RESUMEN
A profound understanding of the molecular interactions between receptors and ligands is important throughout diverse research, such as protein design, drug discovery, or neuroscience. What determines specificity and how do proteins discriminate against similar ligands? In this study, we analyzed factors that determine binding in two homologs belonging to the well-known superfamily of periplasmic binding proteins, PotF and PotD. Building on a previously designed construct, modes of polyamine binding were swapped. This change of specificity was approached by analyzing local differences in the binding pocket as well as overall conformational changes in the protein. Throughout the study, protein variants were generated and characterized structurally and thermodynamically, leading to a specificity swap and improvement in affinity. This dataset not only enriches our knowledge applicable to rational protein design but also our results can further lay groundwork for engineering of specific biosensors as well as help to explain the adaptability of pathogenic bacteria.
Asunto(s)
Escherichia coli K12/química , Proteínas de Escherichia coli/química , Proteínas de Unión Periplasmáticas/química , Receptores de Amina Biogénica/química , Espermidina/química , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Unión Periplasmáticas/genética , Proteínas de Unión Periplasmáticas/metabolismo , Unión Proteica , Receptores de Amina Biogénica/genética , Receptores de Amina Biogénica/metabolismo , Espermidina/metabolismoRESUMEN
Periplasmic binding proteins (PBPs) are ubiquitous receptors in gram-negative bacteria. They sense solutes and play key roles in nutrient uptake. Escherichia coli's putrescine receptor PotF has been reported to bind putrescine and spermidine. We reveal that several similar biogenic polyamines are recognized by PotF. Using isothermal titration calorimetry paired with X-ray crystallography of the different complexes, we unveil PotF's binding modes in detail. The binding site for PBPs is located between two lobes that undergo a large conformational change upon ligand recognition. Hence, analyzing the influence of ligands on complex formation is crucial. Therefore, we solved crystal structures of an open and closed apo state and used them as a basis for molecular dynamics simulations. In addition, we accessed structural behavior in solution for all complexes by 1H-15N HSQC NMR spectroscopy. This combined analysis provides a robust framework for understanding ligand binding for future developments in drug design and protein engineering.