Molecular determinants for substrate specificity of the ligand-binding protein OpuAC from Bacillus subtilis for the compatible solutes glycine betaine and proline betaine.

Compatible solutes play a decisive role in the defense of microorganisms against changes in temperature and increases in osmolarity in their natural habitats. In Bacillus subtilis, the substrate-binding protein (SBP)-dependent ABC-transporter OpuA serves for the uptake of the compatible solutes glycine betaine (GB) and proline betaine (PB). Here, we report the determinants of compatible solute binding by OpuAC, the SBP of the OpuA transporter, by equilibrium binding studies and X-ray crystallography. The affinity of OpuAC/GB and OpuAC/PB complexes were analyzed by intrinsic tryptophan fluorescence and the K(D) values were determined to be 17(+/-1)microM for GB and 295(+/-27)microM for PB, respectively. The structures of OpuAC in complex with GB or PB were solved at 2.0 A and 2.8 A, respectively, and show an SBP-typical class II fold. The ligand-binding pocket is formed by three tryptophan residues arranged in a prism-like geometry suitable to coordinate the positive charge of the trimethyl ammonium group of GB and the dimethyl ammonium group of PB by cation-pi interactions and by hydrogen bonds with the carboxylate moiety of the ligand. Structural differences between the OpuAC/GB and OpuAC/PB complexes occur within the ligand-binding pocket as well as across the domain-domain interface. These differences provide a structural framework to explain the drastic differences in affinity of the OpuAC/GB and OpuAC/PB complexes. A sequence comparison with putative SBP specific for compatible solutes reveals the presence of three distinct families for which the crystal structure of OpuAC might serve as a suitable template to predict the structures of these putative compatible solute-binding proteins.

Arg149 is involved in switching the low affinity, open state of the binding protein AfProX into its high affinity, closed state.

The substrate binding protein AfProX from the Archaeoglobus fulgidus ProU ATP binding cassette transporter is highly selective for the compatible solutes glycine betaine (GB) and proline betaine, which confer thermoprotection to this hyperthermophilic archaeon. A detailed mutational analysis of the substrate binding site revealed the contribution of individual amino acids for ligand binding. Replacement of Arg149 by an Ala residue displayed the largest impact on substrate binding. The structure of a mutant AfProX protein (substitution of Tyr111 with Ala) in complex with GB was solved in the open liganded conformation to gain further insight into ligand binding. In this crystal structure, GB is bound differently compared to the GB closed liganded structure of the wild-type AfProX protein. We found that a network of amino acid side chains communicates the presence of GB toward Arg149, which increases ligand affinity and induces domain closure of AfProX. These results were corroborated by molecular dynamics studies and support the view that Arg149 finalizes the high-affinity state of the AfProX substrate binding protein.

Crystal structures of the choline/acetylcholine substrate-binding protein ChoX from Sinorhizobium meliloti in the liganded and unliganded-closed states.

The ATP-binding cassette transporter ChoVWX is one of several choline import systems operating in Sinorhizobium meliloti. Here fluorescence-based ligand binding assays were used to quantitate substrate binding by the periplasmic ligand-binding protein ChoX. These data confirmed that ChoX recognizes choline and acetylcholine with high and medium affinity, respectively. We also report the crystal structures of ChoX in complex with either choline or acetylcholine. These structural investigations revealed an architecture of the ChoX binding pocket and mode of substrate binding similar to that reported previously for several compatible solute-binding proteins. Additionally the ChoX-acetylcholine complex permitted a detailed structural comparison with the carbamylcholine-binding site of the acetylcholine-binding protein from the mollusc Lymnaea stagnalis. In addition to the two liganded structures of ChoX, we were also able to solve the crystal structure of ChoX in a closed, substrate-free conformation that revealed an architecture of the ligand-binding site that is superimposable to the closed, ligand-bound form of ChoX. This structure is only the second of its kind and raises the important question of how ATP-binding cassette transporters are capable of distinguishing liganded and unliganded-closed states of the binding protein.

