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We present the crystal structure of the pheromone receptor protein PrgZ from Enterococcus faecalis in complex with the heptapeptide cCF10 (LVTLVFV), which is used in signaling between conjugative recipient and donor cells. Comparison of PrgZ with homologous oligopeptide-binding proteins (AppA and OppA) explains the high specificity of PrgZ for hydrophobic heptapeptides versus the promiscuity of peptide binding in the homologous proteins.

Lactococcal oligopeptide-binding protein A (OppA) binds peptides with widely varied lengths and sequences. We previously hypothesized that a hydrophobic pocket in OppA preferentially binds a hydrophobic peptide side chain and thus determines its binding register. Two crystal structures of OppA with different nonapeptides now indeed show binding in different registers.

Substrate-binding proteins (SBP) are associated with a wide variety of protein complexes. The proteins are part of ATP-binding cassette transporters for substrate uptake, ion gradient driven transporters, DNA-binding proteins, as well as channels and receptors from both pro- and eukaryotes. A wealth of structural and functional data is available on SBPs, with over 120 unique entries in the Protein Data Bank (PDB). Over a decade ago these proteins were divided into three structural classes, but based on the currently available wealth of structural data, we propose a new classification into six clusters, based on features of their three-dimensional structure., (Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

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.

Lactococcus lactis is a promising host for (membrane) protein overproduction. Here, we describe a protocol for incorporation of selenomethionine (SeMet) into proteins expressed in L. lactis. Incorporation efficiencies of SeMet in the membrane protein complex OpuA (an ABC transporter) and the soluble protein OppA, both from L. lactis, were monitored by mass spectrometry. Both proteins incorporated SeMet with high efficiencies (>90%), which greatly extends the usefulness of the expression host L. lactis for X-ray crystallography purposes. The crystal structure of ligand-free OppA was determined at 2.4 A resolution by a semiautomatic approach using selenium single-wavelength anomalous diffraction phasing.

The oligopeptide transporter Opp is a five-component ABC uptake system. The extracytoplasmic lipid-anchored substrate-binding protein (or receptor) OppA delivers peptides to an integral membrane complex OppBCDF (or translocator), where, on ATP binding and hydrolysis, translocation across the membrane takes place. OppA and OppBCDF were labeled with fluorescent probes, reconstituted into giant unilamellar vesicles, and the receptor-translocator interactions were investigated by fluorescence correlation spectroscopy. Lateral mobility of OppA was reduced on incorporation of OppBCDF into giant unilamellar vesicles, and decreased even further on the addition of peptide. Fluorescence cross-correlation measurements revealed that OppBCDF distinguished liganded from unliganded OppA, binding only the former. Addition of ATP or its nonhydrolyzable analog AMP-PNP resulted in release of OppA from OppBCDF. In vanadate-trapped "transition state" conditions, OppA also was not bound by OppBCDF. A model is presented in which ATP-binding to OppDF results in donation of peptide to OppBC and simultaneous release of OppA. ATP-hydrolysis would complete the peptide translocation and reset the transporter for another catalytic cycle. Implications in terms of a general transport mechanism for ABC importers and exporters are discussed.

In previous studies, it has been shown that inactivation of opp or even oppA abolishes the capacity of Lactococcus lactis to utilize oligopeptides. We now show that the opp operon has been duplicated in L. lactis MG1363. The nucleotide sequence of the oppA and oppC homologues (appA and appC) and most of the oppB homologue (appB) indicate that the corresponding protein sequences are 83%, 92% and 91% identical, respectively. Inactivation of appA, via homologous recombination, as well as complementation studies were carried out to determine the possible function of appA in peptide utilization. As anticipated from studies with an oppA knock-out, peptide utilization was not impaired in an appA disruption mutant. Importantly, AppA expressed from a plasmid could restore the ability of oppA deletion mutants to utilize Leu-enkephalin, albeit with a lower efficiency than OppA. The differences in the ability to utilize this pentapeptide were not due to differences in expression levels but most likely reflect a different catalytic efficiency in oligopeptide utilization when AppA is used as ligand receptor.

