Dataset Information

Ligand binding and crystal structures of the substrate-binding domain of the ABC transporter OpuA.

ABSTRACT: BACKGROUND:The ABC transporter OpuA from Lactococcus lactis transports glycine betaine upon activation by threshold values of ionic strength. In this study, the ligand binding characteristics of purified OpuA in a detergent-solubilized state and of its substrate-binding domain produced as soluble protein (OpuAC) was characterized. PRINCIPAL FINDINGS:The binding of glycine betaine to purified OpuA and OpuAC (K(D) = 4-6 microM) did not show any salt dependence or cooperative effects, in contrast to the transport activity. OpuAC is highly specific for glycine betaine and the related proline betaine. Other compatible solutes like proline and carnitine bound with affinities that were 3 to 4 orders of magnitude lower. The low affinity substrates were not noticeably transported by membrane-reconstituted OpuA. OpuAC was crystallized in an open (1.9 A) and closed-liganded (2.3 A) conformation. The binding pocket is formed by three tryptophans (Trp-prism) coordinating the quaternary ammonium group of glycine betaine in the closed-liganded structure. Even though the binding site of OpuAC is identical to that of its B. subtilis homolog, the affinity for glycine betaine is 4-fold higher. CONCLUSIONS:Ionic strength did not affect substrate binding to OpuA, indicating that regulation of transport is not at the level of substrate binding, but rather at the level of translocation. The overlap between the crystal structures of OpuAC from L.lactis and B.subtilis, comprising the classical Trp-prism, show that the differences observed in the binding affinities originate from outside of the ligand binding site.

SUBMITTER: Wolters JC

PROVIDER: S-EPMC2861598 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Ligand binding and crystal structures of the substrate-binding domain of the ABC transporter OpuA.

Wolters Justina C JC Berntsson Ronnie P-A RP Gul Nadia N Karasawa Akira A Thunnissen Andy-Mark W H AM Slotboom Dirk-Jan DJ Poolman Bert B

PloS one 20100429 4

<h4>Background</h4>The ABC transporter OpuA from Lactococcus lactis transports glycine betaine upon activation by threshold values of ionic strength. In this study, the ligand binding characteristics of purified OpuA in a detergent-solubilized state and of its substrate-binding domain produced as soluble protein (OpuAC) was characterized.<h4>Principal findings</h4>The binding of glycine betaine to purified OpuA and OpuAC (K(D) = 4-6 microM) did not show any salt dependence or cooperative effects ...[more]

PMID: 20454456

Similar Datasets

Project description:The accumulation of compatible solutes is a common defense of bacteria against the detrimental effects of high osmolarity. Uptake systems for these compounds are cornerstones in cellular osmostress responses because they allow the energy-preserving scavenging of osmostress protectants from environmental sources. Bacillus subtilis is well studied with respect to the import of compatible solutes and its five transport systems (OpuA, OpuB, OpuC, OpuD, and OpuE), for these stress protectants have previously been comprehensively studied. Building on this knowledge and taking advantage of the unabated appearance of new genome sequences of members of the genus Bacillus, we report here the discovery, physiological characterization, and phylogenomics of a new member of the Opu family of transporters, OpuF (OpuFA-OpuFB). OpuF is not present in B. subtilis but it is widely distributed in members of the large genus Bacillus OpuF is a representative of a subgroup of ATP-binding cassette (ABC) transporters in which the substrate-binding protein (SBP) is fused to the transmembrane domain (TMD). We studied the salient features of the OpuF transporters from Bacillus infantis and Bacillus panaciterrae by functional reconstitution in a B. subtilis chassis strain lacking known Opu transporters. A common property of the examined OpuF systems is their substrate profile; OpuF mediates the import of glycine betaine, proline betaine, homobetaine, and the marine osmolyte dimethylsulfoniopropionate (DMSP). An in silico model of the SBP domain of the TMD-SBP hybrid protein OpuFB was established. It revealed the presence of an aromatic cage, a structural feature commonly present in ligand-binding sites of compatible solute importers.IMPORTANCE The high-affinity import of compatible solutes from environmental sources is an important aspect of the cellular defense of many bacteria and archaea against the harmful effects of high external osmolarity. The accumulation of these osmostress protectants counteracts high-osmolarity-instigated water efflux, a drop in turgor to nonphysiological values, and an undue increase in molecular crowding of the cytoplasm; they thereby foster microbial growth under osmotically unfavorable conditions. Importers for compatible solutes allow the energy-preserving scavenging of osmoprotective and physiologically compliant organic solutes from environmental sources. We report here the discovery, exemplary physiological characterization, and phylogenomics of a new compatible solute importer, OpuF, widely found in members of the Bacillus genus. The OpuF system is a representative of a growing subgroup of ABC transporters in which the substrate-scavenging function of the substrate-binding protein (SBP) and the membrane-embedded substrate translocating subunit (TMD) are fused into a single polypeptide chain.

Dataset Information

Ligand binding and crystal structures of the substrate-binding domain of the ABC transporter OpuA.

Publications

Ligand binding and crystal structures of the substrate-binding domain of the ABC transporter OpuA.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets