structural basis for pH-dependent retrieval of ER proteins from the Golgi by the KDEL receptor

Abstract

Selective export and retrieval of proteins between the endoplasmic reticulum (ER) and Golgi apparatus is indispensable for eukaryotic cell function. An essential step in the retrieval of ER luminal proteins from the Golgi is the pH-dependent recognition of a carboxyl-terminal Lys-Asp-Glu-Leu (KDEL) signal by the KDEL receptor. Here, we present crystal structures of the chicken KDEL receptor in the apo ER state, KDEL-bound Golgi state, and in complex with an antagonistic synthetic nanobody (sybody). These structures show a transporter-like architecture that undergoes conformational changes upon KDEL binding and reveal a pH-dependent interaction network crucial for recognition of the carboxyl terminus of the KDEL signal. Complementary in vitro binding and in vivo cell localization data explain how these features create a pH-dependent retrieval system in the secretory pathway.

Fig. 1. Crystal structure of the KDELR.

(A) Representative curves showing normalized binding of [³H]-TAEKDEL peptide to the KDELR at different pH conditions. (B) Crystal structure of KDELR viewed from the Golgi membrane. The transmembrane helices are labeled by number. (Top right) Topology of the fold, highlighting the two triple helix bundles (THB1 and THB2). (C) Electrostatic surface representation of KDELR highlighting the negatively charged band on the cytosolic side of the receptor. (D) View in (A) rotated 90°, showing placement of the receptor with respect to the membrane bilayer (dashed lines). (E) Sliced-through volume representation of (D), highlighting the polar cavity on the luminal side of the receptor.

Fig. 2. Molecular basis for KDEL peptide recognition.

(A) The polar cavity in KDELR is shown with contributing side chains represented as sticks and their electrostatic surface shown. The bound TAEKDEL peptide is shown with the mFo-DFc difference electron density (green mesh) used for model building displayed, contoured at 3σ. The N-terminal threonine was disordered in the maps and not modeled. Water molecules are represented as spheres (red) with hydrogen bonds as dashed lines (yellow). (B) Close-up view of the polar cavity showing the structural movement induced in TM1 and TM6 upon peptide binding. The apo structure is shown in colored helices, the peptide-bound structure in gray. In the top-down view (right), only the C-terminal leucine of the peptide is shown. (C) Close-up view of the polar cavity shown in (A). Water molecules are represented as spheres (red) with hydrogen bonds as dashed lines (yellow). (D) Overlay of the apo (color-coded helices) and peptide-bound structure (gray), highlighting the short hydrogen bond formed between Glu¹²⁷ and Tyr¹⁵⁸ at the base of the polar cavity after peptide binding. Two water molecules coordinate the interaction between the C terminus of the peptide with Tyr¹⁵⁸ and His¹². Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

Fig. 3. Functional characterization of binding-site mutations.

(A) In vitro binding assay using purified chicken KDELR. Asterisk indicates protein that could not be produced. (B) ER retrieval assays were performed in the absence (-) or presence (+) of KDEL^Sec and the Golgi signal for KDELR is plotted as mean ± SEM. (C) KDELRs were tested for KDEL^Sec-induced redistribution from Golgi to ER as in (B). TGN46 was used as a Golgi marker. Scale bar, 10 μm.

Fig. 4. Structural basis for exposure of COPI retrieval signal.

(A) Overlay of the apo (gray) and peptide-bound (colored helices) crystal structures of the KDELR. The movement of TM7 is highlighted, exposing the C-terminal lysine side chains, indicated by the dashed circle. (Right) Cytoplasmic view of the receptor with the movement in TM7 indicated. (B) Electrostatic surface representation of (A). Dashed lines indicate rough position of the Golgi membrane, and the lysine retrieval motif is indicated by the dashed circle. After movement of TM7, a new cavity opens on the cytoplasmic side of the receptor, exposing Asp¹⁹³. (C) Cytoplasmic surface of the receptor, showing the substantial change in distribution of surface charge, most notably the disruption of the negative band present in the apo state. (D) Wild-type, Asp¹⁹³→Asn, and KGKK-dilysine motif mutant KDELRs were tested for KDEL^Sec-induced redistribution from Golgi to ER. TGN46 was used as a Golgi marker. Scale bar, 10 μm. (E) Golgi signal for KDELR in retrieval assays performed as in (D), in the absence (-) or presence (+) of KDEL^Sec is plotted as mean ± SEM.