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 genome sequence of Lactococcus lactis IL1403 revealed the presence of a putative peptide-binding protein-dependent ABC-transporter (Dpp). The genes for two peptide-binding proteins (dppA and dppP) precede the membrane components, which include two transmembrane protein genes (dppB and dppC) and two ATP-binding protein genes (dppD and dppF). In this work, the gene specifying the second peptide-binding protein (DppP) was cloned under the control of the nisin promoter. The protein fused to a carboxyl-terminal histidine tag (DppP-His(6)) was purified and its binding properties were determined by monitoring the intrinsic fluorescence changes observed upon ligand binding. The major features of peptide binding to DppP-His(6) include: (i) a requirement for a free N-terminal alpha-amino group in the ligand; (ii) a high affinity for di-, tri- and tetra-peptides; (iii) affinity constants for peptide binding independent of pH; and (iv) a high affinity for D-isomer-containing peptides. Remarkably, the features (ii), (iii) and (iv) differ from those previously reported for DppA-His(6), suggesting that DppP-His(6) is a more versatile peptide-binding protein that could have additional functions.

For most metabolic pathways, the uptake of the substrate into the cell represents the first step. This transport reaction can exert a large control on the flux through the pathway, in particular when the substrate concentration becomes limiting. Besides serving a role in the uptake of nutrients and the excretion of metabolic (end)products or drugs, transport systems can have one or more other functions in the physiology of the cell. Two of these functions, control of carbohydrate utilization and regulation of cell volume, have been well established in lactic acid bacteria (LAB). The first example concerns the phosphoenolpyruvate-dependent phosphotransferase system (PTS), which serves a role in the transport of sugars into the cell but also regulates the activity of metabolic pathways, either through regulation of transcription and/or (in)activation of transporters and key enzymes already present. The regulation by the PTS results in a hierarchy in the utilization of sugars and/or adjustment of the first step(s) of a metabolic pathway to the metabolic capacity of the cell and the availability of a particular substrate. The second example relates to the activation of transporters (and mechanosensitive channels), which represents the first mechanism of defence against osmotic stress. The activation by osmotic-upshift of the ATP-binding Cassette (ABC) transporter OpuA from Lactococcus lactis is compared with the activation by osmotic-downshift of mechanosensitive channels. The mechanosensitive channels have been best studied in organisms other than LAB, but the presence of similar systems in LAB, and their conservation of structure, suggest that the postulated functions and mechanisms generally hold.

ATP-binding cassette (ABC) transporters use ATP to drive solute transport across biological membranes. Members of this superfamily have crucial roles in cell physiology, and some of the transporters are linked to severe diseases. However, understanding of the transport mechanism, especially of human ABC exporters, is scarce. We reconstituted the human lysosomal polypeptide ABC transporter TAPL, expressed in Pichia pastoris, into lipid vesicles (liposomes) and performed explicit transport measurements. We analyzed solute transport at the single liposome level by monitoring the coincident fluorescence of solutes and proteoliposomes in the focal volume of a confocal microscope. We determined a turnover number of eight peptides per minute, which is two orders of magnitude higher than previously estimated from macroscopic measurements. Moreover, we show that TAPL translocates peptides against a large concentration gradient. Maximal filling is not limited by an electrochemical gradient but by trans-inhibition. Countertransport and reversibility studies demonstrate that peptide translocation is a strictly unidirectional process. Altogether, these data are included in a refined model of solute transport by ABC exporters. [ABSTRACT FROM AUTHOR]

Dimerization and inactivation of ribosomes in Escherichia coli is a two-step process that involves the binding of ribosome modulation factor ( RMF) and hibernation promotion factor ( HPF). Lactococcus lactis MG1363 expresses a protein, YfiALl, which associates with ribosomes in the stationary phase of growth and is responsible for dimerization of ribosomes. We show that full-length YfiALl is necessary and sufficient for ribosome dimerization in L. lactis but also functions heterologously in vitro with E. coli ribosomes. Deletion of the yfiA gene has no effect on the growth rate but diminishes the survival of L. lactis under energy-starving conditions. The N-terminal domain of YfiALl is homologous to HPF from E. coli, whereas the C-terminal domain has no counterpart in E. coli. By assembling ribosome dimers in vitro, we could dissect the roles of the N- and C-terminal domains of YfiALl. It is concluded that the dimerization and inactivation of ribosomes in L. lactis and E. coli differ in several cellular and molecular aspects. In addition, two-dimensional maps of dimeric ribosomes from L. lactis obtained by single particle electron microscopy show a marked structural difference in monomer association in comparison to the ribosome dimers in E. coli. [ABSTRACT FROM AUTHOR]

