Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 217 (2019) 155–163

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Polymyxins interaction to the : A thermodynamic and computational study

A. Poursoleiman a,M.H.Karimi-Jafaria, Z. Zolmajd-Haghighi a,M.Bagheria,T.Haertléb,c, G. Rezaei Behbehani d, A. Ghasemi a, Y.Y. Stroylova e,V.I.Muronetze,A.A.Sabourya,⁎ a Institute of and Biophysics, University of Tehran, Tehran, Iran b Poznan University of Life Sciences, Department of Animal Nutrition, Poznan, Poland c Biopolymers, Interactions, Assemblies, UR 1268, Institute National de la Recherche Agronomique, Nantes, France d Chemistry Department, Imam Khomeini International University, Qazvin, Iran e Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia article info abstract

Article history: Polymyxin B and E (colistin), are a group of cationic charged cyclic antibiotic lipopeptides that are frequently used Received 18 October 2018 in the clinics to treat infections caused by the multidrug-resistant gram-negative bacteria. Since the interactions Received in revised form 18 March 2019 with the blood plasma -transport may play a critical role in determining their pharmacological and Accepted 22 March 2019 pharmacokinetic profiles, we studied the binding properties of polymyxins to the human serum albumin (HSA) Available online 25 March 2019 under simulated physiological conditions by the combination of biophysical approaches, such as isothermal titra- fl Keywords: tion calorimetry (ITC), uorescence anisotropy, circular dichroism (CD) buttressed by computational studies. The ≈ 7 −1 Colistin HSA binding to the polymyxins was relatively strong (Ka 1.0 × 10 M ). Molecular docking indicated that Polymyxin B polymyxins bind to the cleft of HSA between domains I and III via the electrostatic interactions. This evidence Isothermal titration calorimetry (ITC) was further confirmed by the entropy-driven interaction for the polymyxins bound HSA. Far UV-CD experiments Fluorescence anisotropy showed that the secondary structure of HSA doesn't alter and its stable structure is preserved. Collectively, these Simulated-annealing investigations revealed that the polymyxins bind preferentially to the partially unfolded intermediate forms of the structure; however, HSA molecule does not undergo any significant conformational changes upon binding. This is promising as it may limit the unfavorable side effects of the . On the whole, the results provide quantitative and qualitative insight of the binding interaction between HSA and polymyxins, which is important in understanding their effect as therapeutic agents. © 2019 Elsevier B.V. All rights reserved.

1. Introduction untreatable serious infections [5]. Efficacy of these antibiotics can be im- proved by understanding their and pharmacokinet- The increasing emergence of resistant bacteria to available antibi- ics, chemistry, side effects and location of the binding interaction. For otics and the lack of new antimicrobial agents poses a major challenge this purpose, the study of interactions of these peptides with plasma to medical researches [1]. Multidrug-resistant (MDR) gram-negative proteins at the molecular level is seriously needed [6]. bacteria like Pseudomonas aeruginosa, Acinetobacter baumannii,and Polymyxins are a group of antibiotics that are composed of a posi- Klebsiella pneumoniae are on a hit-list of the most dangerous pathogens tively charged cyclic heptapeptide ring (due to the presence of 5-L- and have been identified as one of the 3 greatest threats to human diaminobutyric acid (Dab), and γ-amino groups) and of a tripeptide health, according to the World Health Organization (WHO) and Infec- chain acylated on its N end with a long fatty acid (Fig. 1). Polymyxin B tious Diseases Society of America (IDSA) [2,3]. (PMB) and E (PME) are used currently to treat infections caused by Considering the fact that introduction of new antibiotics active multidrug-resistant (MDR) gram-negative bacteria. Polymyxin B and against gram-negative bacteria is unlikely in the near future [4], it is im- colistin differ in amino acid N° 6, which is DPhe in PMB and DLeu in portant to optimize the activity of already existing , such as poly- PME [7,8]. myxins, which are the last-ditch antibiotics for defense against binding to plasma proteins is a reversible reaction and there is usually an equilibrium between the concentrations of unbound and bound drugs. Among many components in the human blood plasma (e.g., HSA, α-1-acid glycoprotein (AGP), globulins and lipoproteins), ⁎ Corresponding author at: Institute of Biochemistry and Biophysics, University of Tehran, P.O. Box 13145-1384, Tehran, Iran. which interact with drugs, the serum albumin is the most important E-mail address: [email protected] (A.A. Saboury). candidate protein because it can bind drugs in relatively high quantities

