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Selectivity mechanism of a bacterial homolog of the human drug-peptide transporters PepT1 and PepT2

Nature Structural & Molecular Biology volume 21, pages 728–731 (2014)Cite this article

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Abstract

Peptide transporters of the PepT family have key roles in the transport of di- and tripeptides across membranes as well as in the absorption of orally administered drugs in the small intestine. We have determined structures of a PepT transporter from Shewanella oneidensis (PepTSo2) in complex with three different peptides. The peptides bind in a large cavity lined by residues that are highly conserved in human PepT1 and PepT2. The bound peptides adopt extended conformations with their N termini clamped into a conserved polar pocket. A positively charged patch allows differential interactions with the C-terminal carboxylates of di- and tripeptides. Here we identify three pockets for peptide side chain interactions, and our binding studies define differential roles of these pockets for the recognition of different subtypes of peptide side chains.

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Figure 1: Substrate coordination and 2Fo − Fc maps.
Figure 2: PepTSo2 binding site.
Figure 3: PepTSo2 is a peptide transporter exhibiting multisubstrate specificity.

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Acknowledgements

We thank the members of our group for suggestions and comments on the manuscript. This research was performed in P.N.'s research group and was supported by grants to P.N. from the Swedish Research council, the Swedish Cancer Society and the integrated EU project European drug initiative on channels and transporters (EDICT) as well as a Singapore National Research Foundation Competitive Research Program grant (NRF-CRP). We thank Diamond Light Source for access to beamline I03 (proposal mx6603) and BESSY for provision of synchrotron radiation facilities at beamline MX 14.3 in Berlin (proposal 2012_1_111073). We acknowledge the Protein Science Facility at the Karolinska Institutet for providing crystallization infrastructure. The research leading to these results has received further funding to P.N. from the European Community's Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement 783).

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Authors and Affiliations

  1. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

    Fatma Guettou, Esben M Quistgaard, Michael Raba, Per Moberg, Christian Löw & Pär Nordlund

  2. School of Biological Sciences, Nanyang Technological University, Singapore

    Pär Nordlund

  3. Institute of Molecular and Cell Biology, A* STAR, Singapore

    Pär Nordlund

Authors
  1. Fatma Guettou
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  2. Esben M Quistgaard
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  3. Michael Raba
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  4. Per Moberg
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  5. Christian Löw
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Contributions

C.L., F.G., M.R., P.N. and P.M. designed and performed experiments. C.L., E.M.Q., F.G., M.R. and P.N. wrote the manuscript. All authors discussed the results and commented on the manuscript. P.N. supervised the project.

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Correspondence to Pär Nordlund.

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Integrated supplementary information

Supplementary Figure 1 Substrate coordination and Fo – Fc omit maps.

(A-C) PepTSo2 is shown as a cartoon representation with the N- and C-terminal halves colored in pink and blue respectively. The omit densities of the substrates are shown in green at a contour level of 3 σ. Important residues in the binding site are labeled and shown as sticks. The peptides AAA, AY(Br) and AY(Br)A are shown as sticks and colored green, magenta and blue respectively. Black dashes show potential hydrogen interaction between the substrate termini and the binding site residues. (D) PepTSo2 is presented as a grey colored cartoon. Important residues in the binding site are shown as blue sticks. One bromide anomalous density of the AY(Br)A substrate is presented as a green density contoured at 3 σ. The second bromide shows a strong negative Fo-Fc density countered here at 4 σ.

Supplementary Figure 2 Stereo view and samples of electron density maps for the AAA-bound protein.

(A) Stereo view of transmembrane helices 1, 5 and 6. The helices are labeled at the top of the panel and they are shown as sticks. 2Fo-Fc electron density map is contoured at 1.0 σ. (B) 2Fo-Fc electron density maps of helix 1 to 6 contoured at 1.0 σ, green density corresponds to the actual helix.

Supplementary Figure 3 Overall structure and conservation of PepTSo2 binding site.

(A) Overview of the inward open conformation of PepTSo2 viewed from the plane of the membrane. The N- and C-terminal subdomains are colored pink and blue respectively. The protein is in complex with the tripeptide AY(Br)Y, shown as blue sticks with a black Br atom. The binding site residues are colored according to conservation. Red residues are identical while the yellow residues are similar to the human PepT homologues. Corresponding residues are presented in brackets. For creating the figure, alignment from 18 was used. (B) A close-up view of binding residues shown from the periplasmic side. The extended shape of the AY(Br)A peptide is visible, the N-terminus is coordinated by the conserved polar pocket (N151, E329 and E402). The C-terminus is coordinated by K121. The tyrosine side chain is clamped into the conserved hydrophobic pocket formed by F287, F288 and Y291.

Supplementary Figure 4 Superimposition of three peptide-bound structures including the antibacterial compound alafosfalin (4LEP)-bound structure.

The four known structures PepTSo2 are superimposed and shown in cartoon (protein) and sticks representations (peptide substrates). The structures are colored green (AAA), magenta (AY(Br)), blue (AY(Br)A) or yellow (alafosfalin). Average RMSD is 0.3 Å over 3000 atoms. There are minor variations in the C-terminal parts of H10 and H11.

Supplementary Figure 5 Wall-eyed stereo image of binding site and thermal aggregation assay versus uptake assay in proteoliposomes.

(A) The coordination of AY(Br)A is presented in a surface model where the binding site is divided into three pockets. Pocket 1 is colored in green, pocket 2 in yellow and pocket 3 in magenta. Important residues for substrate coordination are shown as sticks. (B) Peptide induced stabilization of PepTSo2. Thermal stability of PepTSo2 in presence of various peptides (5 mM) plotted as TΔ°C relative to the thermal stability of apo PepTSo2. The melting temperatures were derived from stargazer static light scattering unfolding curves. (C) Inhibition of [3H]-L-AlaAla uptake in the presence of various tripeptides. Error bars represent the standard deviation of triplicate measurements.

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Supplementary Figures 1–5 and Supplementary Tables 1 and 2 (PDF 2681 kb)

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Guettou, F., Quistgaard, E., Raba, M. et al. Selectivity mechanism of a bacterial homolog of the human drug-peptide transporters PepT1 and PepT2. Nat Struct Mol Biol 21, 728–731 (2014). https://doi.org/10.1038/nsmb.2860

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