The compatible-solute-binding protein OpuAC from Bacillus subtilis: ligand binding, site-directed mutagenesis, and crystallographic studies

Abstract

In the soil bacterium Bacillus subtilis, five transport systems work in concert to mediate the import of various compatible solutes that counteract the deleterious effects of increases in the osmolarity of the environment. Among these five systems, the ABC transporter OpuA, which catalyzes the import of glycine betaine and proline betaine, has been studied in detail in the past. Here, we demonstrate that OpuA is capable of importing the sulfobetaine dimethylsulfonioacetate (DMSA). Since OpuA is a classic ABC importer that relies on a substrate-binding protein priming the transporter with specificity and selectivity, we analyzed the OpuA-binding protein OpuAC by structural and mutational means with respect to DMSA binding. The determined crystal structure of OpuAC in complex with DMSA at a 2.8-A resolution and a detailed mutational analysis of these residues revealed a hierarchy within the amino acids participating in substrate binding. This finding is different from those for other binding proteins that recognize compatible solutes. Furthermore, important principles that enable OpuAC to specifically bind various compatible solutes were uncovered.

FIG. 1.

Chemical structures of the OpuAC substrates used in this study.

FIG. 2.

Osmoprotective effects of the compatible solutes glycine betaine and DMSA for B. subtilis. (A) The OpuA⁺ (OpuB⁻ OpuC⁻ OpuD⁻) strain RMKB34 was grown in SMM with 1.2 M NaCl (▪), 1.2 M NaCl with 1 mM glycine betaine (•), and 1.2 M NaCl with 1 mM DMSA (▴). (B) The OpuA⁻ (OpuB⁻ OpuC⁻ OpuD⁻) strain RMKB24 was grown in SMM with 1.2 M NaCl (▪), 1.2 M NaCl with 1 mM glycine betaine (▴), and 1.2 M NaCl with 1 mM DMSA (•). Cultures (20 ml) were inoculated to an OD₅₇₈ of 0.1 from overnight cultures pregrown in SMM with 0.4 M NaCl and were propagated in 100-ml Erlenmeyer flasks in a shaking water bath (220 rpm) at 37°C. Cell growth was monitored over time by measuring the OD₅₇₈.

FIG. 3.

Ligand binding of OpuAC with DMSA. (A) Emission spectra of the protein in the absence (solid line) or presence (dashed line) of 1 mM substrate. (B) Equilibrium binding titration experiments with DMSA. a.u., arbitrary units.

FIG. 4.

View of the ligand-binding pocket of the OpuAC-DMSA complex. Interactions between the OpuAC protein and its ligand DMSA are highlighted by dashed lines. Highlighted are the three tryptophans (Trp⁷², Trp¹⁷⁸, and Trp²²⁵) and the histidine residue (His²³⁰), which constitute the binding pocket. Amino acids given in single-letter code in parentheses indicate the mutations performed in this study.

FIG. 5.

View of the superpositioning of the ligand-binding sites of the OpuAC-glycine betaine, OpuAC-proline betaine, and OpuAC-DMSA complexes. Residues involved in glycine betaine coordination are shown in green, residues involved in proline betaine binding are shown in orange, and residues involved in DMSA binding are shown in purple. For simplicity, the backbone contacts of the ligands with Gly²⁶ and Ile²⁷ have been omitted from the representation.

FIG. 6.

Domain organization of glycine betaine binding proteins related to OpuAC from B. subtilis. Data base searches using the BLAST program showed that there are four classes of ligand-binding proteins that are related to OpuAC from B. subtilis. The OpuAC protein from B. subtilis is shown with the residues involved in binding of the trimethylammonium head group of glycine betaine (W72, W178, W225) and the carboxylate of glycine betaine (G26, I27, H230). Group 1 contains those proteins that align directly with the OpuAC protein. An example is the glycine betaine binding protein GbuC from Listeria monocytogenes (29). Group 2 is composed of proteins that align with the OpuAC protein when the N- and C-terminal domains are inverted. An example is the glycine betaine binding protein OtaC from the archaeon Methanosarcina mazei (41). Binding protein domains that are fused to the transmembrane domain of the ABC transport system and contain the domain inversion form group 3. An example is the glycine betaine binding/transmembrane protein OpuBC (also referred to as BusAB) from Lactococcus lactis (36, 44). Finally, group 4 of the OpuAC related proteins contain those examples where the transmembrane domain is fused to a duplicated binding protein domain both of which contain the domain inversion. This type of fusion protein was first noticed by van der Heide and Poolman (43). An example of this group of OpuAC-related proteins is present in Streptomyces coelicolor (NP_625895). But in contrast to the other mentioned glycine betaine binding proteins, the substrate specificity of this fused binding protein has not been experimentally assessed. For the various alignments, the N-terminal and C-terminal domains of OpuAC were split between the amino acids 168/169 as initially described by Horn et al. (20).