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Oligopeptide-binding protein A (OppA) from Lactococcus lactis binds peptides of an exceptionally wide range of lengths (4-35 residues), with no apparent sequence preference. Here, we present the crystal structures of OppA in the open-and closed-liganded conformations. The structures directly explain the protein's phenomenal promiscuity. A huge cavity allows binding of very long peptides, and a lack of constraints for the position of the N and C termini of the ligand is compatible with binding of peptides with varying lengths. Unexpectedly, the peptide's amino-acid composition (but not the exact sequence) appears to have a function in selection, with a preference for proline-rich peptides containing at least one isoleucine. These properties can be related to the physiology of the organism: L. lactis is auxotrophic for branched chain amino acids and favours proline-rich caseins as a source of amino acids. We propose a new mechanism for peptide selection based on amino-acid composition rather than sequence.

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Energy coupling factor (ECF) proteins are ATP-binding cassette transporters involved in the import of micronutrients in prokaryotes. They consist of two nucleotide-binding subunits and the integral membrane subunit EcfT, which together form the ECF module and a second integral membrane subunit that captures the substrate (the S component). Different S components, unrelated in sequence and specific for different ligands, can interact with the same ECF module. Here, we present a high-resolution crystal structure at 2.1 Å of the biotin-specific S component BioY from Lactococcus lactis. BioY shares only 16% sequence identity with the thiaminspecific S component ThiT from the same organism, of which we recently solved a crystal structure. Consistent with the lack of sequence similarity, BioY and ThiT display large structural differences (rmsd ¼ 5.1 Å), but the divergence is not equally distributed over the molecules: The S components contain a structurally conserved N-terminal domain that is involved in the interaction with the ECF module and a highly divergent C-terminal domain that binds the substrate. The domain structure explains how the S components with large overall structural differences can interact with the same ECF module while at the same time specifically bind very different substrates with subnanomolar affinity. Solitary BioY (in the absence of the ECF module) is monomeric in detergent solution and binds D-biotin with a high affinity but does not transport the substrate across the membrane. membrane transport | biotin transport | vitamine uptake E nergy coupling factor (ECF) proteins are an abundant class of ATP-binding cassette (ABC) transporters involved in the import of vitamins and transition metal ions in prokaryotes (1-4). Like all ABC transporters, ECF transporters consist of two cytosolic nucleotide-binding domains (NBDs), which are associated with integral membrane subunits that form the translocation pore. In ECF transporters the two NBDs (EcfA and EcfA', which may be identical or homologous) and a single membrane subunit (EcfT) form a so-called energizing or ECF module. A second integral membrane protein (the S component) binds the substrate and forms a complex with the ECF module to create a functional transporter. This organization is typical for ECF transporters (3-5), because other ABC importers utilize a soluble substrate-binding protein to capture ligands (6, 7). In many ECF transporters multiple S components (specific for different substrates) can interact with the same energizing module (3, 5). Strikingly, S components from a single organism, which interact with the same ECF module, are generally not homologous at the sequence level.To gain insight in the characteristic modularity of ECF transporters, one needs to compare crystal structures of different S components that interact with the same ECF module (i.e., S components from a single organism). Crystal structures of the S components ThiT from Lactococcus lactis (thiamin-specific) and RibU from Staphylococcus aureus (riboflavin-spe...

The first biochemical and spectroscopic characterization of a purified membrane transporter for riboflavin (vitamin B 2 ) is presented. The riboflavin transporter RibU from the bacterium Lactococcus lactis was overexpressed, solubilized, and purified. The purified transporter was bright yellow when the cells had been cultured in rich medium. We used a detergent-compatible matrix-assisted laser desorption ionization time-of-flight mass spectrometry method (Cadene, M., and Chait, B. T. Riboflavin (vitamin B 2 ) is a water-soluble vitamin that is converted by flavokinases and FAD synthases to the cofactors FMN and FAD. These cofactors are indispensable for all living organisms, and they are involved in a wide range of reactions (1).