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

Abstract

Tel: +41 21 692 5630. 14 15 Running title: Ligand binding pocket redesign of Ribose-binding protein 16 Keywords: Rosetta, RbsB, ribose, 1,3-cyclohexanediol, reporter bacteria, fluorescence-assisted bead 17 sorting, protein folding 18 19 20 2 21 Bacterial periplasmic-binding proteins have been acclaimed as general biosensing platform, but 22 their range of natural ligands is too limited for optimal development of chemical compound 23 detection. Computational redesign of the ligand-binding pocket of periplasmic-binding proteins 24 may yield variants with new properties, but, despite earlier claims, genuine changes of specificity 25 to non-natural ligands have so far not been achieved. In order to better understand the reasons of 26 such limited success, we revisited here the Escherichia coli RbsB ribose-binding protein, aiming to 27 achieve perceptible transition from ribose to structurally related chemical ligands 1,3-28 cyclohexanediol and cyclohexanol. Combinations of mutations were computationally predicted for 29 nine residues in the RbsB binding pocket, then synthesized and tested in an E. coli reporter chassis. 30 Two million variants were screened in a microcolony-in-bead fluorescence-assisted sorting 31 procedure, which yielded six mutants no longer responsive to ribose but with 1.2-1.5 times 32 induction in presence of 1 mM 1,3-cyclohexanediol, one of which responded to cyclohexanol as 33 well. Isothermal microcalorimetry confirmed 1,3-cyclohexanediol binding, although only two 34 mutant proteins were sufficiently stable upon purification. Circular dichroism spectroscopy 35 indicated discernable structural differences between these two mutant proteins and wild-type 36 RbsB. This and further quantification of periplasmic-space abundance suggested most mutants to 37 be prone to misfolding and/or with defects in translocation compared to wild-type. Our results 38 thus affirm that computational design and library screening can yield RbsB mutants with 39 recognition of non-natural but structurally similar ligands. The inherent arisal of protein instability 40 or misfolding concomitant with designed altered ligand-binding pockets should be overcome by 41 new experimental strategies or by improved future protein design algorithms. 42 43 Periplasmic binding proteins (PBPs) form a versatile superfamily of proteins with a conserved protein 44 structure, named the bilobal structural fold 1,2 . PBPs facilitate nutrient and trace mineral scavenging 45 for bacterial cells, by binding the ligand in the periplasmic space at high affinity and delivering the 46 bound-ligand to a specific membrane-spanning transport channel 2 . Some PBPs are additionally 47 involved in chemotactic sensing and interact in the ligand-bound state with a membrane-located 48 chemoreceptor 3 . The crystal structures of several PBPs have been determined, showing two domains 49 connected by a hinge region, with the binding pocket located between the two domains 3 . Both 50 structure and nuclear-magnetic resonance data indicate that PBPs switch betw...