Crystal structure of the ligand-binding protein EhuB from Sinorhizobium meliloti reveals substrate recognition of the compatible solutes ectoine and hydroxyectoine.

In microorganisms, members of the binding-protein-dependent ATP-binding cassette transporter superfamily constitute an important class of transport systems. Some of them are involved in osmoprotection under hyperosmotic stress by facilitating the uptake of "compatible solutes". Currently, the molecular mechanisms used by these transport systems to recognize compatible solutes are limited to transporters specific for glycine betaine and proline betaine. Therefore, this study reports a detailed analysis of the molecular principles governing substrate recognition in the Ehu system from Sinorhizobium meliloti, which is responsible for the uptake of the compatible solutes ectoine and hydroxyectoine. To contribute to a broader understanding of the molecular interactions underlying substrate specificity, our study focused on the substrate-binding protein EhuB because this protein binds the ligand selectively, delivers it to the translocation machinery in the membrane and is thought to be responsible for substrate specificity. The crystal structures of EhuB, in complex with ectoine and hydroxyectoine, were determined at a resolution of 1.9 A and 2.3 A, respectively, and allowed us to assign the structural principles of substrate recognition and binding. Based on these results, site-directed mutagenesis of amino acids involved in ligand binding was employed to address their individual contribution to complex stability. A comparison with the crystal structures of other binding proteins specific for compatible solutes revealed common principles of substrate recognition, but also important differences that might be an adaptation to the nature of the ligand and to the demands on protein affinity imposed by the environment.

Biochemical and structural analysis of the Bacillus subtilis ABC transporter OpuA and its isolated subunits.

Adaptation of microorganisms to changing osmotic conditions is a prerequisite for survival and cellular vitality for most microorganisms. In the Gram-positive soil bacterium Bacillus subtilis, five transport systems catalyze the uptake of compatible solutes across the plasma membrane that allow the growth of B. subtilis over a wide range of osmotic conditions. Focus of this review is the osmoprotectant uptake A (OpuA) transporter, a member of the family of substrate-binding protein (SBP)-dependent ATP-binding cassette (ABC) transporters that mediates the uptake of the compatible solutes glycine betaine and proline betaine. OpuA is composed of three subunits: a nucleotide-binding domain (OpuAA) located in the cytosol, a transmembrane domain (OpuAB), and a SBP (OpuAC), which binds glycine betaine and proline betaine with high specificity and targets it to OpuAB for ATP-dependent translocation across the plasma membrane. After a brief introduction in the field of bacterial osmoadaptation, we will summarize our recent findings about the biochemical and structural analysis of the components of the OpuA systems. Our studies covered both the isolated subunits of the OpuA transporter and initial investigations of the whole transporter in vitro.

Ectoine-induced proteins in Sinorhizobium meliloti include an Ectoine ABC-type transporter involved in osmoprotection and ectoine catabolism.

To understand the mechanisms of ectoine-induced osmoprotection in Sinorhizobium meliloti, a proteomic examination of S. meliloti cells grown in minimal medium supplemented with ectoine was undertaken. This revealed the induction of 10 proteins. The protein products of eight genes were identified by using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Five of these genes, with four other genes whose products were not detected on two-dimensional gels, belong to the same gene cluster, which is localized on the pSymB megaplasmid. Four of the nine genes encode the characteristic components of an ATP-binding cassette transporter that was named ehu, for ectoine/hydroxyectoine uptake. This transporter was encoded by four genes (ehuA, ehuB, ehuC, and ehuD) that formed an operon with another gene cluster that contains five genes, named eutABCDE for ectoine utilization. On the basis of sequence homologies, eutABCDE encode enzymes with putative and hypothetical functions in ectoine catabolism. Analysis of the properties of ehuA and eutA mutants suggests that S. meliloti possesses at least one additional ectoine catabolic pathway as well as a lower-affinity transport system for ectoine and hydroxyectoine. The expression of ehuB, as determined by measurements of UidA activity, was shown to be induced by ectoine and hydroxyectoine but not by glycine betaine or by high osmolality.