https://doi.org/10.1016/j.saa.2019.03.077 1386-1425/© 2019 Elsevier B.V. All rights reserved. 156 A. Poursoleiman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 217 (2019) 155–163

Fig. 1. Chemical structures of PMB and PME. These peptides contain 10 amino acids displayed by the three-letter code for amino acids, and DAB is 1,3-diamino butyric acid. R in PMB is D- Phe and in PME is D-Leu. DAB4 has an additional amide linkage between its 3-amino group and the C-terminal carboxylate of Thr10 to form a cycle. The N terminus of DAB1 is end-capped with a long alkyl chain.

[9]. The HSA has a molecular weight of 66.5 kDa with a usual concentra- 2. Materials and methods tion of around 42 mg/mL. It is involved in binding, transportation, deposition, distribution and metabolism of a range of endogenous 2.1. Materials and exogenous (e.g. drugs) ligands, including fatty acids, amino acids, nutrients, steroids, hormones, enzymes, metal ion and numer- Albumin from human serum lyophilized powder, essentially fatty ous pharmaceuticals [10]. The crystal structure of the HSA revealed a acid free and globulin-free (N99% purity), dialysis membranes (cut-off, single non-glycosylated chain protein with 585 amino acids residue 12,000 Da), polymyxin E, and polymyxin B were purchased from the composed of three structurally homologous α-helical domains (I, II, Sigma-Aldrich (USA). Dimethyl sulfoxide (DMSO) and Nile Red were andIII),eachconsistingoftwosubdomains(namedAandB).Also, from Merck (Germany). Fluorescein 5-isothiocyanate (FITC) was ob- the HSA consists of an equilateral triangle with sides of about 80 Å tained from Thermo-Fisher Scientific(USA). and a depth of about 30 Å with 17 disulfide bridges, one free thiol (Cys34) and a single tryptophan residue (Trp214), which are located 2.2. Preparation of solutions in domain IIA [11]. Crystallographic studies revealed several drug binding sites in the HSA including: sites 1, 2 and 3 (located in The HSA stock solution (20 μM) was freshly prepared in the phos- subdomains IIA, IIIA and IB, respectively), site 4 (between phate buffer of pH 7.4. The pH was adjusted with a suitably standardized subdomains IIB and IIA), site 5 (between subdomains IB and IIA) using Orion-868 pH meter (Thermo Scientific, USA) at room tempera- and site 6 (on the interface between subdomains IIIA and IIIB). ture. The concentration of HSA was measured by NanoDrop 2000/ Also, there is a cleft between domains I and III. Besides, nine fatty 2000c spectrophotometer (Thermo Fisher Scientific, USA) using the ex- acid sites (FAs) are localized on the HSA [12,13]. tinction coefficients of (36,500 M−1 cm−1) at 280 nm [22]. The poly- Determination of the equilibrium (drug binding) constants is one of myxins' working solution was prepared by dissolving appropriate the major aspects of pharmacokinetic and pharmacodynamics studies amounts of the peptides in the same buffer. [14] because only the free drug fraction is able to exert the biological ac- tion and/or to show pharmacological and toxicological effects [9,15]. As 2.3. Isothermal titration calorimetry (ITC) disruption of biological activity and the possible side effect would be the result of irreversible structural changes in the protein molecule, further Experiments on binding interaction of the polymyxins with HSA research and thorough knowledge around plasma protein and drugs in- were performed using a VP-ITC Microcalorimeter (MicroCal, LLC, teractions, is extremely needed to find the optimal dose of drug admin- USA). The sample cell was filled with 1.8 mL of protein solution istration [16]. (0.21 mM) and the syringe was filled with 300 μLofpolymyxinsolution As the of polymyxins is complex and there is an ev- (2 mM). The samples were degassed under vacuum prior to using for ident scarcity of pharmacological and pharmacokinetic knowledge ITC runs. The reference cell was filled with 0.1 M phosphate buffer about the behavior and fate of polymyxins [5,17,18]. Therefore, (pH 7.4). The system was allowed to equilibrate and a stable baseline re- deeper understanding of the plasma protein binding of polymyxins corded before initiating an automated titration. The titration of the pro- is badly needed. Several studies of the binding of cationic peptides tein with polymyxins involved 30 consecutive injections of 10 μL by plasma proteins have been reported for example binding of poly- polymyxin solution with 3-min intervals. The first injection was set to myxins by BSA (Bovine Serum Albumin) and AGP and binding of 5 μL. The cell content was stirred constantly at 307 rpm. The ITC titra- short cationic antimicrobial peptides by HSA [19,20]. Investigations tions were performed at 25 °C. The enthalpies of colistin + HSA interac- of polymyxins interactions with plasma proteins in rats show that tions, ΔH, and the enthalpies of dilution for colistin in the buffer over 50% of polymyxins is bound by plasma proteins and the circulat- solution, ΔHdilut, are listed in Supporting information Table S1. ing drug is a minor fraction of the total concentration of the drug [19,21]. No study to date has examined the conformational changes 2.4. Circular dichroism (CD) and mechanism of the interactions of polymyxins with the HSA. For this reason, we have studied these interactions using isothermal ti- The circular dichroic spectra were recorded on Aviv 215 Spectropo- tration calorimetry, circular dichroism spectroscopy, fluorescence larimeter (USA) in the far-UV region (190–260 nm) using a 1-mm anisotropy and molecular modeling. quartz cells at 25 °C. The molar ratio of polymyxin analogs to HSA was A. Poursoleiman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 217 (2019) 155–163 157 changed from 17 to 50. The signal of buffer solution as reference was DAB4 and the C-terminal of Thr10 (see Fig. 1 and Supporting informa- subtracted from the sample spectra. The results were expressed as the tion Fig. S2). This attachment necessitated some new parameters and 2 −1 molar ellipticity in [θ]λ (deg cm dmol ), which is based on a mean all of them were extracted by analogy from other amino acids including amino acid residue weight of about 114 (MRW). The molar ellipticity alanine, glycine and threonine (see Supporting information Fig. S2). An- was determined as [θ]λ=( θobs × MRW / 10 dc), where θobs is the ob- other CHARMM topology patch was also defined for capping of the N- served ellipticity (in degrees) at a given wavelength, c is the protein terminal (DAB1) by a fatty acid chain (6-methyloctanoyic acid). All of concentration (in units of g/mL) and d is the length of the light path the new topologies and parameter definitions are provided in (cm) [23]. Percentage of secondary structures was performed using Supporting materials. The CHARMM-compatible recipes for PMB and the CDNN CD spectra deconvolution software (version 2.1) [24]. PME peptides were used in subsequent MD and SAMD simulations.

