Abstract
Compatible solutes such as glycine betaine and proline betaine serve as protein stabilizers because of their preferential exclusion from protein surfaces. To use extracellular sources of this class of compounds as osmo-, cryo-, or thermoprotectants, Bacteria and Archaea have developed high affinity uptake systems of the ATP-binding cassette type. These transport systems require periplasmic- or extracellular-binding proteins that are able to bind the transported substance with high affinity. Therefore, binding proteins that bind compatible solutes have to avoid the exclusion of their ligands within the binding pocket. In the present study we addressed the question to how compatible solutes can be effectively bound by a protein at temperatures around 83 degrees C as this is done by the ligand-binding protein ProX from the hyperthermophilic archaeon Archaeoglobus fulgidus. We solved the structures of ProX without ligand and in complex with both of its natural ligands glycine betaine and proline betaine, as well as in complex with the artificial ligand trimethylammonium. Cation-pi interactions and non-classical hydrogen bonds between four tyrosine residues, a main chain carbonyl oxygen, and the ligand have been identified to be the key determinants in binding the quaternary amines of the three investigated ligands. The comparison of the ligand binding sites of ProX from A. fulgidus and the recently solved structure of ProX from Escherichia coli revealed a very similar solution for the problem of compatible solute binding, although both proteins share only a low degree of sequence identity. The residues involved in ligand binding are functionally equivalent but not conserved in the primary sequence.
Highlights
Compatible solutes such as glycine betaine and proline betaine serve as protein stabilizers because of their preferential exclusion from protein surfaces
Binding proteins that bind compatible solutes have to avoid the exclusion of their ligands within the binding pocket
In the present study we addressed the question to how compatible solutes can be effectively bound by a protein at temperatures around 83 °C as this is done by the ligand-binding protein ProX from the hyperthermophilic archaeon Archaeoglobus fulgidus
Summary
Bacterial Strains—The E. coli strain BL21 (Codon Plus RIL), which harbors a plasmid encoding rare tRNAs for arginine, isoleucine, and. We observed that a substantial portion of the ProX protein heterologously produced in E. coli still carried an unprocessed OmpA signal sequence.2 To remove both the unprocessed OmpA signal sequence and the StrepTag affinity tag from the recombinant A. fulgidus ProX, the protein preparation was treated with the protease factor Xa using the cleavage site of the pASK-IBA6 vector between the Strep-Tag and ProX. To obtain A. fulgidus ProX preparations that were free of its ligands GB and PB, the overproducing strain BL21 (Codon Plus RIL) (pGH26) was grown in a chemically defined minimal medium [45]. Using a different setup, unliganded ProX crystallized in hanging drops equilibrated against a reservoir containing 0.1 M ZnAc2, 0.1 M MES, pH 6.5, 25–30% (v/v) ethylene glycol These crystals grew within 4 weeks, reached a final size of 200 ϫ 150 ϫ 20 m3, and belong to space group P212121 (crystal form IV). The accession codes are 1SW1, 1SW2, 1SW4, and 1SW5 for ProX-PB, ProX-GB, ProX-TM, and ProX ligand free, respectively
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