The Binding Specificity of OppA Determines the Selectivity of the Oligopeptide ATP-binding Cassette Transporter

Abstract

The purification and functional reconstitution of a five-component oligopeptide ATP-binding cassette transporter with a remarkably wide substrate specificity are described. High-affinity peptide uptake was dependent on liganded substrate-binding protein OppA, which interacts with the translocator OppBCDF with higher affinity than unliganded OppA. Transport screening with combinatorial peptide libraries revealed that (i) the Opp transporter is not selective with respect to amino acid side chains of the transported peptides; (ii) any peptide that can bind to OppA is transported via Opp, including very long peptides up to 35 residues long; and (iii) the binding specificity of OppA largely determines the overall transport selectivity.

The specificity of oligopeptide transport by Streptococcus thermophilus resembles that of Lactococcus lactis and not that of pathogenic streptococci

“…However, the role of OppA in the determination of the specificity of peptide transport is clearly established (Charbonnel et al, 2003;Doeven et al, 2004). A (Charbonnel et al, 2003), although this point is controversial (Doeven et al, 2004). Extensive analysis of specificities of both peptide binding and peptide transport displayed by single and multiple negative mutants for the binding proteins is required to solve this intriguing question.…”

“…From these data, it is clear that peptide transport specificity cannot be deduced from the overall sequence similarity of peptidebinding proteins. However, the role of OppA in the determination of the specificity of peptide transport is clearly established (Charbonnel et al, 2003;Doeven et al, 2004). A (Charbonnel et al, 2003), although this point is controversial (Doeven et al, 2004).…”

“…Three positively charged residues (R 439 , H 397 and K 331 ) interact with the C-terminal carboxyl group of the liganded tri-, tetraand pentapeptide, respectively, whereas a negatively charged residue (D 445 ) forms a salt bridge with the N-terminal aamino group of the bound peptide (Tame et al, 1994(Tame et al, , 1995. The Opp system of L. lactis MG1363 transports oligopeptides containing from four to 35 amino acids (Doeven et al, 2004), with preference for hydrophobic, cationic peptides (Juillard et al, 1998). The Opt system of L. lactis transports peptides of up to nine amino acids at least, depending on their amino acid sequence (Lamarque et al, 2004).…”

“…Intact cells of L. lactis are able to transport peptides containing up to at least 18 amino acid residues (Detmers et al, 1998). Moreover, the Opp system functionally reconstituted in OppA artificial membranes has the (up to now) unique capacity to bind and transport peptides containing up to at least 35 amino acid residues (Doeven et al, 2004). Recently, a second oligopeptide transport system, Opt, has been characterized in L. lactis IL1403 (Lamarque et al, 2004).…”

Functional reconstitution and osmoregulatory properties of the ProU ABC transporter fromEscherichia coli

“…Residual detergent was removed by incubation of the (proteo) liposomes with Biobeads. The ATP regenerating system (ARS), composed of 10 mM Mg-ATP, 24 mM phosphocreatine plus 2.4 mg/ml creatine kinase in 50 mM KPI, pH 7.0, was included in the proteoliposomes by two cycles of freezing in liquid nitrogen and thawing at room temperature (Doeven et al 2004, Geertsma et al 2008a (Figure 1B). …”