Change in protein-ligand specificity through binding pocket grafting
Ulrike Scheib1, Sooruban Shanmugaratnam1, José Arcadio Farías-Rico1
1Max Planck Institute for Developmental Biology, Spemannstr. 35, 72076 Tübingen, Germany.
Journal of Structural Biology
|June 25, 2013
Summary
Researchers engineered a biosensor by modifying a periplasmic binding protein (PBP). This modification successfully swapped its binding specificity for polyamines, demonstrating how key residues control molecular recognition.
Area of Science:
- Biochemistry
- Molecular Biology
- Synthetic Biology
Background:
- Small molecule recognition is vital for biological processes and synthetic biology applications like biosensor development.
- Periplasmic binding proteins (PBPs) are promising scaffolds for biosensors due to their ligand-induced structural changes.
- PotF and PotD are homologous polyamine-binding PBPs with distinct specificities for putrescine and spermidine.
Purpose of the Study:
- To elucidate the molecular determinants of binding specificity between PotF and PotD.
- To engineer a PBP variant with altered ligand-binding specificity.
- To investigate the role of binding pocket residues in controlling polyamine recognition.
Main Methods:
- Grafting the binding site of PotD onto the PotF scaffold.
- Introducing seven mutations into the PotF binding pocket.
- Isothermal titration calorimetry (ITC) to confirm binding profile changes.
- X-ray crystallography (1.7Å resolution) to determine the structure of the variant complex.
Main Results:
- The engineered PotF variant exhibited a swapped binding profile, recognizing spermidine over putrescine.
- Crystallographic analysis revealed the specific interactions of mutated residues and a water network.
- Specificity-determining residues were identified within the first shell of the binding pocket.
Conclusions:
- Binding specificity in PotF is primarily encoded by residues in the first shell of the binding pocket.
- Transplantation of these residues can successfully alter and swap PBP binding specificity.
- This study provides a foundation for engineering novel biosensors with de novo ligand affinities.
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