Change in protein-ligand specificity through binding pocket grafting

Scheib, U.; Shanmugaratnam, S.; Farías-Rico, José Arcadio; Höcker, Birte

doi:10.1016/j.jsb.2013.06.002

Cited by 22 publications

(24 citation statements)

References 27 publications

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“…Table 4 collects ligand-binding data for selected PBPs that have been characterized calorimetrically (a comprehensive compilation of ITC data is presented in Table S1). Clearly, the thermodynamic signatures observed in the PBP family are widely diverse (Davies et al, 1999;Sleigh et al, 1999;Cadieux et al, 2002;Bevers et al, 2006;Abbott and Boraston, 2007;Prajapati et al, 2007;Zhang et al, 2007;Schreier et al, 2009;Mattle et al, 2010;Stamp et al, 2011;Aryal et al, 2012;Ortega et al, 2012;Hu et al, 2013;Salmon et al, 2013;Scheib et al, 2014). Tungsten transport protein A and periplasmic heme binding protein show the largest affinities; for both proteins, picomolar affinity is achieved on the basis of a largely favorable entropy change.…”

Section: Enthalpy and Entropy Changesmentioning

confidence: 99%

On the molecular basis of the high affinity binding of basic amino acids to LAOBP, a periplasmic binding protein from Salmonella typhimurium

Pulido

Silva

Téllez

et al. 2015

J of Molecular Recognition

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The rational designing of binding abilities in proteins requires an understanding of the relationship between structure and thermodynamics. However, our knowledge of the molecular origin of high-affinity binding of ligands to proteins is still limited; such is the case for l-lysine-l-arginine-l-ornithine periplasmic binding protein (LAOBP), a periplasmic binding protein from Salmonella typhimurium that binds to l-arginine, l-lysine, and l-ornithine with nanomolar affinity and to l-histidine with micromolar affinity. Structural studies indicate that ligand binding induces a large conformational change in LAOBP. In this work, we studied the thermodynamics of l-histidine and l-arginine binding to LAOBP by isothermal titration calorimetry. For both ligands, the affinity is enthalpically driven, with a binding ΔCp of ~-300 cal mol(-1) K(-1) , most of which arises from the burial of protein nonpolar surfaces that accompanies the conformational change. Osmotic stress measurements revealed that several water molecules become sequestered upon complex formation. In addition, LAOBP prefers positively charged ligands in their side chain. An energetic analysis shows that the protein acquires a thermodynamically equivalent state with both ligands. The 1000-fold higher affinity of LAOBP for l-arginine as compared with l-histidine is mainly of enthalpic origin and can be ascribed to the formation of an extra pair of hydrogen bonds. Periplasmic binding proteins have evolved diverse energetic strategies for ligand recognition. STM4351, another arginine binding protein from Salmonella, shows an entropy-driven micromolar affinity toward l-arginine. In contrast, our data show that LAOBP achieves nanomolar affinity for the same ligand through enthalpy optimization.

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Section: Enthalpy and Entropy Changesmentioning

confidence: 99%

On the molecular basis of the high affinity binding of basic amino acids to LAOBP, a periplasmic binding protein from Salmonella typhimurium

Pulido

Silva

Téllez

et al. 2015

J of Molecular Recognition

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“…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

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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.

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“…Such a division between a stabilizing and a variable part can also be found in other binders with broad ligand spectra. For example, the periplasmic binding protein PotF shows affinity to different polyamines 3 . In this case an invariable aromatic box stabilizes the hydrophobic middle part of the ligands.…”

Section: "Indole-based" Promiscuitymentioning

confidence: 99%

“…A molecular understanding of how different ligands bind to the same receptor provides insight into how nature tunes affinity and modulates biological functioning 1 . In addition, such promiscuity is a vantage point for rationally designing protein specificity to molecules of interest [2][3][4] . For the tryptophan repressor (TrpR) such promiscuity is firmly established: Next to its native ligand tryptophan (TRP) TrpR possesses a number of corepressors and pseudo-repressors ( Figure 1A) that share the indole-group but have different substituents 5 resulting in different affinities of the ligand to TrpR and of TrpR to its operator DNA 6 .…”

Section: Introductionmentioning

confidence: 99%

Ligand promiscuity in the tryptophan repressor – from structural understanding towards rational design

Stiel

Shanmugaratnam

Herud-Sikimic

et al. 2020

Preprint

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Receptors that promiscuously bind a range of ligands provide insights into how nature mediates affinity and biological functioning. Moreover, such receptors provide vantage points for the rational design of specific binding for biotechnological applications. Here we describe the molecular details of the ligand binding promiscuity of the well-known tryptophan repressor TrpR. We elucidated high-resolution structures of TrpR bound to the co-repressors 5-methyltryptophan and 5-methyl-tryptamine as well as the pseudo-repressors indole-3-propionic and indole-3-acetic acid. Furthermore, using isothermal titration calorimetry we procure the corresponding thermodynamic parameters. Together this data provides molecular explanations for the strongly varied affinities and biological effects of the ligands providing insights into how nature shapes specificity and affinity. Beyond this we use these insights to exemplarily showcase knowledge-based design of TrpR by swapping its specificity from its native ligand tryptophan to indole-3-acetic acid. Finally, we elucidate the structures of the variant bound to indole-3-acetic and indole-3-propionic acid to retrace our design rationale.

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Change in protein-ligand specificity through binding pocket grafting

Cited by 22 publications

References 27 publications

On the molecular basis of the high affinity binding of basic amino acids to LAOBP, a periplasmic binding protein from Salmonella typhimurium

On the molecular basis of the high affinity binding of basic amino acids to LAOBP, a periplasmic binding protein from Salmonella typhimurium

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

Ligand promiscuity in the tryptophan repressor – from structural understanding towards rational design

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