title: Change in protein-ligand specificity through binding pocket grafting.
abstract: Recognition and discrimination of small molecules are crucial for biological processes in living systems. Understanding the mechanisms that underlie binding specificity is of particular interest to synthetic biology, e.g. the engineering of biosensors with de novo ligand affinities. Promising scaffolds for such biosensors are the periplasmic binding proteins (PBPs) due to their ligand-mediated structural change that can be translated into a physically measurable signal. In this study we focused on the two homologous polyamine binding proteins PotF and PotD. Despite their structural similarity, PotF and PotD have different binding specificities for the polyamines putrescine and spermidine. To elucidate how specificity is determined, we grafted the binding site of PotD onto PotF. The introduction of 7 mutations in the first shell of the binding pocket leads to a swap in the binding profile as confirmed by isothermal titration calorimetry. Furthermore, the 1.7Å crystal structure of the new variant complexed with spermidine reveals the interactions of the specificity determining residues including a defined water network. Altogether our study shows that specificity is encoded in the first shell residues of the PotF binding pocket and that transplantation of these residues allows the swap of the binding specificity.
authors.affiliation: Max Planck Institute for Developmental Biology, Spemannstr. 35, 72076 Tübingen, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Scheib
authors.affiliation: Max Planck Institute for Developmental Biology, Spemannstr. 35, 72076 Tübingen, Germany.
authors.forename: Sooruban
authors.initials: S
authors.lastname: Shanmugaratnam
authors.affiliation: Max Planck Institute for Developmental Biology, Spemannstr. 35, 72076 Tübingen, Germany.
authors.forename: José Arcadio
authors.initials: JA
authors.lastname: Farías-Rico
authors.affiliation: Max Planck Institute for Developmental Biology, Spemannstr. 35, 72076 Tübingen, Germany. Electronic address: birte.hoecker@tuebingen.mpg.de.
authors.forename: Birte
authors.initials: B
authors.lastname: Höcker
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D008024:Ligands; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D033902:Periplasmic Binding Proteins / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011700:Putrescine / Q000737:chemistry; D017982:Receptors, Biogenic Amine / Q000737:chemistry* / Q000235:genetics; D013095:Spermidine / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D029968:Escherichia coli Proteins; D008024:Ligands; D026901:Membrane Transport Proteins; D033902:Periplasmic Binding Proteins; C099274:PotD protein, E coli; D017982:Receptors, Biogenic Amine; C049683:potF protein, E coli; D013095:Spermidine; D011700:Putrescine
keywords: Periplasmic binding protein; Polyamine binding; Protein engineering; Putrescine; Rational design; Spermidine