2.5. Fluorescence anisotropy measurements 2.7.2. Sampling of ligand conformations in water Full consideration of the flexibility of ligand and protein is a major Lysine residues on the surface of HSA molecules were labeled ac- challenge in calculation of energetic and structure of interactions. This cording to the previously mentioned protocol [25]. The labeling was issue becomes more critical in the case of peptide structures with achieved as follows: protein was dissolved in phosphate buffer many torsional degrees of freedom and results in the so called combina- (pH 9.0). Subsequently, the FITC was dissolved in a few drops of di- torial explosion of search space in docking problem [31–33]. To avoid methyl sulfoxide and added to the protein solution. Then, tube was this problem, a conformational search of PMB and PME in water was wrapped in foil and incubated at room temperature in the dark under considered to obtain stable conformations of these peptides and the di- gentle stirring during 8 h. Then, the solution was first extensively dia- verse set of generated structures were used separately in docking simu- lyzed against 0.1 M phosphate buffer (pH 7.0) to remove free unreacted lations. The SAMD simulations of peptides were performed in a cubic dyes using dialysis membrane (cut-off, 12,000 Da) and immediately box of TIP3P water models [34] with box size of 52 × 52 × 52 Å. Each used. Dialysis was considered complete when free FITC fluorescence in system was neutralized by addition of 5 chloride ions and minimized the outer solution was no longer detectable [25]. Fluorescence spectra for 10,000 steps of conjugated gradient method. and steady-state emission anisotropies of all HSA samples were deter- All MD simulations were performed with NAMD 2.10 package using mined with a Cary Eclipse fluorimeter (Agilent, Palo Alto, CA). Anisot- the CHARMM27 force field for proteins with the above mentioned ex- ropy was measured with a manual polarizer and the G factor tensions made to support PMB and PME peptides. An equilibration correction was done manually, with a reference fluorophore. phase was then performed for 200 ps of NPT dynamics at 300 K and Stoichiometry of labeling for HSA-FITC was determined by measur- 1 atm. A time step of 2 fs was taken with SHAKE algorithm [35]to ing the absorbance of the solutions at 280 and 490 nm and using the keep frozen all hydrogen-heavy atom bonds. Berendsen pressure bath same extinction coefficients as above for proteins and FITC. The F/P coupling [36] and Langevin dynamics [37,38] were used to maintain ratio of labeled HSA samples was computed according to standard pressure and temperature around their reference values, respectively. methods [25–27]. The anisotropy measurement of the labeled protein A cutoff of 12 Å was used for non-bonded interactions with a switching was then done in the presence of ligands (PMB and PME). (All the scheme started at 10 Å. The particle mesh Ewald method [39]wasused data are the average of three repeated measurements of anisotropy ± in calculation of long-range electrostatic interactions over adopted peri- SD). odic boundary conditions. After equilibration, five rounds of SAMD were conducted separately that each of them includes 100 cycles of cooling 2.6. Anisotropy of Nile Red the system from 800 to 300 K with a rate of 5 K/ps and minimization of the final structure for 1000 steps of conjugated gradient. Each cycle To investigate the binding interaction of the peptides and HSA, the was started by a new set of randomized velocities to enhance the diver- peptides were labeled with Nile Red and the anisotropy of the com- sity of conformational search and the final geometry was checked to en- pounds was measured using a Cary Eclipse fluorimeter (Agilent, Palo sure correct chirality of all amino acids. The whole procedure results in Alto, CA) equipped with a pair of manual polarizers. The anisotropy of 500 conformations for each of the PMB and PME systems and all of them Nile Red solution in ethanol was measured. The solution was then were used in subsequent docking simulations. added to the peptides and the anisotropy was measured again. After- wards, the labeled peptides were added to the protein and the anisot- 2.7.3. Docking of PMB and PME conformational ensemble ropy was measured again after 3 min of incubation. All the data are Initial binding modes of PMB and PME to the HSA were searched by the average of five repeated measurements of anisotropy ± SD. docking of all 500 stable conformations of ligands to the protein. A sin- gle chain complete structure of HSA was extracted from reported X-ray 2.7. Computational section structure of protein (PDB ID 1AO6 [40]). Missing hydrogen atoms were added to the structure using the REDUCE program [41] which also de- 2.7.1. CHARMM Recipes for PMB and PME cides on preferred protonation states of amino acids. The protein was The initial 3D models of the peptides were built from scratch since then solvated in a box of water and minimized for 20,000 steps of con- there is not any experimentally determined geometry for