Fine-tuning spermidine binding modes in the putrescine binding protein PotF

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.

Retracing the evolution of a modern periplasmic binding protein

“…These differences in the architecture of type I and II PBP‐like fold proteins could explain why one of the isolated lobes from type II proteins such as LAO, HisJ, and ArgBP is able to bind their respective ligand while none of the individual lobes from type I PBPs have been shown to be competent by themselves. Variations in ligand affinity and promiscuity for some of the studied PBPs (Chu et al, 2013 ; Kröger, Shanmugaratnam, Ferruz, et al, 2021 ; Kröger, Shanmugaratnam, Scheib, et al, 2021 ; Vergara et al, 2020 ) indicate that possibly the PBP ancestor was able to bind some ligands but with considerably lower affinity, similarly to what has been reported for enzyme evolution (Copley, 2020 ; Khersonsky & Tawfik, 2010 ; Tawfik, 2020 ). In a plausible scenario, after duplication and fusion of the flavodoxin‐like fold ancestor (Figure 1a ), type I PBP‐like fold proteins were able to evolve obtaining increased selectivity and affinity for specific compounds but still sharing almost equally the ligand binding residues between both domains, as has been observed for RBP.…”

“…This common binding mechanism is reflected in PBPs that bind similar molecules with very different selectivities and affinities at the same binding site (Kröger, Shanmugaratnam, Ferruz, et al, 2021 ). For these reasons, PBPs have been used in several engineering and design approaches, especially creating highly sensitive biosensors and molecular switches (Dwyer & Hellinga, 2004 ; Jeffery, 2011 ; Medintz & Deschamps, 2006 ; Steffen et al, 2016 ), and designing new binding properties (Banda‐Vázquez et al, 2018 ; Kröger, Shanmugaratnam, Scheib, et al, 2021 ; Scheib et al, 2014 ).…”

A fluorescent biosensor for the visualization of agmatine

“…From this screen, A327L/E328I of the N-linker emerged as the most favorable variant while the C-linker remaining unaltered (N332/P333). Since the screen was based on measuring fluorescence changes after PUT addition to crude extract of E. coli, we introduced two further mutations (S87Y, F276W) as they reduce initial PUT affinity from the native nanomolar to the micromolar range 23 to mitigate potential confounding effects of intracellular ligand. Next, we substituted the initially used cpGFP with the circular permuted variant of superfolder GFP 19 (sfcpGFP).…”

“…In the sensor the large conformational change of the PBP upon ligand binding is transmitted to the fluorescent protein by altering the chromophore environment and thus triggering a change in signal 20,21 . We chose PotF due to its promiscuous binding of biogenic amines characterized in prior studies 22,23 . Our engineering employed a semi-rational approach combined with a medium throughput fluorescence screening to optimize linker positions and improve AGM specificity.…”

Retracing the evolution of a modern periplasmic binding protein

“…This common binding mechanism is reflected in PBPs that bind similar molecules with very different selectivities and affinities at the same binding site (Kröger, 2021a). For these reasons, PBPs have been used in several engineering and design approaches, especially creating highly sensitive biosensors and molecular switches (Steffen, 2016;Jeffery, 2011;Medintz, 2006;Dwyer, 2004), and designing new binding properties (Banda-Vázquez, 2018; Scheib, 2014, Kröger, 2021b.…”