Probing the Putative Active Site of YjdL: An Unusual Proton-Coupled Oligopeptide Transporter from E. coli

Abstract

YjdL from E. coli is an unusual proton-coupled oligopeptide transporter (POT). Unlike prototypical POTs, dipeptides are preferred over tripeptides, in particular dipeptides with a positively charged C-terminal residue. To further understand this difference in peptide specificity, the sequences of YjdL and YdgR, a prototypical E. coli POT, were compared in light of the crystal structure of a POT from Shewanella oneidensis. Several residues found in the putative active site were mutated and the activities of the mutated variants were assessed in terms of substrate uptake assays, and changes in specificity in terms of uptake inhibition. Most strikingly, changing the YjdL specific Asp392 to the conserved Ser in YjdL obliterated the preference for a positively charged C-terminal residue. Based on this unique finding and previously published results indicating that the dipeptide N-terminus may interact with Glu388, a preliminary orientation model of a dipeptide in the YjdL cavity is presented. Single site mutations of particularly Ala281 and Trp278 support the presented orientation. A dipeptide bound in the cavity of YjdL appears to be oriented such that the N-terminal side chain protrudes into a sub pocket that opens towards the extracellular space. The C-terminal side chain faces in the opposite direction into a sub pocket that faces the cytoplasm. These data indicated a stabilizing effect on a bulky N-terminal residue by an Ala281Phe variant and on the dipeptide backbone by Trp278. In the presented orientation model, Tyr25 and Tyr58 both appear to be in proximity of the dipeptide backbone while Lys117 appears to be in proximity of the peptide C-terminus. Mutational studies of these conserved residues highlight their functional importance.

Structural insights into substrate recognition in proton‐dependent oligopeptide transporters

“…Hence, they can interact favourably in both hydrophobic and hydrophilic environments, which could be a key aspect for the broad substrate specificity of POTs. The principle binding mode of the ligand is consistent with the mutagenesis data on PEPT1, the prokaryotic POTs PepT So , PepT St and two transporters from Escherichia coli [9,10,12,13,15,18,19], indicating that these invariant residues have a key role in the recognition of the Nand C-terminus of peptide substrates. For the recognition of a large variety of di-and tripeptides, further interactions could be formed with the invariant backbone CO-and NH-groups of the peptide via the tyrosine cluster consisting of residues 29, 147 and 291.…”

Peptide Selectivity of the Proton-Coupled Oligopeptide Transporter from <b><i>Neisseria meningitidis</i></b>

“…The chain length specificity conforms to prototypical POTs [Jensen et al, 2012b], but surprisingly a strong inability to accommodate the positively charged peptide in the C-terminal position was observed for both di-and tripeptides. Molecular modeling and phylogenetic analysis suggest that a unique hydrophobic patch of residues in the active site may explain this behavior.…”

“…NmPOT was expressed Ala >10 >11.14 >10 >10.14 Ala-Ala 0.39 ± 0.04 2 1.52 ± 0.04 0.14 ± 0.02 0.12 ± 0. with a hexahistidine tag enabling detection by Western blotting ( fig. 1 b), and exhibited expression after 3 h of induction as observed for YjdL and YdgR previously using the same expression system [Jensen et al, 2012a;Jensen et al, 2012b]. A previously documented substrate of several bacterial POTs, β-Ala-Lys-N-7-amino-4 methylcoumarin-3-acetic acid (β-Ala-Lys-AMCA) was used as a reporter substrate and was also tested to measure the specific uptake of the substrate by NmPOT ( fig.…”

“…The expression constructs for NmPOT and YdgR were created as described in Ernst et al [2009] and Jensen et al [2012b].…”

Peptide transporter DtpA has two alternate conformations, one of which is promoted by inhibitor binding

“…The crystal structures of bacterial peptide transporters from Shewanella oneidensis (PepT So and PepT So2 ), Streptococcus thermophilus (PepT St ), and Geobacillus kaustophilus (GkPOT) revealed 14 TMHs (10,(20)(21)(22). In Escherichia coli, four PTR family members have been characterized: dipeptide and tripeptide permease A (DtpA, formerly named YdgR or TppB) (3,4,23), DtpB (formerly YhiP) (3), DtpC (formerly YjdL) (11,(24)(25)(26), and DtpD (formerly YbgH) (27). Among these transporters, DtpA shows peptide selectivity very similar to that of hPEPT1 (3,4).…”