Abstract
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.
Highlights
The binding-induced change of the overall structure of periplasmic binding protein (PBP) might be the prerequisite for the adaptability and large coverage of different ligands by PBPs
Despite great similarities among their binding pockets, PotF and PotD differ in their polyamine-binding profiles: PotF is promiscuous for PUT (KD ≈ 68 nM) and SPD (KD ≈ 30 μM) [10], whereas PotD binds exclusively SPD (KD ≈ 6 nM) [9]
PotF/D does not reveal the specific interactions that direct the ligand-binding profiles of PotF and PotD for the different polyamines
Summary
Despite great similarities among their binding pockets, PotF and PotD differ in their polyamine-binding profiles: PotF is promiscuous for PUT (KD ≈ 68 nM) and SPD (KD ≈ 30 μM) [10], whereas PotD binds exclusively SPD (KD ≈ 6 nM) [9]. The PotF_Abox_Prox variant (S38T, D39E, D247S, and F276W) showed no binding of PUT similar to PotF_Prox but restored a very low SPD affinity (Table 1) This illustrates the power of F276W in relation to SPD binding and the importance of the proximal residues that form interactions with the N1 primary amines of the respective ligands for polyamine binding in general. When introduced in PotF/D, this Tyr turns outward and disrupts the salt bridge between E184 and R91 (Fig. 3C) This observation explains the negative influence of S87Y on polyamine binding in general as well as the lack of affinity improvement in PotF/D, as the Tyr does disrupt the salt bridge and thereby obstructs complete closure and is not able to coordinate N3 of SPD. This was expected as we reintroduced the wildtype-like proximal primary amine–binding site in these variants
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