Crystal Structures of the Extracellular Domain from PepT1 and PepT2 Provide Novel Insights into Mammalian Peptide Transport

Abstract

Mammals obtain nitrogen via the uptake of di- and tri-peptides in the gastrointestinal tract through the action of PepT1 and PepT2, which are members of the POT family of proton-coupled oligopeptide transporters. PepT1 and PepT2 also play an important role in drug transport in the human body. Recent crystal structures of bacterial homologs revealed a conserved peptide-binding site and mechanism of transport. However, a key structural difference exists between bacterial and mammalian homologs with only the latter containing a large extracellular domain, the function of which is currently unknown. Here, we present the crystal structure of the extracellular domain from both PepT1 and PepT2 that reveal two immunoglobulin-like folds connected in tandem, providing structural insight into mammalian peptide transport. Functional and biophysical studies demonstrate that these domains interact with the intestinal protease trypsin, suggesting a role in clustering proteolytic activity to the site of peptide transport in eukaryotic cells.

Graphical abstract

Figure 1

Topology of Mammalian Peptide Transporters Topology diagram of the human plasma membrane peptide transporter PepT1. Conserved PTR2/POT family signature motifs are indicated along with predicted N-linked glycosylation sites, three of which are in the extracellular domain. Inset: Crystal structure of the bacterial homolog PepT_St (PDB: 4D2C). The N- (light blue) and C-terminal (wheat) domains are shown as cylinders, with the bound peptide indicating the location of the central peptide-binding site conserved between mammalian and bacterial proteins.

Figure 2

Crystal Structure of the Extracellular Domain from PepT1 and PepT2 (A) The asymmetric unit of MmPepT1^ECD containing two monomers related by a two-fold non-crystallographic symmetry axis (black oval). One monomer is rainbow colored from the N terminus to the C terminus; the second is shown in gray with the secondary structure labeled from β1 to β16. (B) Structure of the RnPepT2^ECD colored from the N (blue) to the C terminus (red) and with the secondary structure components labeled as for (A). (C) The s⁰_20,W values of MmPepT1^ECD and RnPepT2^ECD from the AUC analysis are 2.16 and 2.22, respectively, consistent with both proteins migrating as a 20-kDa monomer in solution. Inset: the Lamm equation fit profiles for MmPepT1^ECD and RnPepT2^ECD.

Figure 3

Salt Bridges Stabilize the Interface between the Two Immunoglobulin-like Domains in PepT1^ECD and PepT2^ECD (A) Structure of PepT1^ECD illustrating the two salt bridges, K398 and D574 and R490 and D476, that form an interaction between the lobes. (B) Comparative view in RnPepT2^ECD, where a single salt bridge is observed between Asp505 and Arg518. (C) Size-exclusion chromatography traces from the MmPepT1^ECD-3CX experiment. The cleaved MmPepT1^ECD-3CX constructs elute at the same volume as wild-type, showing that the lobes still interact in solution even after the two lobes are separated. The cleaved MmPepT1^ECD-3CX-D574A construct, however, elutes in a larger volume consistent with disruption of the interaction. (D) DAMMIN envelopes of MmPepT1^ECD (dark purple) and RnPepT2^ECD (light purple) calculated from the SAXS data, which show lengths of 48 and 61 Å, respectively, and illustrate the more dynamic behavior of PepT2^ECD. For scale, a black and white outline of A is overlaid on the MmPepT1^ECD envelope.

Figure 4

PepT1 and PepT2 are Modular Proteins with Functionally Distinct Domains (A) Homology model of the human PepT1 transporter generated using the crystal structure of MmPepT1^ECD (colored blue to red as in Figure 1B) and the recently determined bacterial homolog PepT_So representing the transmembrane portion of the transporter (shown in gray). The peptide-binding site is highlighted (magenta). (B) Kinetic analysis of Gly-Sar uptake in human PepT1 and PepT2 using the TEVC method. (C) Kinetic analysis of Gly-Sar uptake in the PepT2^ΔECD, PepT2^T1ECD, and PepT1^D573A constructs. (D) K_i values for the different constructs for lysyl-lysine and cefaclor are shown, indicating no effect of removing the ECD on peptide or drug uptake in PepT2.

Figure 5

Trypsin Interacts with a Di-acidic Motif on the Extracellular Domain of PepT1 and PepT2 (A) SPR analysis of the MmPepT1^ECD interaction with trypsin. Inset: SPR sensorgram used to determine the binding constant. RU, response units. Error bars show the SEM (n = 3). (B) The binding experiment in (A) was repeated with the RnPepT2^ECD protein. (C) MST binding analysis reveals no interaction with the Gly-Sar peptide and abolition of trypsin interaction in the presence of high salt. (D and E) Surface representation of (D) MmPepT1^ECD and (E) RnPepT2^ECD with the sequence conservation from cow, dog, chicken, human, mouse, and rat species mapped from blue to red. A highly conserved patch (indicated by the white dashed ellipse) was identified. Insets: MST binding analysis reveals an important role for D550 and E573 in MmPepT1^ECD, and D576 and E599 in RnPepT2^ECD, in mediating the electrostatic interaction with trypsin.

Figure 6

A Model for the Interaction between Trypsin and the Mammalian Peptide Transporters During protein digestion in the small intestine, trypsin transiently docks onto the conserved di-acidic motif on the trypsin-binding domain, localizing the protease to the main site of peptide import on the brush border membrane. Localization would create an increase in the local concentration of arginine- and lysine-containing peptides (shown here as blue circles), which would be expected to increase the efficiency of their uptake into the cell.