Substrate-binding protein-dependent secondary transporters are widespread in prokaryotes and are represented most frequently by members of the tripartite ATP-independent periplasmic (TRAP) transporter family. Here, we report the membrane reconstitution of a TRAP transporter, the sialic acid-specific SiaPQM system from Haemophllus influenzae, and elucidate its mechanism of energy coupling. Uptake of sialic acid via membrane-reconstituted SiaQM depends on the presence of the sialic acid-binding protein, SiaP, and is driven by the electrochemical sodium gradient. The interaction between SiaP and SiaQM is specific as transport is not reconstituted using the orthologous sialic acid-binding protein VC1779. Importantly, the binding protein also confers directionality on the transporter, and reversal of sialic acid transport from import to export is only possible in the presence of an excess of unliganded SiaP. [ABSTRACT FROM AUTHOR]

The Major Facilitator Superfamily lactose transport protein (LacS) undergoes reversible self-association in the detergent-sotubilized state, and is present in the membrane as a dimer. We determined the functional unit for proton motive force (Δp)-driven lactose uptake and tactose/methyl-β-D-galactopyranoside equilibrium exchange in a proteoliposomat system in which a single cysteine mutant, LacS-C67, defective in Δp-driven uptake, was co-reconstituted with fully functional cysteine-less protein, LacS-cl. From the quadratic relationship between the uptake activity and the ratio of LacS-C67/LacS-cl, we conclude that the dimeric state of LacS is required for Δp-driven uptake. N-ethylmaleimide (NEM) treatment of proteotiposomes abolished the LacS-C67 exchange activity but left the LacS-cl unaffected. After NEM treatment, the exchange activity decreased linearly with increasing ratios of LacS-C67/LacS-cl, suggesting that the monomeric state of LacS is sufficient for this mode of transport. We propose that the two subunits of LacS are functionally coupled in the step associated with conformational reorientation of the empty binding site, a step unique for Δp-driven uptake. [ABSTRACT FROM AUTHOR]

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 (OppALI) 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 OppALI, 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 flint file 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 OppALI 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. [ABSTRACT FROM AUTHOR]

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 (C[sub 10]E[sub 8]), 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 TX[sub 100], C[sub 12]E[sub 8] 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 TX[sub 100]-mediated membrane protein reconstitution, the orientation of the DtpT protein in the membrane was random with DDM and inside-in when T[sub 100] was used. The methodology to determine the orientation of membrane-reconstituted proteins from the accessibility of cysteines for thiol-specific reagents is critically evaluated. [ABSTRACT FROM AUTHOR]

Evidence is presented in this report for the presence of two sets of dithiols associated with proline transport activity in Escherichia coli. One set is located at the outer surface, the other at the inner surface of the cytoplasmic membrane. Treatment of right-side-out membrane vesicles from E. coli ML 308–225 with the membrane-impermeable oxidant ferricyanide resulted in inhibition of L-proline uptake without having significant effect on the magnitude of the Δ&mu¯H+. Subsequent addition of reducing agents restored proline transport activity. The membrane-impermeable SH-reagent glutathione hexane maleimide inhibited proline transport in right-side-out membrane vesicles irreversibly. Pretreatment of the vesicles with ferricyanide protected the carrier against inactivation by glutathione hexane maleimide. Electron transfer in the respiratory chain of right-side-out vesicles led to the generation of a Δμ¯H+, interior negative and alkaline, and the conversion of a disulphide to a dithiol in the proline carrier as is shown by the increased inhibition of proline transport by the membrane impermeable dithiol reagent 4-(2-arsonophenyl)azo-3-hydroxy-2,7-naphthalene disulphonic acid (thorin). The inhibition exerted by thorin was completely reversed by dithiothreitol. Pretreatment of the vesicles with thorin protected against glulathione hexane maleimide inhibition, indicating that both reagents react with the same group. Treatment of inside-out membrane vesicles with ferricyanide inactivated the proline transport system reversibly. The oxidizing effect of ferricyanide in inside-out vesicles resulted in protection against inhibition by glutathione hexane muleimide. Imposition in these vesicles of a Δμ¯H+) interior positive and acid, also protected the proline carrier against glutathione hexane maleimide inactivation, indicating that a dithiol is converted to udisulphide upon energization. [ABSTRACT FROM AUTHOR]

