Original Research Article

Specific Interactions between Bovine Serum Albumin and Monensin A: Molecular Description Through Theoretical Calculations

Dr. Otávio Augusto Chaves,

Otávio Augusto Chaves1,2,*, Romulo Correia Ferreira1, Robson Pacheco Pereira2

1 Chemistry Department, Universidade Federal Rural do Rio de Janeiro (UFRRJ), Rodovia BR-465, Km 7, 23970-000, Seropédica-RJ, Brazil. 

2 Instituto Militar de Engenharia (IME), Praça General Tibúrcio 80, Urca, 22290-270, Rio de Janeiro-RJ, Brazil. 

*Corresponding author: otavio_ufrrj@hotmail.com

The interaction between Bovine Serum Albumin (BSA), one of the standard proteins used to study the bioavailability of biological molecules in the bloodstream, and Monensin A (MONA), a commercial drug used in veterinary medicine, was studied by molecular docking and semi-empirical calculations.

*Corresponding author:

Corresponding author information
Dr Otávio Augusto Chaves,Chemistry Department, Universidade Federal Rural do Rio de Janeiro (UFRRJ), Rodovia BR-465, Km 7, 23970-000, Seropédica-RJ, Brazil, Instituto Militar de Engenharia (IME), Praça General Tibúrcio 80, Urca, 22290-270, Rio de Janeiro-RJ, Brazil.

 Email: otavio_ufrrj@hotmail.com



Bovine serum albumin, ionophore, monensin A, molecular docking

Monensin A (4-[2-[5-ethyl-5-[5-[6-hydroxy-6-(hydroxymethyl)-3,5-dimethyl-oxan-2-yl]-3-methyl-oxolan-2-yl]oxolan-2-yl]-9-hydroxy-2,8-dimethyl-1,6-dioxaspiro[4,5]dec-7-yl]-3-methoxy-2-methyl-pentanoic acid – MONA, Figure 1) is a representative of a large group of natural polyether ionophore antibiotic, with empirical formula C36H62O11 and pKa = 4.30 [1]. It can be isolated from Streptomyces cinnamonensis bacteria and is related to the crown ethers with a preference to form complexes with monovalent cations such as: Li+, Na+, K+, Rb+, Ag+ and Ti+,  being able to form pseudomacrocyclic complexes to transport some cations across lipid membranes [2]. Antibiotics are commonly used as growth promoters in animal husbandry worldwide. This practice has been linked to the emergence of particular antibiotic-resistant bacteria, and is now controversial [3]. Ionophores were approved by the United States Food and Drug Administration in the mid-1970s as feed additives for livestock, and since then their use has become routine in the feeding of growing ruminants [4]. MONA is representative of a large group of naturally occurring polyether ionophorous antibiotics [1], commonly used as coccidiostat for poultry and nonhormonal growth promoter agent for cattle [5]. MONA can increase the rumen proportion of propionate and decrease rumen proportions of acetate, butyrate and lactate of lactating cows fed forage and grain diets [6].

Bovine serum albumin (BSA) is a protein present in the bovine circulatory system, accounting for approximately 60% of the total plasma proteins, being the main responsible for the distribution and excretion of several endogenous and exogenous compounds, e.g. fatty acids, prostaglandins, steroids, cholesterol, hormones, vitamins, and drugs [7]. BSA is a single-chain protein formed by 582 amino acids with 2 tryptophan residues (Trp-134 and Trp-212). The BSA structure consists of three domains (I, II and III) and each domain is divided in two sub-domains (A and B) [8]. Extensive investigations on the interaction between serum albumin and natural or synthetic compounds showing biological activity have been reported [9-11]. Since serum albumin serves as a carrier for drugs and plays a dominant role on their bioavailability (exhibiting a significant impact on drug pharmacokinetics), the study of albumin:drug interactions is of extreme importance. Therefore, the effectiveness of these compounds as pharmacologically active molecules depends on their binding ability [12]. Despite the reports of medicinal importance and activities of MONA and its derivatives, in the treatment of sick animals [1,3-6], there are no reports on the BSA:MONA interactions. In order to offer a molecular level description for these interactions involving BSA and MONA, by estimating the role of specific chemical groups and their interactions in the complex stabilization, molecular docking and semi-empirical calculations were carried out. These theoretical studies will provide relevant aspects for the proper and comprehensive understanding the biodistribution of MONA, a commercially available drug used in veterinary medicine.

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Figure 1: (A) Molecular structure and (B) Potential surface of Monensin A (MONA). Both structures were built and energy-minimized using the Density Functional Theory (DFT) method B3LYP/6-31G*, at pH = 7.4, available in the Spartan'14 software (Wavefunction, Inc.). Elements colors: carbon, oxygen and hydrogen represented in black; red and white, respectively.

