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Alteration of Protein Binding Affinities by Aqueous Two-Phase Systems Revealed by Pressure Perturbation.

ABSTRACT: Interactions between proteins and ligands, which are fundamental to many biochemical processes essential to life, are mostly studied at dilute buffer conditions. The effects of the highly crowded nature of biological cells and the effects of liquid-liquid phase separation inducing biomolecular droplet formation as a means of membrane-less compartmentalization have been largely neglected in protein binding studies. We investigated the binding of a small ligand (ANS) to one of the most multifunctional proteins, bovine serum albumin (BSA) in an aqueous two-phase system (ATPS) composed of PEG and Dextran. Also, aiming to shed more light on differences in binding mode compared to the neat buffer data, we examined the effect of high hydrostatic pressure (HHP) on the binding process. We observe a marked effect of the ATPS on the binding characteristics of BSA. Not only the binding constants change in the ATPS system, but also the integrity of binding sites is partially lost, which is most likely due to soft enthalpic interactions of the BSA with components in the dense droplet phase of the ATPS. Using pressure modulation, differences in binding sites could be unravelled by their different volumetric and hydration properties. Regarding the vital biological relevance of the study, we notice that extreme biological environments, such as HHP, can markedly affect the binding characteristics of proteins. Hence, organisms experiencing high-pressure stress in the deep sea need to finely adjust the volume changes of their biochemical reactions in cellulo.

SUBMITTER: Oliva R

PROVIDER: S-EPMC7228918 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Alteration of Protein Binding Affinities by Aqueous Two-Phase Systems Revealed by Pressure Perturbation.

Oliva Rosario R Banerjee Sudeshna S Cinar Hasan H Ehrt Christiane C Winter Roland R

Scientific reports 20200515 1

Interactions between proteins and ligands, which are fundamental to many biochemical processes essential to life, are mostly studied at dilute buffer conditions. The effects of the highly crowded nature of biological cells and the effects of liquid-liquid phase separation inducing biomolecular droplet formation as a means of membrane-less compartmentalization have been largely neglected in protein binding studies. We investigated the binding of a small ligand (ANS) to one of the most multifuncti ...[more]

PMID: 32415277

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Project description:The rapid growth of structural information for G-protein-coupled receptors (GPCRs) has led to a greater understanding of their structure, function, selectivity, and ligand binding. Although novel ligands have been identified using methods such as virtual screening, computationally driven lead optimization has been possible only in isolated cases because of challenges associated with predicting binding free energies for related compounds. Here, we provide a systematic characterization of the performance of free-energy perturbation (FEP) calculations to predict relative binding free energies of congeneric ligands binding to GPCR targets using a consistent protocol and no adjustable parameters. Using the FEP+ package, first we validated the protocol, which includes a full lipid bilayer and explicit solvent, by predicting the binding affinity for a total of 45 different ligands across four different GPCRs (adenosine A2AAR, β1 adrenergic, CXCR4 chemokine, and δ opioid receptors). Comparison with experimental binding affinity measurements revealed a highly predictive ranking correlation (average spearman ρ = 0.55) and low root-mean-square error (0.80 kcal/mol). Next, we applied FEP+ in a prospective project, where we predicted the affinity of novel, potent adenosine A2A receptor (A2AR) antagonists. Four novel compounds were synthesized and tested in a radioligand displacement assay, yielding affinity values in the nanomolar range. The affinity of two out of the four novel ligands (plus three previously reported compounds) was correctly predicted (within 1 kcal/mol), including one compound with approximately a tenfold increase in affinity compared to the starting compound. Detailed analyses of the simulations underlying the predictions provided insights into the structural basis for the two cases where the affinity was overpredicted. Taken together, these results establish a protocol for systematically applying FEP+ to GPCRs and provide guidelines for identifying potent molecules in drug discovery lead optimization projects.

Dataset Information

Alteration of Protein Binding Affinities by Aqueous Two-Phase Systems Revealed by Pressure Perturbation.

Publications

Alteration of Protein Binding Affinities by Aqueous Two-Phase Systems Revealed by Pressure Perturbation.

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