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

Bioreporters consist of genetically modified living organisms that respond to the presence of target chemical compounds by production of an easily measurable signal. The central element in a bioreporter is a sensory protein or aptamer, which, upon ligand binding, modifies expression of the reporter signal protein. A variety of naturally occurring or modified versions of sensory elements has been exploited, but it has proven to be challenging to generate elements that recognize non-natural ligands. Bacterial periplasmic binding proteins have been proposed as a general scaffold to design receptor proteins for non-natural ligands, but despite various efforts, with only limited success. Here, we show how combinations of randomized mutagenesis and reporter screening improved the performance of a set of mutants in the ribose binding protein (RbsB) of Escherichia coli, which had been designed based on computational simulations to bind the non-natural ligand 1,3-cyclohexanediol (13CHD). Randomized mutant libraries were constructed that used the initially designed mutants as scaffolds, which were cloned in an appropriate E. coli bioreporter system and screened for improved induction of the GFPmut2 reporter fluorescence in presence of 1,3-cyclohexanediol. Multiple rounds of library screening, sorting, renewed mutagenesis and screening resulted in 4.5-fold improvement of the response to 1,3-cyclohexanediol and a lower detection limit of 0.25 mM. All observed mutations except one were located outside the direct ligand-binding pocket, suggesting they were compensatory and helping protein folding or functional behavior other than interaction with the ligand. Our results thus demonstrate that combinations of ligand-binding-pocket redesign and randomized mutagenesis can indeed lead to the selection and recovery of periplasmic-binding protein mutants with non-natural compound recognition. However, current lack of understanding of the intermolecular movement and ligand-binding in periplasmic binding proteins such as RbsB are limiting the rational production of further and better sensory mutants.

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

Scientific Reports ◽

10.1038/s41598-019-53507-5 ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Diogo Tavares ◽

Artur Reimer ◽

Shantanu Roy ◽

Aurélie Joublin ◽

Vladimir Sentchilo ◽

...

Keyword(s):

Escherichia Coli ◽

Ligand Binding ◽

Binding Proteins ◽

Binding Pocket ◽

Wild Type ◽

Ligand Binding Pocket ◽

Mutant Proteins ◽

Periplasmic Binding Proteins ◽

Natural Ligands ◽

Ribose Binding Protein

AbstractBacterial 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.

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Computational redesign of theEscherichia coliribose-binding protein ligand binding pocket for 1,3-cyclohexanediol and cyclohexanol

10.1101/559328 ◽

2019 ◽

Author(s):

Diogo Tavares ◽

Artur Reimer ◽

Shantanu Roy ◽

Aurélie Joublin ◽

Vladimir Sentchilo ◽

...

Keyword(s):

Ligand Binding ◽

Binding Proteins ◽

Binding Protein ◽

Computational Design ◽

Binding Pocket ◽

Wild Type ◽

Ligand Binding Pocket ◽

Mutant Proteins ◽

Periplasmic Binding Proteins ◽

Natural Ligands

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 theEscherichia coliRbsB 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 anE. colireporter 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.

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Subcellular localization defects characterize ribose-binding mutant proteins with new ligand properties in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.02117-21 ◽

2021 ◽

Author(s):

Diogo Tavares ◽

Jan R. van der Meer

Keyword(s):