2.1. Molecular docking

The crystallographic structure of bovine serum albumin (with a 2.47 Å resolution) was obtained from the Protein Data Bank (PDB) with access code 4F5S [13]. MONA molecular structure was built and energy-minimized using the Density Functional Theory (DFT) hybrid method B3LYP with 6-31G* basis set, at pH = 7.4, available for the Spartan'14 software (Wavefunction, Inc.).

Molecular docking was performed with the GOLD 5.2 software (CCDC). Hydrogen atoms were added to the protein according to data inferred by the software on the ionization and tautomeric states [14]. As described in the literature, the main binding sites of BSA for different classes of molecules are: Trp-134-containing binding site (subdomain IB) and Trp-212-containing binding site (subdomain IIA) [15-17]. In order to identify, in the present work, the main binding site on the protein, a spherical domain of 10 Å radius was defined around each tryptophan residue. Docking calculations were performed with the ligand (MONA) inside each of those domains, considered as the two possible interaction sites in the system. The score of each pose identified is calculated as the negative of the sum of a series of energy terms involved in the protein-ligand interaction process. In the GOLD 5.2 software a very positive fitness score corresponds to a strong binding and a less positive or even negative score corresponds to a weak or non-existing bond [18]. The number of genetic operations (crossover, migration, mutation) in each docking run used for the search procedure was set to 100,000. The software optimizes hydrogen-bond geometries by rotating all hydroxyl and amino groups of the amino acid side chains. The scoring functions used in the described procedures were ChemPLP, GoldScore, ChemScore and ASP, however, the best results were obtained with ChemPLP function, which is the default function of the GOLD 5.2 software [19]. The Student's t-test was used to determine if the difference between any two data sets was statistically significant. The figures of the docking poses to the largest docking score value were generated with the PyMOL Delano Scientific LLC software.

2.2. Semi-empirical calculations

Although molecular docking procedure has proven to be an efficient method for determination of binding geometries, resulting in geometries in good agreement to the corresponding crystallographic data, the docking scores are not comparable to experimental data [18]. A semi-empirical molecular orbital method was employed to evaluate the interaction enthalpy associated to the best docking geometry. In order to determine the theoretical interaction enthalpy, the Trp-134-containing binding site was initially defined by selecting, for the highest-ranked BSA:MONA docking complex, all residues located into a 5 Å radius sphere around the ligand, using the DeepView-Swiss-PdbViewer 4.1 software (Swiss Institute of Bioinformatics, Lausanne, Switzerland). The selected Trp-134-containing binding site was composed by the following amino-acid residues: Leu-14, Gly-15, Glu-16, Glu-17, His-18, Phe-126, Lys-127, Ala-128, Asp-129, Glu-130, Lys-131, Trp-134, Asn-158, Asn-161, Gly-162, Gln-165, Leu-282 and Leu-283.

The BSA:MONA complex, BSA binding site and MONA molecular structures were subsequently optimized with the semi-empirical molecular orbital PM7 method [20], which is available from the MOPAC2012™ software (Molecular Orbital PACkage, Stewart Computational Chemistry, Colorado Springs, CO, USA). For BSA:MONA complex, BSA cavity and free MONA, the total charge values were -3, -2 and -1, respectively. From the molecular docking results, part of the ligand structure was more exposed to aqueous phase than trapped inside the protein cavity. Since the Trp-134-containing binding site is a pocket located in a cavity near the protein surface, the dielectric constant (ε=78.4) used to the continuum model should probably be the best approximation for this description.

After a previous geometry optimization of all hydrogen atoms of each structure, the amino acid side chains and ligand geometries were completely optimized to obtain the enthalpy values for the BSA:MONA complex, BSA binding site and MONA. In order to obtain the theoretical interaction enthalpy, the difference between the complex enthalpy and the sum of BSA empty site and MONA enthalpy values was calculated.

BSA contains only two tryptophan residues: Trp-134 located at the protein surface, in the sub-domain IB and Trp-212 inside a protein cavity, located in the sub-domain IIA. Table 1 shows all the molecular docking fitness scores for Trp-134 and Trp-212-containing binding site (obtained for ChemPLP, GoldScore, ChemScore and ASP functions). The less positive fitness scores obtained for GoldScore (Trp-212-containing binding site), ChemScore and ASP functions, or even negatives fitness scores obtained for GoldScore function (Trp-212-containing binding site) correspond to a weak or non-existing binding. On the other hand, very positive fitness scores, which resulted in the largest docking score average, were obtained with ChemPLP function, therefore indicating ChemPLP as the best scoring function for the BSA:MONA interaction description.