A new family of homologous membrane proteins that transport galactosides--pentoses--hexuronides (GPH) is described. By analysing the aligned amino acid sequences of the GPH family, and by exploiting their different specificities for cations and sugars, we have designed mutations that yield novel insights into the nature of ligand binding sites in membrane proteins. Mutants have been isolated/constructed in the melibiose transport proteins of Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium, and the lactose transport protein of Streptococcus thermophilus which facilitate uncoupled transport or have an altered cation and/or substrate specificity. Most of the mutations map in the amino-terminal region, in or near amphipathic a-helices II and IV, or in interhelix-loop 10-11 of the transport proteins. On the basis of the kinetic properties of these mutants, and the primary and secondary structure analyses presented here, we speculate on the cation binding pocket of this family of transporters. The regulation of the transporters through interaction with, or phosphorylation by, components of the phosphoenolpyruvate:sugar phosphotransferase system is also discussed. [ABSTRACT FROM AUTHOR]

Transport of hydrophilic dipeptides and tripeptides into Lactococcus lactis is mediated by a protonmotive-force-driven peptide-transport protein (DtpT) that shares similarity to eukaryotic peptide transporters, e.g. from yeasts, plants, and the kidney and small intestine of rabbit, man and rat. The expression level of DtpT protein in L. lactis was increased (20-40-fold) to approximately 10% of total integral membrane protein by means of a low-copy-number vector and selecting the appropriate growth conditions. Membrane vesicles bearing the DtpT-His6 protein (containing a C-terminal factor-Xa cleavage site and a six-histidine-tag) showed a Pro-Ala uptake activity that was half that of membranes containing the wild-type protein. The activity in the DtpT-His6 membrane vesicles increased at least 50% upon removal of the His6 tag from the protein. More than 95% DtpT was solubilized from L. lactis membranes in the presence of 1% (mass/vol.) n-dodecyl-β-D-maltoside, and approximately 2 mg DtpT-His6 was purified by Ni2+-chelate affinity chromatography from 100 mg membrane protein. Purified DtpT-His6 was reconstituted unidirectionally into detergent-saturated formed liposomes, which were prepared from Escherichia coli phospholipid and egg phosphatidylcholine; the detergent was removed by adsorption to polystyrene beads. The highest uptake activities were obtained when DtpT was incorporated into liposomes that were treated with a low amount of n-dodecyl-β-D-maltoside (onset of liposome solubilization). The uptake activity could be improved by addition of NaCl (200 mM) and lipids (2 mg/ml) during the solubilization, purification and reconstitution steps. [ABSTRACT FROM AUTHOR]

ATP-binding cassette (ABC) transport proteins catalyze the translocation of substrates at the expense of hydrolysis of ATP, but the actual ATP/substrate stoichiometry is still controversial. In the osmoregulated ABC transporter (OpuA) from Lactococcus lactis, ATP hydrolysis and substrate translocation are tightly coupled, and the activity of right-side-in and inside-out reconstituted OpuA can be determined accurately. Although the ATP/substrate stoichiometry determined from the uptake of glycine betaine and intravesicular ATP hydrolysis tends to increase with decreasing average size of the liposomes, the data from inside-out reconstituted OpuA indicate that the mechanistic stoichiometry is 2. Moreover, the two orientations of OpuA in proteoliposomes allowed possible contributions from substrate (glycine betaine) inhibition on the trans-side of the membrane and inhibition by ADP to be determined. Here we show that OpuA is not inhibited by up to 400 mM glycine betaine on the trans-side of the membrane. ADP is an inhibitor, but accumulation of ADP was negligible in the assays with inside-out-oriented OpuA, and potential effects of the ATP/ADP ratio on the ATP/substrate stoichiometry determinations could be eliminated. [ABSTRACT FROM AUTHOR]

Presents a study that examined functions associated with the oligomeric organization of secondary transport proteins. Background on primary and secondary transport systems; Techniques used to study the quaternary structure of membrane proteins in detergent solutions and in cell membranes; Functions related to the quaternary structure of transport proteins; Details on the structural and functional oligomeric state of secondary transport proteins.