PMB and jugated gradient method while harmonic restraints were imposed on PME. All residues were built in natural L form with the exception of position of backbone atoms. The minimized structure was used in Phe6 or Leu6 which were considered as D-amino acids in PMB and docking simulations. Since there was no predefined binding site for PME, respectively. Available topologies and parameters in CHARMM27 PMB and PME peptides, the whole structure of protein was considered force field [28,29] (NAMD 2.10 program) [30] were extended to support to be searched by defining three docking boxes with enough overlap the unnatural structural components available in considered cyclic pep- (see Supporting information Fig. S3). Using 500 conformations of pep- tides. By analogy with lysine, a new CHARMM residue was defined for tide, this results in 1500 docking experiments for each of the PMB or the unusual amino acid DAB. As seen in Supporting information PME systems. The Autodock Vina docking software [42]wereused Fig. S1, two middle charge groups of lysine that contain CG and CD with an exhaustiveness value of 200. Input files of docking were pre- were deleted and atoms of the last charged group were renamed to cre- pared automatically with Python scripts provided by MGLTools [43]. ate a new topology for DAB. A new CHARMM topology patch was also Predicted complex geometries with best binding affinity on each defined to tackle the unusual cyclic structure of peptides which is not docking experiment were collected together. This results in a complex a normal N- to C-terminal attachment. Instead, the cyclic topology is ensemble of 1500 peptide-protein structures. The RMSD of alpha carbon formed via an amide bond between the amine group in side-chain of atoms of peptide structures within a cutoff of 10 Å were used to cluster 158 A. Poursoleiman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 217 (2019) 155–163 binding poses and to identify the most important binding sites of HSA Table 1 for PMB and PME. Ten structures with highest affinity over the complex Binding parameters for polymyxins-HSA interactions computed using Eq. (1). ensemble were selected for subsequent refinement with SAMD HSA-PME HSA-PMB simulations. ρ 0.76 0.86 ° δA 0.66 ± 0.02 0.61 ± 0.02 ° 2.7.4. Refinement of complexes with SAMD δB 0.50 ± 0.01 0.91 ± 0.01 − Geometry of 10 best scored complexes from docking ensemble were ΔH°/kJ mol 1 0.38 ± 0.04 −0.55 ± 0.04 Δ ° −1 used as initial structures in SAMD simulations that aim to depict a more T S /kJ mol 41.21 ± 0.33 41.00 ± 0.33 ΔG°/kJ mol−1 −40.58 ± 0.43 −40 ± 0.43 refined picture of the atomistic details of the interaction of HSA with −1 7 7 Ka/mol L 1.31 × 10 ± 1200 1.00 × 10 ± 1100 PMB and PME peptides. Such simulations were reported to be successful g (number of binding sites) 3 1.25 in the case of protein-ligand complexes [44]. The whole procedure in- cludes multiple cycles of cooling the system from high temperatures in order to reach more stable minima over the interaction energy land- binding of one molecule at one site of HSA reduces the affinity for other ' scape. Details of SAMD simulations are the same as those used for con- ligands. The xB can be expressed as follows: formational search of peptides with following differences. The solvent ν effects were included by Generalized Born Implicit Solvent (GBIS) 0 ¼ pxB ¼ ð Þ xB þ 2 model [45–48]. Non-bonded interactions were gradually switched to xA pxB g zero over a range of 14.0–16.0 Å. In GBIS model, the maximum distance between atom pairs was set to 14 Å in calculation of Born radius. Using where xB is the molar ratio of either PMB or PME that is obtained from an RMSD collective variable [49] on alpha carbon atoms, the structure of the concentration of peptide ligands after each injection ([L]) divided HSA was restrained to not deviate drastically from the minimized ge- by their maximum concentration upon saturation of all HSA ([L]max) ometry while the peptide geometry was free to change. The SAMD pro- as follows: cedure includes cooling from randomized velocities at 500 K to 50 K ½L with a rate of 5 K/ps. For each complex, 100 separate SAMD cycles ¼ ; ¼ − ð Þ xB ½ xA 1 xB 3 were performed and final geometries were minimized. All structures L max were analyzed and visualized by VMD 1.9 package [50]. and xA is the fraction of unbound HSA. In general, there will be “g” sites ν fi 3. Results and discussion for binding of polymyxins per HSA molecule and is de ned as the av- erage moles of bound polymyxins per mole of total HSA. The LA and LB 3.1. Experimental section are the relative contributions of the free and bound ligands in the en- thalpies of dilution with the exclusion of HSA. They can be calculated Δ 3.1.1. Isothermal titration calorimetry (ITC) from the enthalpies of dilution of the ligands in buffer, Hdilut as follows: We have previously shown that the heats of the biomolecules and li-   ∂ΔH ∂ΔH gands interactions in the aqueous solvent systems can be described by L ¼ ΔH þ x dilut ; L ¼ ΔH −x dilut ð4Þ A dilut B ∂ B dilut A ∂ Eq. (1) [51–54]. xB xB