Escherichia Coli ◽

Subcellular Localization ◽

Ligand Binding ◽

Binding Proteins ◽

Binding Protein ◽

Binding Pocket ◽

Binding Properties ◽

General Properties ◽

Periplasmic Binding Proteins ◽

Ribose Binding Protein

Periplasmic-binding proteins have been previously proclaimed as a general scaffold to design sensor proteins with new recognition specificities for non-natural compounds. Such proteins can be integrated in bacterial bioreporter chassis with hybrid chemoreceptors to produce a concentration-dependent signal after ligand binding to the sensor cell. However, computationally designed new ligand-binding properties ignore the more general properties of periplasmic binding proteins, such as their periplasmic translocation, dynamic transition of open and closed forms, and interactions with membrane receptors. In order to better understand the roles of such general properties in periplasmic signaling behaviour, we study here the subcellular localization of ribose-binding protein (RbsB) in Escherichia coli in comparison to a recently evolved set of mutants designed to bind 1,3-cyclohexanediol. As proxies for localization we calibrate and deploy C-terminal end mCherry fluorescent protein fusions. Whereas RbsB-mCherry coherently localized to the periplasmic space and accumulated in (periplasmic) polar regions depending on chemoreceptor availability, mutant RbsB-mCherry expression resulted in high fluorescence cell-to-cell variability. This resulted in higher proportions of cells devoid of clear polar foci and of cells with multiple fluorescent foci elsewhere, suggesting poorer translocation, periplasmic autoaggregation and mislocalization. Analysis of RbsB mutants and mutant libraries at different stages of directed evolution suggested overall improvement to more RbsB-wild-type-like characteristics, which was corroborated by structure predictions. Our results show that defects in periplasmic localization of mutant RbsB proteins partly explains their poor sensing performance. Future efforts should be directed to predicting or selecting secondary mutations outside computationally designed binding pockets that take folding, translocation and receptor-interactions into account. Importance Biosensor engineering relies on transcription factors or signaling proteins to provide the actual sensory functions for the target chemicals. Since for many compounds there are no natural sensory proteins, there is a general interest in methods that could unlock routes to obtaining new ligand-binding properties. Bacterial periplasmic-binding proteins (PBPs) form an interesting family of proteins to explore to this purpose, because there is a large natural variety suggesting evolutionary trajectories to bind new ligands. PBPs are conserved and amenable to accurate computational binding pocket predictions. However, studying ribose-binding protein in Escherichia coli we discovered that designed variants have defects in their proper localization in the cell, which can impair appropriate sensor signaling. This indicates that functional sensing capacity of PBPs cannot be obtained solely through computational design of the ligand-binding pocket, but must take other properties of the protein into account, which are currently very difficult to predict.

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Tryptophan-scanning mutagenesis of the ligand binding pocket in Thermotoga maritima arginine-binding protein

Biochimie ◽

10.1016/j.biochi.2013.12.011 ◽

2014 ◽

Vol 99 ◽

pp. 208-214 ◽

Cited By ~ 11

Author(s):

Lindsay J. Deacon ◽

Hilbert Billones ◽

Anne A. Galyean ◽

Teraya Donaldson ◽

Anna Pennacchio ◽

...

Keyword(s):

Ligand Binding ◽

Thermotoga Maritima ◽

Binding Protein ◽

Binding Pocket ◽

Ligand Binding Pocket

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An improved reversibly dimerizing mutant of the FK506-binding protein FKBP

10.1101/053751 ◽

2016 ◽

Author(s):

Juan J. Barrero ◽

Effrosyni Papanikou ◽

Jason C. Casler ◽

Kasey J. Day ◽

Benjamin S. Glick

Keyword(s):

Ligand Binding ◽

Secretory Pathway ◽

Binding Protein ◽

Binding Pocket ◽

Ligand Binding Pocket ◽

Fk506 Binding Protein ◽

Ligand Addition

FK506-binding protein (FKBP) is a monomer that binds to FK506, rapamycin, and related ligands. The F36M substitution, in which Phe36 in the ligand-binding pocket is changed to Met, leads to formation of antiparallel FKBP dimers, which can be dissociated into monomers by ligand binding. This FKBP(M) mutant has been employed in the mammalian secretory pathway to generate aggregates that can be dissolved by ligand addition to create cargo waves. However, when testing this approach in yeast, we found that dissolution of FKBP(M) aggregates was inefficient. An improved reversibly dimerizing FKBP formed aggregates that dissolved more readily. This FKBP(L,V) mutant carries the F36L mutation, which increases the affinity of ligand binding, and the I90V mutation, which accelerates ligand-induced dissociation of the dimers. The FKBP(L,V) mutant expands the utility of reversibly dimerizing FKBP.

