Computational redesign of the Escherichia coli ribose-binding protein ligand binding pocket for 1,3-cyclohexanediol and cyclohexanol

Abstract

Bacterial periplasmic-binding proteins have been acclaimed as general biosensing platform, but their range of natural ligands is too limited for optimal development of chemical compound detection. Computational redesign of the ligand-binding pocket of periplasmic-binding proteins may yield variants with new properties, but, despite earlier claims, genuine changes of specificity to non-natural ligands have so far not been achieved. In order to better understand the reasons of such limited success, we revisited here the Escherichia coli RbsB ribose-binding protein, aiming to achieve perceptible transition from ribose to structurally related chemical ligands 1,3-cyclohexanediol and cyclohexanol. Combinations of mutations were computationally predicted for nine residues in the RbsB binding pocket, then synthesized and tested in an E. coli reporter chassis. Two million variants were screened in a microcolony-in-bead fluorescence-assisted sorting procedure, which yielded six mutants no longer responsive to ribose but with 1.2–1.5 times induction in presence of 1 mM 1,3-cyclohexanediol, one of which responded to cyclohexanol as well. Isothermal microcalorimetry confirmed 1,3-cyclohexanediol binding, although only two mutant proteins were sufficiently stable upon purification. Circular dichroism spectroscopy indicated discernable structural differences between these two mutant proteins and wild-type RbsB. This and further quantification of periplasmic-space abundance suggested most mutants to be prone to misfolding and/or with defects in translocation compared to wild-type. Our results thus affirm that computational design and library screening can yield RbsB mutants with recognition of non-natural but structurally similar ligands. The inherent arisal of protein instability or misfolding concomitant with designed altered ligand-binding pockets should be overcome by new experimental strategies or by improved future protein design algorithms.

Ribose-Binding Protein Mutants With Improved Interaction Towards the Non-natural Ligand 1,3-Cyclohexanediol

“…We previously selected six RbsB mutants (named DT001, DT002, DT011, DT013, DT015, and DT016) that had lost the capacity to bind ribose, and instead had gained primitive affinity to 13CHD as new ligand (Tavares et al, 2019). Purified mutant proteins, however, displayed severe misfolding, poor stability and poor translocation into the periplasmic space (Tavares et al, 2019).…”

“…We previously selected six RbsB mutants (named DT001, DT002, DT011, DT013, DT015, and DT016) that had lost the capacity to bind ribose, and instead had gained primitive affinity to 13CHD as new ligand (Tavares et al, 2019). Purified mutant proteins, however, displayed severe misfolding, poor stability and poor translocation into the periplasmic space (Tavares et al, 2019). In order to potentially improve mutant protein functionality, we used the respective rbsB-DT mutant genes as individual templates to produce random mutagenesis libraries (RML) using error prone PCR (ep-PCR).…”

“…RMLs produced from each starting RbsB-variant were transformed into the E. coli bioreporter strain carrying the Trz1-ompR-ompC':gfpmut2 signaling chain (Reimer et al, 2014). Individual clones were encapsulated in alginate beads and grown to microcolonies, which were incubated with 1 mM 13CHD to induce GFP formation (Tavares et al, 2019). Some 10 million beads, covering three times the estimated sizes of the RML002 and RML016 (derivatives of DT002 and DT016, respectively) were screened by fluorescence-activated cell sorting (FACS), separating beads with an 13CHD-induced fluorescence above the 98th percentile.…”

“…We previously described six novel RbsB mutants with loss of binding to ribose and moderate but significant response to 1,3-cyclohexanediol (13CHD). These mutants were obtained through a combination of computational prediction of binding pocket mutations in RbsB and screening for gain of GFPmut2 fluorescence output in an E. coli bioreporter system (Tavares et al, 2019). However, all mutant proteins showed signs of poor stability, mis-or unfolding and potentially translocation problems compared to wild-type RbsB, suggesting that the poor inducibility by 13CHD may partly be due to protein instability caused by the introduced binding pocket substitutions.…”

“…The goal of this work was to understand whether primitive binding of 13CHD by designed RbsB mutants (Tavares et al, 2019) can be improved by both rational and directed evolution approaches. We started with six mutant rbsB templates that were previously obtained (Tavares et al, 2019), which were used for random or site-directed mutagenesis, cloned into the E. coli GFPmut2 bioreporter strain and extensively screened by fluorescence-assisted cell sorting for improved GFPmut2 induction in presence of 13CHD. Potential gain-of-function mutants were separated and used for new rounds of mutagenesis and screening.…”