Journal Article

PUBDB-2022-05004

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Cryo-EM Structure of an Atypical Proton-Coupled Peptide Transporter: Di- and Tripeptide Permease C

Killer, M. ; Finocchio, G. ; Mertens, H. D. T.EMBL* ; Svergun, D. I.EMBL* ; Pardon, E. ; Steyaert, J. ; Loew, C. (Corresponding author)CSSB*

2022
Frontiers Lausanne

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Please use a persistent id in citations: doi:10.3389/fmolb.2022.917725  doi:10.3204/PUBDB-2022-05004

Abstract: Proton-coupled Oligopeptide Transporters (POTs) of the Major Facilitator Superfamily (MFS) mediate the uptake of short di- and tripeptides in all phyla of life. POTs are thought to constitute the most promiscuous class of MFS transporters, with the potential to transport more than 8400 unique substrates. Over the past two decades, transport assays and biophysical studies have shown that various orthologues and paralogues display differences in substrate selectivity. The E. coli genome codes for four different POTs, known as Di- and tripeptide permeases A-D (DtpA-D). DtpC was shown previously to favor positively charged peptides as substrates. In this study, we describe, how we determined the structure of the 53 kDa DtpC by cryogenic electron microscopy (cryo-EM), and provide structural insights into the ligand specificity of this atypical POT. We collected and analyzed data on the transporter fused to split superfolder GFP (split sfGFP), in complex with a 52 kDa Pro-macrobody and with a 13 kDa nanobody. The latter sample was more stable, rigid and a significant fraction dimeric, allowing us to reconstruct a 3D volume of DtpC at a resolution of 2.7 Å. This work provides a molecular explanation for the selectivity of DtpC, and highlights the value of small and rigid fiducial markers such as nanobodies for structure determination of low molecular weight integral membrane proteins lacking soluble domains.

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Contributing Institute(s):

1. EMBL-User (EMBL-User)
2. EMBL (EMBL)
3. CSSB-EMBL-CL (CSSB-EMBL-CL)

Research Program(s):

1. 6G3 - PETRA III (DESY) (POF4-6G3) (POF4-6G3)

Experiment(s):

1. PETRA Beamline P12 (PETRA III)
2. PETRA Beamline P13 (PETRA III)
3. PETRA Beamline P14 (PETRA III)

Appears in the scientific report 2022

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