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.
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).
This study describes the characterization of the riboflavin transport protein RibU in the lactic acid bacterium Lactococcus lactis subsp. cremoris NZ9000. RibU is predicted to contain five membrane-spanning segments and is a member of a novel transport protein family, not described in the Transport Classification Database. Transcriptional analysis revealed that ribU transcription is downregulated in response to riboflavin and flavin mononucleotide (FMN), presumably by means of the structurally conserved RFN (riboflavin) element located between the transcription start site and the start codon. An L. lactis strain carrying a mutated ribU gene exhibits altered transcriptional control of the riboflavin biosynthesis operon ribGBAH in response to riboflavin and FMN and does not consume riboflavin from its growth medium. Furthermore, it was shown that radiolabeled riboflavin is not taken up by the ribU mutant strain, in contrast to the wild-type strain, directly demonstrating the involvement of RibU in riboflavin uptake. FMN and the toxic riboflavin analogue roseoflavin were shown to inhibit riboflavin uptake and are likely to be RibU substrates. FMN transport by RibU is consistent with the observed transcriptional regulation of the ribGBAH operon by external FMN. The presented transport data are consistent with a uniport mechanism for riboflavin translocation and provide the first detailed molecular and functional analysis of a bacterial protein involved in riboflavin transport.
Improved in-gel approaches to generate peptide maps of integral membrane proteins with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry van Montfort, B.A.; Canas, B.; Duurkens, R.H.T.; Godovac-Zimmermann, J.; Robillard, G.T. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. This paper reports studies of in-gel digestion procedures to generate MALDI-MS peptide maps of integral membrane proteins. The methods were developed for the membrane domain of the mannitol permease of E. coli. In-gel digestion of this domain with trypsin, followed by extraction of the peptides from the gel, yields only 44% sequence coverage. Since lysines and arginines are seldomly found in the membrane-spanning regions, complete tryptic cleavage will generate large hydrophobic fragments, many of which are poorly soluble and most likely contribute to the low sequence coverage. Addition of the detergent octyl-b-glucopyranoside (OBG), at 0.1% concentration, to the extraction solvent increases the total number of peptides detected to at least 85% of the total protein sequence. OBG facilitates the recovery of hydrophobic peptides when they are SpeedVac dried during the extraction procedure. Many of the newly recovered peptides are partial cleavage products. This seems to be advantageous since it generates hydrophobic fragments with a hydrophilic solubilizing part. In-gel CNBr cleavage resulted in 5-10-fold more intense spectra, 83% sequence coverage, fully cleaved fragments and no effect of OBG. In contrast to tryptic cleavage sites, the CNBr cleavage sites are found in transmembrane segments; cleavage at these sites generates smaller hydrophobic fragments, which are more soluble and do not need OBG. With the results of both cleavages, a complete sequence coverage of the membrane domain of the mannitol permease of E. coli is obtained without the necessity of using HPLC separation. The protocols were applied to two other integral membrane proteins, which confirmed the general applicability of CNBr cleavage and the observed effects of OBG in peptide recovery after tryptic digestion. Copyright 2002 John Wiley & Sons, Ltd. KEYWORDS: membrane protein; peptide mapping; octyl glucoside; matrix-assisted laser desorption/ionization; sodium dodecyl sulfate polyacrylamide gel electrophoresis Abbreviations: MALDI, matrix-assisted laser desorption/ionization; TOF, time-of-flight; MS, mass spectrometry; OBG, octyl-ˇ-glucopyranoside; SDS, sodium dodecyl sulfate; PAGE, polyacrylamidegel electrophoresis; CNBr, cyanogen bromide; HPLC, high-performance liquid chromatography; C domain, the membrane domain of the mannitol transporter without the soluble A and B domain; MscL, mechanosensitive channel of l...
D-Mannitol is taken up by Bacillus stearothermophilusand phosphorylated via a phosphoenolpyruvatedependent phosphotransferase system (PTS). Transcription of the genes involved in mannitol uptake in this bacterium is regulated by the transcriptional regulator MtlR, a DNA-binding protein whose affinity for DNA is controlled by phosphorylation by the PTS proteins HPr and IICB mtl . The mutational and biochemical studies presented in this report reveal that two domains of MtlR, PTS regulation domain (PRD)-I and PRD-II, are phosphorylated by HPr, whereas a third IIA-like domain is phosphorylated by IICB mtl . An involvement of PRD-I and the IIA-like domain in a decrease in affinity of MtlR for DNA and of PRD-II in an increase in affinity is demonstrated by DNA footprint experiments using MtlR mutants. Since both PRD-I and PRD-II are phosphorylated by HPr, PRD-I needs to be dephosphorylated by IICB mtl and mannitol to obtain maximal affinity for DNA. This implies that a phosphoryl group can be transferred from HPr to IICB mtl via MtlR. Indeed, this transfer could be demonstrated by the phosphoenolpyruvate-dependent formation of [ 3 H]mannitol phosphate in the absence of IIA mtl . Phosphoryl transfer experiments using MtlR mutants revealed that PRD-I and PRD-II are dephosphorylated via the IIA-like domain. Complementation experiments using two mutants with no or low phosphoryl transfer activity showed that phosphoryl transfer between MtlR molecules is possible, indicating that MtlRMtlR interactions take place. Phosphorylation of the same site by HPr and dephosphorylation by IICB mtl have not been described before; they could also play a role in other PRD-containing proteins.Many bacteria transport D-mannitol and other carbohydrates via a phosphoenolpyruvate-dependent phosphotransferase system (PTS) 1 (1-3). Recently, the mannitol operon of Bacillus stearothermophilus was cloned (4) and shown to consist of four genes, mtlA, mtlR, mtlF, and mtlD, coding for the mannitol transporter IICB mtl , the transcriptional regulator MtlR, the phosphotransferase IIA mtl , and mannitol-1-phosphate dehydrogenase, respectively. Analysis of the mannitol promoter revealed a catabolite response element overlapping the mannitol promoter, indicating that this operon is sensitive to catabolite repression. When favorable catabolites like glucose are utilized, HPr is phosphorylated by a kinase on a specific serine (5) that forms a complex with the CcpA repressor. Binding of this complex to catabolite response element sites located in or near the promoter regions of catabolic operons will prevent expression of these operons (6). In addition to catabolite repression, the expression of the mannitol operon is probably also regulated by the mannitol regulator MtlR (7). Domains in this protein show similarity to domains of two types of transcriptional regulators: DNA-binding proteins and anti-terminators. A helix-turn-helix motif is situated at the N terminus that is similar to those of DNA-binding transcriptional regulators of the DeoR family. The ...
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