Docking score average suggests that MONA interacts more favorably with BSA into the site containing the Trp-134 residue (docking score average 47.7) than in the site containing the Trp-212 residue (docking score average 36.4). Since a t-test is any statistical hypothesis test in which the test statistic follows a Student's t-distribution under the null hypothesis, it can be used to determine if two sets of data are significantly different from each other. In order to give a statistically significant difference for the theoretical results, a Student's t-test was performed: as the p value (1.10x10-3) is less than 0.05 (95% confidence interval), the null hypothesis might be rejected, therefore there is a statistically significant difference between the two protein binding sites [21]. Generally, ligands which interact preferentially with the site IB are dicarboxylic acids and/or bulky heterocyclic molecules with a negative charge or containing azo and/or sulfur groups (e.g. phenylbutazone, azapropazone, tolbutamide, bucolome and sulfisoxazole), whose structural characteristics are similar to those in MONA structure [21, 22]. Furthermore, site IB is the main binding site in BSA due the high kinetic volume of the ligand which inhibits its docking inside the other protein cavity (site IIA).

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Figure 2: (A) Representation of molecular surface of BSA site IB. (B) Best score pose and van der Waals surface for MONA in BSA (site IB), obtained after molecular docking (ChemPLP function). Carbon: pink (MONA), light blue (selected amino acid residues), green (BSA), hydrogen: white; oxygen: red; and nitrogen: dark blue.



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Figure 3 shows the intermolecular interaction between MONA and the amino acid residues at site IB. The molecular docking suggest that the peptidic NH hydrogen of the Glu-17 residue interacts via hydrogen bonding with the carboxylic group of MONA, with a distance of 3.09 Å and the peptidic NH hydrogen of Glu-130 is a donor for hydrogen bonding to the hydroxyl group of the ligand, with a distance of 2.94 Å. Finally, Lys-131 residue interacts with MONA via hydrogen bonding by the peptidic NH hydrogen interacting with one hydroxyl group of the ligand with a distance of 2.13 Å and the other hydroxyl group of MONA structure interacting with the amino group of the amino acid residue, with a distance of 1.75 Å.

Figure 3: Best score pose for the BSA:MONA interaction in the site IB, obtained after molecular docking (ChemPLP function). Carbon: pink (MONA), light blue (selected amino acids residues), green (BSA), hydrogen: white; oxygen: red; and nitrogen: dark blue.



The semi-empirical calculations were employed to evaluate the interaction enthalpy associated to the best docking geometry in the Trp-134-containing binding site. According to the following representation of the complex formation and Equation 1, the interaction enthalpy change value was determined:

ΔHint = HBSA:MONA – (HBSAcavity + HMONA) (Eq.1)


in which HBSA:MONA is the enthalpy value for the complex (-6418 kJ/mol), HBSA is the enthalpy value for the Trp-134-containing binding site (-3503 kJ/mol) and HMONA is the enthalpy value for the ligand (-2895 kJ/mol). The calculated interaction enthalpy change value was -20.0 kJ/mol, suggesting that the binding process is exothermic and probably enthalpy driven [18]. According to Ross and Subramanian theory [23], the ΔH<0 also might suggest that hydrogen bonding formation is a specific interaction responsible for the stabilization of the BSA:MONA complex. The molecular docking results, as previously discussed, suggested this specific interaction between the ligand with Glu-17, Glu-130 and Lys-131.
Overall, computational results suggest that MONA interacts with BSA through site IB, due the hydrogen bonding formation in the BSA:MONA complex. Comparison between MONA binding ability with other ligands in the literature that have high kinetic volume, such as plumeran indole alkaloid [21] and trans-dehydrocrotonin [24] points to site IB as the main binding site in BSA. Therefore, the serum albumin can be an effective carrier for MONA biodistribution in bovine bloodstream. The present study does not intend to establish the ultimate description of BSA:MONA specific interactions, only to point out some of the main structural characteristics involved in this system. For further and deep understanding of this system, the application of spectroscopic methods (UV-Vis absorption, steady state, time-resolved, synchronous and 3D fluorescence, circular dichroism and Fourier transform infrared) are required, in order to provide experimental details on the BSA:MONA association.



The molecular docking studies suggest that MONA can interact with the main carrier of molecules in the bovine bloodstream (BSA). The best docking function to study the BSA:MONA interaction is <em>ChemPLP</em> function and the main binding site for this interaction is the site IB, where the Trp-134 residue is found. Part of MONA molecular structure is more exposed to the aqueous media (external to the protein) than inside the protein cavity, however part of ligand structure can interact with Glu-17, Glu-130 and Lys-131 residues by hydrogen bonding. The theoretical predicted interaction enthalpy change value (ΔHint = -20.0 kJ/mol) suggests that the binding process is exothermic and probably enthalpy driven. Overall, theoretical results suggest that BSA can be an efficient carrier for biodistribution of MONA in the bovine bloodstream.


The authors gratefully acknowledge the financial support from the Brazilian agencies: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ). The authors also acknowledge Prof. Dr. Carlos Mauricio R. Sant’Anna (UFRRJ) for the computational facilities and Prof.ª Dr.ª Cristiane M. Cardoso (UFRRJ) for the generous critical of this work.

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Published: 06 January 2017

Reviewed By : Dr. Kiran K Solingapuram Sai.Dr. Yafeng Qiu.


Copyright: Copyright: © 2016 Otávio Augusto Chaves. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.