 ° 0 0 0 0 0 0 The enthalpies of HSA and the colistin interactions (ΔH values, ΔH ° ¼ ΔH x −δ ° x L þ x L − δ °−δ ° x L þ x L x ð1Þ max B A A A B B B A A A B B B Supporting information Table S1) were fitted to Eq. (1) over the whole concentrations. Enthalpies of PMB-HSA interactions are not ° shown. In the fitting procedure, the only adjustable parameter (p) was ΔH are the enthalpies of polymyxins - HSA interactions, and ΔHmax is the heat value at the saturation limit with respect to HSA. changed until the best agreement between the experimental and calcu- δ° δ° The parameters δ° and δ° reflect the net effect of ligands on the HSA lated data was attained (Fig. 2). A and B parameters are calculated A B fi conformational changes in the low and high concentrations, respec- from the coef cients of the second and third terms of Eq. (1). Binding ° ° parameters for the interactions between the peptides and HSA recov- tively. The negative values for δA or δB indicate that the ligands form destabilized PME-HSA or PMB-HSA complexes. The p b 1 indicates neg- ered from Eq. (1) are reported in Table 1.TheKa is the apparent associ- ative cooperatively of the HSA molecule in binding of the peptides. The ation constant as a function of polymyxins concentrations. The Ka values was computed according to specified equation [48]. The Gibbs energies can be obtained as follows:

