Frank C. Lanfermeijer

Peptides and ATP binding cassette peptide transporters.

In this review our knowledge of ATP binding cassette (ABC) transporters specific for peptides is discussed. Besides serving a role in nutrition of the cell, the systems participate in various signaling processes that allow (micro)organisms to monitor the local environment. In bacteria, these include regulation of gene expression, competence development, sporulation, DNA transfer by conjugation, chemotaxis, and virulence development, and the role of ABC transporters in each of these processes is discussed. Particular attention is paid to the specificity determinants of peptide receptors and transporters in relation to their structure and to the mechanisms of peptide binding.

On the binding mechanism of the peptide receptor of the oligopeptide transport system of Lactococcus lactis

Lactococcus lactis degrades exogenous proteins such as β‐casein to peptides of 4–30 amino acids, and uses these as nitrogen sources. The binding protein or receptor (OppALl) of the oligopeptide transport system (Opp) of L.lactis has the unique capacity to bind peptides from five up to at least 20 residues. To study the binding mechanism of OppALl, nonameric peptides were used in which the cysteine at position 1, 3, 4, 5, 6, 7 or 9 was selectively labeled with either bulky and non‐fluorescent or bulky and fluorescent groups. Also, nonameric peptides with a non‐natural residue, azatryptophan, at positions 3 or 7 were used. The fluorescence of azatryptophan reports on the polarity of the environment. The studies indicate that the binding protein encloses the first six amino acids of the peptide, whereas the remaining residues stick out and interact with the surface of the binding protein. The peptide binding mechanism of OppALl is discussed in relation to known three‐dimensional structures of members of this class of proteins, and an adaptation of the general binding mechanism is proposed.

Specificity mutants of the binding protein of the oligopeptide transport system of Lactococcus lactis

The kinetic properties of wild-type and mutant oligopeptide binding proteins of Lactococcus lactis were determined. To observe the properties of the mutant proteins in vivo, the oppA gene was deleted from the chromosome of L. lactis to produce a strain that was totally defective in oligopeptide transport. Amplified expression of the oppA gene resulted in an 8- to 12-fold increase in OppA protein relative to the wild-type level. The amplified expression was paralleled by increased bradykinin binding activity, but had relatively little effect on the overall transport of bradykinin via Opp. Several site-directed mutants were constructed on the basis of a comparison of the primary sequences of OppA from Salmonella enterica serovar Typhimurium and L. lactis, taking into account the known structure of the serovar Typhimurium protein. Putative peptide binding-site residues were mutated. All the mutant OppA proteins exhibited a decreased binding affinity for the high-affinity peptide bradykinin. Except for OppA(D471R), the mutant OppA proteins displayed highly defective bradykinin uptake, whereas the transport of the low-affinity substrate KYGK was barely affected. Cells expressing OppA(D471R) had a similar K(m) for transport, whereas the V(max) was increased more than twofold as compared to the wild-type protein. The data are discussed in the light of a kinetic model and imply that the rate of transport is determined to a large extent by the donation of the peptide from the OppA protein to the translocator complex.

Genetic and functional characterization of dpp genes encoding a dipeptide transport system in Lactococcus lactis

Abstract. The genes encoding a binding-protein-dependent ABC transporter for dipeptides (Dpp) were identified in Lactococcus lactis subsp. cremoris MG1363. Two (dppA and dppP) of the six ORFs (dppAdppPBCDF) encode proteins that are homologous to peptide- and pheromone-binding proteins. The dppP gene contains a chain-terminating nonsense mutation and a frame-shift that may impair its function. The functionality of the dpp genes was proven by the construction of disruption mutants via homologous recombination. The expression of DppA and various other components of the proteolytic system was studied in synthetic and peptide-rich media and by using isogenic peptide-transport mutants that are defective in one or more systems (Opp, DtpT, and/or Dpp). In peptide-rich medium, DppA was maximally expressed in mutants lacking Opp and DtpT. DppA expression also depended on the growth phase and was repressed by tri-leucine and tri-valine. The effect of tri-leucine on DppA expression was abolished when leucine was present in the medium. Importantly, the Dpp system also regulated the expression of other components ofthe proteolytic system. This regulation was achieved via the internalization of di-valine, which caused a 30–50% inhibition in the expression of the proteinase PrtP and the peptidases PepN and PepC. Similar to the regulation of DppA, the repressing effect was no longer observed when high concentrations of valine were present. The intricate regulation of the components of the proteolytic system by peptides and amino acids is discussed in the light of the new and published data.

Manipulation of activity and orientation of membrane-reconstituted di-tripeptide transport protein DtpT of Lactococcus lactis.

The di-tripeptide transport system (DtpT) of Lactococcus lactis was purified to apparent homogeneity by pre-extraction of crude membrane vesicles with octaethylene glycol monodecyl ether (C10E8), followed by solubilization with n-dodecyl-beta-D-maltoside (DDM) and chromatography on a Ni-NTA resin. The DtpT protein was reconstituted into detergent-destabilized preformed liposomes prepared from E. coli phospholipid/phosphatidylcholine. A variety of detergents were tested for their ability to mediate the membrane reconstitution of DtpT and their effectiveness to yield proteoliposomes with a high transport activity. The highest activities were obtained with TX100, C12E8 and DM, whereas DDM yielded relatively poor activities, in particular when this detergent was used at concentrations beyond the onset of solubilization of the preformed liposomes. Parallel with the low activity, significant losses of lipid were observed when the reconstitution was performed at high DDM concentrations. This explained at least part of the reduced transport activity as the DtpT protein was highly dependent on the final lipid-to-protein ratios in the proteoliposomes. Consistent with the difference in mechanism of DDM- and TX100-mediated membrane protein reconstitution, the orientation of the DtpT protein in the membrane was random with DDM and inside-in when TX100 was used. The methodology to determine the orientation of membrane-reconstituted proteins from the accessibility of cysteines for thiol-specific reagents is critically evaluated.