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

Abstract

Peptide transport in living organisms is facilitated by either primary transport, hydrolysis of ATP, or secondary transport, cotransport of protons. In this study, we focused on investigating the ligand specificity of the Neisseria meningitidis proton-coupled oligopeptide transporter (NmPOT). It has been shown that the gene encoding this transporter is upregulated during infection. NmPOT conformed to the typical chain length preference as observed in prototypical transporters of this family. In contrast to prototypical transporters, it was unable to accommodate a positively charged peptide residue at the C-terminus position of the substrate peptide. Sequence analysis of the active site of NmPOT displayed a distinctive aromatic patch, which has not been observed in any other transporters from this family. This aromatic patch may be involved in providing NmPOT with its atypical preferences. This study provides important novel information towards understanding how these transporters recognize their substrates.

Functional Characterization of the Putative POT from Clostridium perfringens

“…The prototypical transporter YdgR was subjected to the same assay for comparative analysis. The YdgR-mediated uptake of β-Ala-Lys-AMCA was not outcompeted by A and AAAA, but its uptake was significantly decreased in the presence of AA and AAA which is in agreement with previous studies (Figure 1) [26,31]. CPEPOT was tested for its ability to recognize and translocate β-Ala-Lys-AMCA and this was compared with E. coli cells expressing an empty vector (pTTQ18) (Figure 1).…”

Multispecific Substrate Recognition in a Proton-Dependent Oligopeptide Transporter

“…Thus, in PepT St [apo] and PepT St [Phe-Ala] where there is either nothing or just a very small residue present in P2, the pocket is narrow, while in PepT St [Ala-Leu] and PepT St [Ala-Phe] where P2 instead harbors a bulky residue, the position of Tyr-68 is shifted to substantially widen it ( Figure 3 D). P1 and P2 are overall less well conserved than the residues interacting with the peptide backbone ( Figures S2 N–S2P), but this is not surprising considering that POTs have broad substrate preferences ( Boggavarapu et al., 2015 , Brandsch et al., 2008 , Chiang et al., 2004 , Ernst et al., 2009 , Fang et al., 2000 , Guettou et al., 2014 , Ito et al., 2013 , Sharma et al., 2016 , Solcan et al., 2012 ). Tyr-68 is, however, very well conserved.…”

“…The substrate range of POTs is generally very wide; for example, human PepT1 and PepT2 can probably transport almost any di- and tripeptide ( Brandsch et al., 2008 ). However, the preferences for different peptide side chains vary significantly among different POTs ( Boggavarapu et al., 2015 , Brandsch et al., 2008 , Chiang et al., 2004 , Ernst et al., 2009 , Fang et al., 2000 , Guettou et al., 2014 , Ito et al., 2013 , Sharma et al., 2016 , Solcan et al., 2012 ), and some strongly prefer dipeptides to tripeptides ( Boggavarapu et al., 2015 , Ernst et al., 2009 , Solcan et al., 2012 ). Structures have been determined for several different bacterial POTs in both apo and substrate-bound forms ( Boggavarapu et al., 2015 , Doki et al., 2013 , Fowler et al., 2015 , Guettou et al., 2013 , Guettou et al., 2014 , Lyons et al., 2014 , Newstead et al., 2011 , Quistgaard et al., 2017 , Solcan et al., 2012 , Zhao et al., 2014 ).…”

Recent advances in understanding proton coupled peptide transport via the POT family

“…The importance of the polar pocket in determining peptide specificity in PepT St was recently demonstrated through computational docking and supported by in vitro transport assays [32]. The importance of specificity pockets would also explain the preferences for different peptides observed between members of the POT family that have been reported [33,34,19,20,23,30].…”