° ΔG ¼ −RTlnKa ð5Þ

Table 2 Fraction of secondary structure elements as calculated from the respective CD spectra of polymyxin B and polymyxin E.

PME PMB

58.0% 57.7% 57.2% %Helix 58.0% 58.1% 57.7%

22.2% 23.0% 23.8% %Beta-Sheet 22.2% 22.0% 22.2%

19.8% 19.3% 19.0% %Random.Coil Fig. 2. Comparison between the experimental heats, ΔH°, (dots) for HSA-polymyxins 19.8% 19.9% 20.1% interaction and calculated data (lines) via Eq. (1). A. Poursoleiman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 217 (2019) 155–163 159

Fig. 3. CD spectra of PMB–HSA and PME-HSA system; at the molar ratios of 0,17 and 50 polymyxins/HSA.

where T is absolute temperature (298 K) and R is the gas constant the interaction between the HSA and polymyxins is driven by entropy. (8.314 J mol−1 K−1). Moreover the TΔS° values were calculated from The changes in the molar enthalpy of the interactions are negligible the Gibbs energies. The agreement between the calculated and experi- and the ΔS° is positive for both colistin and polymyxin B binding to mental results was striking and gave considerable support to the use the HSA that points to the significant role of the electrostatic interac- of Eq. (1) (Fig. 2). tions as the major stabilizing forces in the lipopeptide–protein complex ° ° ° ° Very close δA and δB values indicate that there is no significant formation [55]. The negative values for ΔH and ΔS values are consid- changes in the HSA structure. p b 1 indicates that colistin and polymyxin ered for the van der Waals force and hydrogen bonding formation in B bind preferentially to the partially unfolded forms of the HSA low dielectric media, thus, the thermodynamic data obtained for structure. polymyxins–HSA system clearly excluded the participation of The values of the thermodynamic parameters shed light on the in- hydrophobic interactions [56]. Taken together, the binding constants termolecular forces involved in the interaction between the polymyxins showed the strong interactions between the peptides and HSA (Ka 7 −1 7 −1 and HSA. The negative sign of Gibbs energies value suggested spontane- ≈ 1.0 × 10 M for PMB, and Ka ≈ 1.31 × 10 M for PME; see ous nature of the binding reaction. The magnitude and sign of ΔS° and Table 1); however, minor changes of HSA structure observed from ΔH° are used in the predicting kind of forces that may take place in var- structural analysis by the CD and fluorescence spectroscopies suggests ious ligand–protein binding processes, as described below. In overall, the possible role of the electrostatic interactions as the main driving

