Charakterisierung der molekularen Determinanten zur hochaffinen Bindung der kompatiblen Soluten Glycin Betain, Prolin Betain, Ectoin und Hydroxyectoin durch Substratbindeproteine von bakteriellen ABC-Transportern

Die Akkumulation von kompatiblen Soluten ist ein weit verbreiteter Schutzmechanismus gegen variierende Umweltbedingungen und wird in vielen Spezies der Bacteria und Archaea verwendet. Das einige kompatible Solute nicht nur osmoprotektive Wirkung haben, sondern generell Protein-stabilisierende Substa...

Full description

Saved in:
Bibliographic Details
Main Author: Sohn-Bösser, Linda
Contributors: Bremer, Erhard (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2006
Online Access:PDF Full Text
Tags: Add Tag
No Tags, Be the first to tag this record!
Table of Contents: The accumulation of compatible solutes is a widely distributed mechanism for coping with varying environmental conditions and it is used by a variety of species of Bacteria and Archaea. Beside their osmoprotective property some of these compatible solutes possess also general protein stabilizing properties, which has been shown by several in vitro experiments. This protein-stabilizing effect is described by the preferential exclusion model which predicts the preferential exclusion of compatible solutes from the vicinity of proteins because of their thermodynamic unfavourable interactions with the peptide backbone. In this present work, the substrate binding proteins of two high affinity ABC-transporters for the compatible solutes glycine betaine and proline betaine have been studied at the molecular level in order to understand the molecular determinants for binding of preferential excluded solutes by proteins. For this purpose we established the high resolution crystal structures of the substrate binding proteins ProX (EcProX 1.6Å) of the Escherichia coli ProU-System and the ProX protein of the Archaeoglobus fulgidus ProU system (AfProX 1,9Å). The crystal structure of the EcProX protein demonstrated that the quaternary ammonium group of the bound glycine betaine is coordinated by three tryptophane residues (Trp-65, Trp-140 and Trp-188); the carboxyl group of glycine betaine is stabilized by hydrogen bonds. Using site directed mutagenesis I established that cation-pi interactions are a key determinant in the high affinity binding of glycine betaine. It could be demonstrated that the three tryptophane residues show a different importance in substrate binding. Trp-188 is essential for the binding of glycine betaine whereas Trp-140 is of minor importance. Nevertheless Trp-188 needs Trp-140 and Trp-65 for the stabilization of the quaternary Ammonium group of glycine betaine for high affinity binding. The AfProX protein shows an especially high binding affinity to glycine betaine and proline betaine. Compared to the EcProX protein the KD of AfProX at 50°C (KD=0,01 µM) is 100 times lower as it is the case for EcProX at room temperature (KD=1 µM) and 1000 times lower compared to the KD of the OpuAC protein (KD=17 µM), a glycine betaine and proline betaine binding protein from Bacillus subtilis. The crystal structure of AfProX showed a substrate binding site with another composition of aromatic amino acids. The quaternary ammonium group of glycine betaine is coordinated by four tyrosine residues, Tyr-63, Tyr-111, Tyr-190 and Tyr-214 (tyrosine-girdle) and an aspartate residue, the carboxyl group is stabilized by hydrogen bonds and salt bridges. Mutational analysis of the Tyr-girdle again characterized cation-pi interactions as an important force for high affinity binding of glycine betaine. The results demonstrated that the particular architecture of the Tyr-girdle, together with the amino acid residues responsible for the stabilization of the carboxyl group of glycine betaine is relevant for the strong binding affinity. Depending on the position of the tyrosine residue an exchange against an alanine residue causes a drop of binding affinity that is comparable to the EcProX protein, the BsOpuAC protein or lower. An exchange of two tyrosine residues against alanine residues always cause a complete loss of binding affinity. Database research with amino acid sequences of EcProX, AfProX and BsOpuAC showed a wide distribution of their conserved motifs and in the case of BsOpuAC a significant conservation of the tryptophanes that are involved in substrate binding and a domain swap compared to other glycine betaine binding proteins. The discovery of a substrate binding dependend ABC transporter (Ehu) for ectoine and hydroxyectoine in S. meliloti allowed the analysis of ectoine binding mediated by a substrate binding protein. Overproduction, purification and characterization of the high affinity ectoin binding protein EhuB and its subsequent crystallization with bound ectoine (and hydroxyectoine) lead to a high resolution structure (2,1 Å). The substrate binding site of EhuB consists of two phenylalanine residues and one tyrosine residue forming a box-like configuration, which bind the delocalised positive charge of the molecule. The pyrimidine ring and the carboxyl group of ectoine are stabilized by salt bridges and hydrogen bonds. Cation-pi interactions are a key determinant in the high affinity binding of glycine betaine and proline betaine, but also in case of ectoine and hydroxyectoine and seems to be widely distributed in Bacteria and Archaea.