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Locostatin Disrupts Association of Raf Kinase Inhibitor Protein With Binding Proteins by Modifying a Conserved Histidine Residue in the Ligand-Binding Pocket

Forum on Immunopathological Diseases and Therapeutics ◽

10.1615/forumimmundisther.v2.i1.60 ◽

2011 ◽

Vol 2 (1) ◽

pp. 47-58 ◽

Cited By ~ 9

Author(s):

Anwar B. Beshir ◽

Christian E. Argueta ◽

Lochana C. Menikarachchi ◽

Jose A. Gascon ◽

Gabriel Fenteany

Keyword(s):

Ligand Binding ◽

Binding Proteins ◽

Kinase Inhibitor ◽

Binding Pocket ◽

Histidine Residue ◽

Inhibitor Protein ◽

Ligand Binding Pocket ◽

Raf Kinase ◽

Raf Kinase Inhibitor Protein

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Mechanical Unfolding of Ribose Binding Protein and Its Comparison with Other Periplasmic Binding Proteins

The Journal of Physical Chemistry B ◽

10.1021/jp507463q ◽

2014 ◽

Vol 118 (39) ◽

pp. 11449-11454 ◽

Cited By ~ 6

Author(s):

Hema Chandra Kotamarthi ◽

Satya Narayan ◽

Sri Rama Koti Ainavarapu

Keyword(s):

Binding Proteins ◽

Binding Protein ◽

Periplasmic Binding Proteins ◽

Ribose Binding Protein

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Gene Regulatory Potential of Nonsteroidal Vitamin D Receptor Ligands

Molecular Endocrinology ◽

10.1210/me.2004-0417 ◽

2005 ◽

Vol 19 (8) ◽

pp. 2060-2073 ◽

Cited By ~ 36

Author(s):

Mikael Peräkylä ◽

Marjo Malinen ◽

Karl-Heinz Herzig ◽

Carsten Carlberg

Keyword(s):

Ligand Binding ◽

Cellular Proliferation ◽

Bone Mineralization ◽

Binding Pocket ◽

Drug Candidate ◽

Hydroxyl Groups ◽

Ligand Binding Pocket ◽

Natural Ligand

Abstract The seco-steroid 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] is a promising drug candidate due to its pleiotropic function including the regulation of calcium homeostasis, bone mineralization and cellular proliferation, differentiation, and apoptosis. We report here a novel class of nonsteroidal compounds, represented by the bis-aromatic molecules CD4409, CD4420, and CD4528, as ligands of the 1α,25(OH)2D3 receptor (VDR). Taking the known diphenylmethane derivative LG190178 as a reference, this study provides molecular evaluation of the interaction of nonsteroidal ligands with the VDR. All four nonsteroidal compounds were shown to induce VDR-retinoid X receptor heterodimer complex formation on a 1α,25(OH)2D3 response element, stabilize the agonistic conformation of the VDR ligand-binding domain, enable the interaction of VDR with coactivator proteins and contact with their three hydroxyl groups the same residues within the ligand-binding pocket of the VDR as 1α,25(OH)2D3. Molecular dynamics simulations demonstrated that all four nonsteroidal ligands take a shape within the ligand-binding pocket of the VDR that is very similar to that of the natural ligand. CD4528 is mimicking the natural hormone best and was found to be in vitro at least five times more potent than LG190178. In living cells, CD4528 was only two times less potent than 1α,25(OH)2D3 and induced mRNA expression of the VDR target gene CYP24 in a comparable fashion. At a noncalcemic dose of 150 μg/kg, CD4528 showed in vivo a clear induction of CYP24 expression and therefore may be used as a lead compound for the development of therapeutics against psoriasis, osteoporosis, and cancer.

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