Fig. 4. Profile of dihedral energy changes in the course of reaching to the first five local minima during the SAMD sampling (a). Agglomerative clustering of generated conformations of PMB in water (b). Number of conformers in each cluster was shown in each box with their representative structure depicted below them. More similar clusters are grouped in five main sets highlighted in green. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 160 A. Poursoleiman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 217 (2019) 155–163

Fig. 5. Potential energy distribution of PMB and PME conformations in water. Representative structures were depicted for selected bins in the lowest, highest and middle energy values. force for the peptide-protein association. The above results were further the literature [14]. In the presence of different concentrations of drugs supported by our molecular modeling results, as described in the rele- at the molar ratios of 0,17 and 50 drugs/HSA (the recommended daily vant section. dose of this type of product [58]), the content of α-helix, β-sheet, turns and random coil structures remains without notable change 3.1.2. Circular dichroism (CD) (Fig. 3). Circular dichroism (CD) spectroscopy can be helpful to illustrate the Several previous studies also indicate that conformational changes geometrical structure of a protein [57]. The far-UV CD spectroscopy can occur in HSA due to interaction with drug compounds, for example, be used to find changes in the secondary structure of the protein. binding of the Taxol, Lomefloxacin, Gefitinib, Chlorogenic acid, Ferulic Changes in the secondary-structure contents leads to protein structural acid, , Camptothecin, Idarubicin, , , alterations, and causes a deprivation of their biological activities. The re- Palladium(II) complexes of Imidazole and etc. with HSA [6,56,59–62]. sults of the quantitative analysis using the CDNN program [24] are given These interactions result in major protein secondary structural changes, in Table 2. The far UV-CD spectrum for the intact protein shows two known as the side effects of such drugs. In our experiment, the far UV- minima at 208 and 222, which are consistent with available data in CD shows that there is a little modification in the secondary structure

Fig. 6. The results of docking simulations clustered by their pose of interaction. The first five clusters with higher interaction energies were shown in different colors. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) A. Poursoleiman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 217 (2019) 155–163 161 upon complexation of polymyxins. The electrostatic interaction be- the peptides was interpreted as a slight conformational change in the tween polymyxins and HSA, can be one of the drugs advantages, since protein [26,27]. it may cause no change in the second and third structures of the protein. It may also prevent the consequent effects ( and other side ef- 3.1.4. FITC labeled HSA and its interaction with the peptides fects) of the conformational changes as the protein keeps its stable The labeled FITC to protein at ratio of 1.5 showed that the protein structure [16]. was only slightly labeled with FITC. The anisotropy of the solution of the labeled protein was 0.26 ± 0.001 at 512 nm as compared with the values 0.23 ± 0.006 and 0.21 ± 0.001 in the presence of PMB and 3.1.3. Anisotropy of Nile Red PME, respectively. The moderate decrease in anisotropies of the samples Nile Red was added to the peptides as a marker to study binding in- could be the sign of resonance energy transfer between the FITC mole- teractions of peptide with proteins [63]. The anisotropy of unbound Nile cules labeled to different proteins. This can be an indication of the pro- Red in ethanol was about 0.05 ± 0.002. Upon addition to peptides, the tein folding in the presence of the polypeptides that had brought the anisotropy increases to 0.10 ± 0.002. The observed increase in anisot- fluorescent dyes to a distance where the chance of resonance energy ropy was attributed to binding of Nile Red to the peptide and the conse- transfer had increased [25–27,67]. quent increase in rotational correlation time of the dye [26,63–66]. Upon addition of Nile Red-peptide solution to the protein, however, 3.2. Computational section the anisotropy changed only slightly and raised to 0.12 ± 0.002. As the peptides masses were only 2% of the molecular weight of the pro- 3.2.1. Structural and energetic diversity of conformations of polymyxins tein, the rotational correlation time of the labeled peptides should The simulated annealing molecular dynamics (SAMD) protocol gen- have more extensively increased if there were a binding interaction be- erates a diverse set of unbound conformations for PMB and PME. In tween the molecules. However, the slight increase in the anisotropy of Fig. 4A variation of dihedral energy as a measure of conformational

