PepT_st (PDB: 4D2C) from Streptococcus thermophiles

Created by: Jacob Malony

PepT_st is a membrane protein of the bacteria, Streptococcus thermophiles. It is primarily alpha-helical and has 391 residues. As a member of the proton-coupled peptide transporters (POT family), its primary function is to selectively transport certain di- and tripeptides across the bacterial membrane. Its tertiary structure consists of 2 psuedosymmetric domains that act as the two halves of the transporter, closing and opening to form different conformations. (1) PepT_st is an interesting protein for research because it is a bacterial homologue to the human POT family transporter, PepT1. PepT1 is a physiologically important protein because peptide transport is the main route in which the body absorbs and retains dietary proteins. Ingested proteins are absorbed across cellular membranes as di- and tri-peptides. (2) While most transport proteins are highly selective, PepT1 is a somewhat unusual protein due to its ability to recognize and transport over 8,000 different di- and tripeptide ligands. This diverse binding is pharmaceutically intriguing, as PepT1 transports an increasing number of antibiotics, antiviral, and anticancer molecules. Due to the pharmaceutical relevance and irregularity of binding selectivity of PepT1, the crystallized structure of the bacterial homologue, PepT_st, is studied to further understand the structure and function of PepT1. (3)

The crystallized structure of PepT_st shows it in an inward open conformation. In this conformation, the peptide is only exposed to reactions with molecules on the inside of the cell. It is blocked from interacting with molecules on the outside of the membrane by certain salt bridge interactions. PepT_st is primarily alpha-helical and possesses a canonical 12-helix, two-domain transmembrane core. The core is characterized by a centralized peptide-binding site surrounded by various hydrophobic pockets that stabilize the nonpolar side chains of di- and tripeptides. To accomplish the job of peptide transport, PepT_st can adopt 3 distinct conformations: an outward-facing state, an occluded state, and an inward-facing state. The outward-facing conformation exposes the internal binding center to molecules outside of the cell. The inward-facing conformation exposes the transported molecule to the inside of the cell. The occluded state is an intermediate that serves to move the transported peptide to and from its binding positioning in the inward and outward conformations.  As a proton-driven peptide symporter, proton activity helps catalyze these changes in conformation. (2)

In order to adopt the outward open conformation to accept the peptide for transport, PepT_st has an extracellular gate that must be opened. This gate consists of close packing of the ends of alpha helix 1 and alpha helix 7. Access to the peptide binding site from the extracellular side requires a significant conformational change in this gate. The gate is stabilized by two separate salt bridge interactions: Arg-53 (H1) with Glu-312 (H7) and Arg-33 (H1 with Glu-300 (H7). By testing the functionality of PepT_st while intentionally mutating the residues of the salt bridges, it was suggested that the Arg-53-Glu-312 salt bridge plays more of a supportive role in the operation of the extracellular gate, while the Arg-33-Glu-300 salt bridge is crucially important to the proper function of the gate in how it pulls the “tops” of the alpha helices together to block access to the binding site from molecules  (2)

The binding site of PepT_st is quite unusual.  It displays the ability to bind di- and tripeptides in both horizontal and vertical orientations. These different orientations utilize different binding modes, interacting with entirely different residues and hydrophobic pockets to stabilize the binding. The horizontal binding mode is well established through study of the co-crystallized structure with the natural dipeptide L-Ala-L-Phe. One important thing to note from the crystallized structure is the presence of a highly conserved ExxERFxYY motif of H1. Despite the highly conserved nature of this motif, the function remains unknown.  This set of residues is conserved over all currently known POT family transporters. This peptide is held in the binding mode by electrostatic interactions between its amino and carboxy termini and both N- and C-terminal side chains. Specifically, the amino terminus interacts with a conserved Glu-400 on H10 and a hydrogen bond to Asn-328 on H8. These 2 residues are crucially important to binding and transport in this binding mode. In accordance with PepT1, the amide nitrogen of the peptide bond does not interact with the binding site. The carbonyl group of the peptide is coordinated to Asn-156 on H5. This interaction forms a bridge between the 2 six-helix bundles. The phenyl ring side chain of the dipeptide fits into a hydrophobic pocket formed by side chains from H2 (Tyr-68), H7 (Trp-296) and H11 (Trp-427, Phe-428, Ser-431). It also forms a pi-pi stacking interaction with the phenyl side chain from Tyr-68. Other larger hydrophobic pockets are present to accommodate larger side chains of different dipeptides.  (3)

Aside from the lateral binding mode of dipeptides, PepT_st can also accommodate tripeptides in a vertically oriented binding mode. It is unknown how exactly the tripeptides are oriented. Experimental data cannot confirm whether the C-terminus faces towards cytoplasmic or periplasmic space, however the data below assumes that the C-terminus faces the periplasm. Compared to the dipeptides binding mode, the tripeptide makes far fewer interactions with PepT_st. The tripeptide sits in an elongated cavity formed by H1 (Tyr-30), H5 (Asn-156), H7 (Glu-299, Glu-300) and H8 (Gln-325, Asn-328). In this binding mode, the extracellular gate is firmly shut. The less compact structure that this conformation forces PepT_st to adopt opens up another smaller hydrophobic pocket to help accommodate the tripeptide. The side chains of Glu-299 and Glu-300 are within hydrogen bond distance to the carbonyl of the C-terminal peptide bond. However, Glu-299 is not a conserved residue. This suggests that different proteins within the POT family will have different residues to help determine their selectivity. (3)

Another conformation change to the open inward conformation is required to release the bound peptide into the cell of the membrane. There is an intracellular gate constructed from conserved side chain interactions between helices H4–H5 and H10–H11. This gate can be opened by the cytoplasmic halves of H7, H10 and H11 swing away from helices H4–H5. Specifically, the gate opens due to bending at Gly-407 and Trp-427 on helices H10 and H11, respectively. Gly-407 and Trp-427 are located at the same point within the H10–H11 helix hairpin, effectively forming a hinge or pivot point, which would control whether the intracellular gate is open or closed between the occluded and inward facing conformation. (2)

Psi-Blast is a program used to find proteins with similar primary structures to a given protein. Psi-Blast assigns an “E value” to subjects that have sequence homology to the given protein. The E value is calculated by looking at the total sequence homology of the protein and assigning gaps. A gap is a section of the subject’s sequence that is not contained in the given protein. Total sequence homology decreases the E value, while gaps increase the E value. An E value of less than .05 is considered significantly comparable. (4)

The Dali Server is a method for finding proteins with similar tertiary structure to a given protein. The Dali Server uses a sum-of-pairs method, which produces a measure of similarity by comparing intramolecular distances. Similarity is measured by a “Z-score”.  Structures with a high Z-score indicate significant similarity. A score over 2 generally indicates similar protein folds. (5)

Expasy is a program that uses a proteins primary structure to identify its isoelectric point and molecular weight. The isoelectric point for the structure of PepTst is 8.96. The molecular weight is 53687.55 amu. (6)

Using Psi-Blast and the Dali Server to compare primary and tertiary structures respectively, the native E. coli YbgH protein (PDB id= 4Q65) was found to be highly similar. Having an E value of 4 x 10^-106, the primary structures are almost identical. A Z-score of 40 indicates that the proteins have highly similar folds. This membrane transporter protein of E. coli functions in almost the same way as PepT_st and the mammalian homologue PepT1. Similarly, it is also predominantly alpha-helical and contains 493 residues. Like PepT_st it also contains two pseudosymmetric domains with a large central cavity formed between the two domains for binding the di- and tripeptides. YbgH also changes conformations like PepT_st from outward-facing, to occluded, to inward-facing. (7,8)