Determines the membrane topology of the peptide transport protein of Lactococcus lactis. Generation of site-specific peptide transport protein (DtpT); Construction of cysteine mutants of DtpT; Presence of transmembrane segments; Location of the deduced topology model of DtpT; Application for eukaryotic peptide transporters.

GlnPQ is an ATP-binding cassette importer with a unique domain organization and intricate transport behavior. The protein has two extracytoplamic substrate-binding domains (SBDs) per membrane subunit, each with different specificity for amino acids and different spacing to the translocator domain. We determined the effect of the length and structure of the linkers, which connect the SBDs to each other and to the membrane-embedded translocator domain, on the transport by GlnPQ. We reveal that varying the linker length impacts transport in a dual manner that depends on the conformational dynamics of the SBD. Varying the linker length not only changes the time for the SBD to find the translocator (docking) but also changes the probability to release the substrate again, thus altering the transport efficiency. On the basis of the experimental data and mathematical modeling, we calculate the docking efficiency as function of linker length and lifetime of the closed conformation. Importantly, not only linker length but also features in the sequence are important for efficient delivery of substrate from SBD to the translocator. We show that the linkers provide a platform for SBD docking and are not merely flexible structures. [ABSTRACT FROM AUTHOR]

The ATP-binding-cassette transporter OpuA from Lactococcus lactis is composed of two ATPase subunits (OpuAA) and two subunits (OpuABC) with the transmembrane domain fused to an extracellular substrate-binding protein. Of the almost 1900 homologues of OpuA known to date, a subset has an amino-terminal amphipathic helix (plus extra transmembrane segment) fused to the core of the transmembrane domain of the OpuABC subunit. FRET measurements indicate that the amphipathic α-helix is located dose to the membrane surface, where its hydrophobic face interacts with the transport protein rather than the membrane lipids. Next, we determined the functional role of this accessory region by engineering the amphipathic α-helix. We analyzed the consequence of the mutations in intact cells by monitoring growth and transport of glycine betaine under normal and osmotic stress conditions. More detailed studies were performed in hybrid membrane vesicles, proteoliposomes, and bilayer nanodiaks. We show that the amphipathic α-helix of OpuA is necessary for high activity of OpuA but is not critical for the biogenesis of the protein or the ionic regulation of transport. [ABSTRACT FROM AUTHOR]

Membrane-bound transport proteins are expected to proceed via different conformational states during the translocation of a solute across the membrane. Tryptophan phosphorescence spectroscopy is one of the most sensitive methods used for detecting conformational changes in proteins. We employed this technique to study substrate-induced conformational changes in the mannitol permease, EnzymeIImtl, of the phosphoenolpyruvate-dependant phosphotransferase system from Escherichia coli. Ten mutants containing a single tryptophan were engineered in the membrane-embedded IICmtl-domain, harboring the mannitol translocation pathway. The mutants were characterized with respect to steady-state and time-resolved phosphorescence, yielding detailed, site-specific information of the Trp microenvironment and protein conformational homogeneity. The study revealed that the Trp environments vary from apolar, unstructured, and flexible sites to buried, highly homogeneous, rigid peptide cores. The most remarkable example of the latter was observed for position 97, because its long sub-second phosphorescence lifetime and highly structured spectra in both glassy and fluid media imply a well defined and rigid core around the probe that is typical of β-sheet-rich structural motifs. The addition of mannitol had a large impact on most of the Trp positions studied. In the case of position 97, mannitol binding induced partial unfolding of the rigid protein core. On the contrary, for residue positions 126, 133, and 147, both steady-state and time-resolved data showed that mannitol binding induces a more ordered and homogeneous structure around these residues. The observations are discussed hi context of the current mechanistic and structural model of EIImtl. [ABSTRACT FROM AUTHOR]