Fig. 7. Refined binding modes of PMB (a) and PME (b) to HSA and hotspot residues involved in specific interactions with ligand. Both binding sites correspond to the cleft of protein. Ligand and protein residues involved in hydrogen bonding are drawn in extended form while those with hydrophobic and van der Waals interactions are depicted in a compact form. 162 A. Poursoleiman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 217 (2019) 155–163 change of peptides was depicted for the first five rounds of annealing. As with the protein was entropy-driven suggesting that electrostatic inter- seen, the system overcomes energy barriers and reaches to different actions may play a dominant role. This evidence was further proved by local minima in the course of sampling. The resulting conformations our results from the modeling studies. The electrostatic interaction of were then clustered hierarchically to obtain patterns of structural simi- the polymyxins-HSA is supposed to mainly be as the result of close con- larities. An agglomerative algorithm with Ward linkage was used. The tact between the side chains of Asp/Glu and Dab residues from the HSA dendrogram of clustering were shown in Fig. 4B and Supporting infor- and polymyxins, respectively. Besides, the hydrogen bonds between mation Fig. S4 for PMB and PME, respectively. Cutting the dendrogram HSA residues as shown in Fig. 7 and the positive nitrogen center on at an appropriate level yields twenty clusters with different populations side chain of DAB residues are seen as a lateral interaction for the (denoted by numbers in leaf boxes), which their representative struc- peptide-protein complex formation [61]. Additionally, the computa- tures shown below them. Main structural similarities were highlighted tional studies revealed that polymyxins bind to the cleft of HSA between in five separate green areas along the clustering hierarchy. Alongside domains I and III. Furthermore, the overall pattern of interaction is very conformational variations in the ring, the relative orientation of the similar for both peptides despite a slight structural difference between tail was important factors of structural variations. While a compact Leu6 and Phe6 where almost had no impact upon the interactions scorpion-like form with the “tail folded on the ring” was more popu- around this position; with exceptions to their interacting partners on lated for the PMB, there seems to be a minor shift towards more ex- proteins that are Lys190 and Pro421 for polymyxin B and Ile115 and tended structures in the PME. The energetic distribution of conformers Ile142 for colistin. was also plotted in Fig. 5. There was N120 kcal/mol energy difference be- Altogether, our results suggested that colistin or polymyxin B binds tween the most stable conformation and that with highest potential en- preferentially to the partially unfolded intermediate forms of HSA, and ergy, what indicates the success of adopted protocol in sampling from the protein does not undergo substantial conformational changes different regions of the energy landscape of the peptides. The extended upon binding. The aforementioned interaction that occurs between ring conformations are preferred energetically to those with twisted polymyxins and HSA is promising as this drug can still be used as the ring geometry. last antibiotic line [16].

3.2.2. Refined binding modes of PMB and PME to HSA Acknowledgements Clustered peptide-HSA complexes predicted from docking simula- tions are presented in Fig. 6. Binding mode of the firstmoststablecom- This work was financially supported by the Research Council of Uni- plexes was shown with different colors. As seen, cluster 1 is more versity of Tehran and Iran National Science Foundation (INSF). preferred not only for its binding affinity, but also for its considerable higher population. This corresponds to the binding of PMB or PME to Appendix A. Supplementary data the cleft of HSA between domains I and III. Ten best scored docked poses were then used in SAMD simulations for further refinement and Supplementary data to this article can be found online at https://doi. checking their binding stability. The interaction energies between pep- org/10.1016/j.saa.2019.03.077. tides and HSA were plotted in Supporting information Fig. S5. 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