Redesign of LAOBP to bind novel <scp>l</scp>‐amino acid ligands

Banda-Vazquez, J.; Shanmugaratnam, S.; Rodríguez‐Sotres, Rogelio; Torres‐Larios, Alfredo; Höcker, Birte; Sosa‐Peinado, Alejandro

doi:10.1002/pro.3403

Cited by 19 publications

(20 citation statements)

References 36 publications

(57 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For this reason, such proteins have been good models to study the structural basis that determine affinity and selectivity in proteins [6,[15][16][17][18][19][20][21][22][23][24][25]. These features have made PBPs a suitable target to develop biosensors [14,[26][27][28][29][30][31] and design new binding abilities [14,32,33].…”

Section: Introductionmentioning

confidence: 99%

The interplay of protein–ligand and water‐mediated interactions shape affinity and selectivity in the LAO binding protein

et al. 2019

View full text Add to dashboard Cite

The study of binding thermodynamics is essential to understand how affinity and selectivity are acquired in molecular complexes. Periplasmic binding proteins (PBPs) are macromolecules of biotechnological interest that bind a broad number of ligands and have been used to design biosensors. The lysine‐arginine‐ornithine binding protein (LAO) is a PBP of 238 residues that binds the basic amino acids l‐arginine and l‐histidine with nm and μm affinity, respectively. It has been shown that the affinity difference for arginine and histidine binding is caused by enthalpy, this correlates with the higher number of protein–ligand contacts formed with arginine. In order to elucidate the structural bases that determine binding affinity and selectivity in LAO, the contribution of protein–ligand contacts to binding energetics was assessed. To this end, an alanine scanning of the LAO‐binding site residues was performed and arginine and histidine binding were characterized by isothermal titration calorimetry and X‐ray crystallography. Although unexpected enthalpy and entropy changes were observed in some mutants, thermodynamic data correlated with structural information, especially, the binding heat capacity change. We found that selectivity is conferred by several residues rather than exclusive arginine–protein interactions. Furthermore, crystallographic structures revealed that protein–ligand contributions to binding thermodynamics are highly influenced by the solvent. Finally, we found a similar backbone conformation in all the closed structures obtained, but different structures in the open state, suggesting that the binding site residues of LAO play an important role in stabilizing not only the holo conformation, but also the apo state. Database Structural data are available in the Protein Data Bank database under the accession numbers http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6MLE, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6MLN, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6MLG, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6MKX, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6MLI, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6MLA, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6MKU, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6MKW, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6ML0, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6MLD, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6MLV, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6MLO, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6MLP, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6ML9, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6MLJ.

show abstract

Section: Introductionmentioning

confidence: 99%

The interplay of protein–ligand and water‐mediated interactions shape affinity and selectivity in the LAO binding protein

et al. 2019

View full text Add to dashboard Cite

show abstract

“…4B, C). This is indicative for poor binding, but a K D of 5 μM is in the range of measured affinity (1.6 μM) of a grafted L-glutamine-binding domain on the Salmonella typhimurium LAO periplasmic binding protein 15 . Finally, circular dichroism spectroscopy and secondary structure fold-decomposition using a recent new approach 24 indicated structural changes to occur in DT016 upon 13CHD addition (Fig.…”

Section: Discussionmentioning

confidence: 88%

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

Tavares

Reimer

Roy

et al. 2019

Preprint

View full text Add to dashboard Cite

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...

show abstract

“…There are numerous examples of new functions being engineered into natural proteins using these methods [74][75][76], including opioid binders [77], an amino-acid binder [78] and Schiff-base-forming enzyme [79]. A related approach mimics nature by combining larger protein fragments [80] and has proven successful for generating non-functional de novo proteins [81,82].…”

Section: Fragment-based Computational Design Beyond Protein Engineeringmentioning

confidence: 99%

Towards functional de novo designed proteins

Dawson

Rhys

Woolfson

2019

Current Opinion in Chemical Biology

View full text Add to dashboard Cite

Our ability to design completely de novo proteins is improving rapidly. This is true of all three main approaches to de novo protein design, which we define as: minimal, rational and computational design. Together, these have delivered a variety of protein scaffolds characterised to high resolution. This is truly impressive and a major advance from where the field was a decade or so ago. That all said, significant challenges in the field remain. Chief amongst these is the need to deliver functional de novo proteins. Such designs might include selective and/or tight binding of specified small molecules, or the catalysis of entirely new chemical transformations. We argue that, whilst progress is being made, solving such problems will require more than simply adding functional side chains to extant de novo structures. New approaches will be needed to target and build structure, stability and function simultaneously. Moreover, if we are to match the exquisite control and subtlety of natural proteins, design methods will have to incorporate multi-state modelling and dynamics. This will require more than black-box methodology, specifically increased understanding of protein conformational changes and dynamics will be needed.

show abstract

Redesign of LAOBP to bind novel l‐amino acid ligands

Cited by 19 publications

References 36 publications

The interplay of protein–ligand and water‐mediated interactions shape affinity and selectivity in the LAO binding protein

The interplay of protein–ligand and water‐mediated interactions shape affinity and selectivity in the LAO binding protein

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

Towards functional de novo designed proteins

Contact Info

Product

Resources

About