Simulation of Peroxiredoxin II and Brain-type Creatine Kinase protein-protein interaction using the on-line docking server ClusPro 2 . 0

Article history: Received on: 28/04/2015 Revised on: 17/05/2015 Accepted on: 22/06/2015 Available online: 28/08/2015 Importance of dedicated web servers and specialized software for simulations of protein-protein interactions is well established. The purpose of our study was to examine the protein-protein interaction that occurred under physiological and stress conditions between peroxiredoxin II and the creatine kinase brain-type using proteindocking server ClusPro 2.0. To predict the particular site of amino acid docking, computer software analyzes various protein conformations and chooses the most profitable energy state, therefore selecting a number of possible combinations that would fit the correct profile. By co-immunoprecipitation assay, we demonstrated that two molecules Prx II and CKBB have interacted with further attenuation of this specific binding by pretreatment with selected stress factors. In previous study, we showed that the enzymatic activity of CKBB was recovered by different concentration ratios of Prx II. The specific binding models were generated by ClusPro 2.0 protein docking server and studied using PyMol software. It was shown that a number of amino acid residues including Lys 11, Arg 13, Ala 204, Arg 209for creatine kinase, and Asp 181, Glu 192, Lys 196, Glu 162, Gln 163 for Prx II have participated in the complex formation throughout the first ten conformations.


INTRODUCTION
Mammalian peroxiredoxin II (Prx II, EC 1.11.1.15)with two conserved cysteine residues is a member of peroxiredoxin antioxidant family that eliminates endogenous hydrogen peroxide and exhibits chaperonic activity towards target proteins (Schroder et al., 2000;Jang et al., 2004).Peroxiredoxin II belongs to the typical 2-Cys Prx subfamily, where the basic active unit is a homodimer, with catalytically active disulfide bond formed between two subunits with peroxidatic (C P ) and reducing (C R ) cysteins (Rhee and Woo, 2011;Hall et al., 2009).Sulfenic acid Cys-S P OH that generated in a result of neutralization of peroxide molecule reduced to its normal active state by the thioredoxin family proteins (Hanschmann et al., 2013;Hall et al., 2011).However, rapid disulfide formation can be impeded by the distance between the sulfur atoms of C P and C R (13Å) what thought to be a substantial obstacle for rapid disulfide formation (Hirotsu et al., 1999).Thus, further hyperoxidation of C P -SOH to protein sulfinic acid C P -SO 2 H is possible.During this type of catalytic hyperoxidation, the Prx II overcomes a structural transition from a homodimer to a doughnutshape decamer, and aggregated form of the enzyme gains protein chaperone activity (Moon et al., 2005).
On the other hand, creatine kinase (CK, creatine Nphosphatransferase, EC 2.7.3.2), as the cytoplasmic forms of phosphagen kinases, has a conventional dimeric structure consisting of two identical 42kDa subunits (Tanaka and Suzuki, 2004).CK catalyzes the reversible reaction of phosphocreatine formation (P/Cr)with consequential ATP consumption.Generated P/Cr, therefore will be used to regenerate ATP molecule during the energy depletion (Wallimann et al., 2011).Cytosolic brain-type creatine kinase (CKBB) is a one of three CK enzyme isoforms that mainly expressed in brain, spermatozoa, kidney, and retina.This enzyme plays a crucial role in energy supplementation of the brain tissue, decrease of its functional activity is one of the biochemical markers of the CNS cell damage in age-related neurodegenerative disorders, including AD, and Huntington diseases (Hensley et al., 1995;Aksenov et al., 1998;Aksenova et al., 1999;Kim et al., 2010).
The crystal structure of CKBB combined with the ADP-Mg 2+ -NO 3-creatine transition-state analogue was resolved by Bong laboratory.The study presented that negatively charged aminoacid residues are responsible for substrate binding in the active site of CKBB (Asp, Arg, Glu).Hydrophobic interaction are the primary interaction within the molecule.Five positively charged Arg residues are composing the phosphate-binding pocket of CKBB (Bong et al., 2008).Whereas, the first crystallographic study of decameric Prx II has been completed on the protein purified from human erythrocytes (Schroder et al., 2000).The study has determined that the structure is a toroid, comprising of five dimers linked predominantly by hydrophobic interactions.The monomer subunit of Prx II homodimer comprised of domain I (residues 2-169) and a C-terminal arm (residues 170-198).The dimer configuration of peroxiredoxin II is organized by two intertwined monomer with the C-terminal arm of one subunit folds over domain I of the other monomer (Schroder et al., 2000).
Automated docking server ClusPro 2.0 was implemented to predict possible interaction sites between Prx II and CKBB proteins.ClusPro 2.0 is a web server that filters docked conformation with good surface complementarity, therefore selects complexes with lowest desolvation and electrostatic energies (Comeau et al., 2004).Methods of computational protein docking generally yield near-native structures however due to the reasons of the intrinsic uncertainty they are far from the native complexes.Therefore, were introduced new approaches that evaluate docked structures based on the chemical affinity between the molecules (Camacho et al., 2000;Norel et al., 2001;Comeau et al., 2004).Filtering algorithm of ClusPro 2.0 include the use of empirical free energy evaluation methods that select the conformations with the lowest desolvation and electrostatic energies (Comeau et al., 2004).
Results from CAPRI (Critical Assessment of Predicted Interaction) experiment have demonstrated that the automatic protein docking server ClusPro 2.0 was the best in the server category.This means that the results generated by ClusPro 2.0 on predicting protein interaction sites are comparable to that of the top human predictor groups (Kozakov et al., 2013).
Recent proteomic studies revealed that members of peroxiredoxin family Prx I and Prx II have numerous target proteins in vitro (Rhee and Woo, 2011).Prx I interacts with apoptosis signal-regulating kinase 1 (ASK1), the Myc box II domain of c-Myc, macrophage migration inhibitory factor (MIF) etc.; where Prx II was identified as a partner of cyclin dependent kinase 5 (Cdk5), and associated with platelet-derived growth factor (PDGF) (Kim et al., 2008;Jung et al., 2001;Mu et al., 2002;Qu et al., 2007;Choi et al., 2005).Computer simulation of the complex formed between Prx I and Srx have demonstrated that the essential substrate of Srxunder physiological conditions is likely to be HMW species of Prx I (Lowther and Haynes, 2011).
Here we confirm with protein-docking software interaction of two proteins, what were identified previously by the immunoprecipitation assay: human brain-type creatine kinase (CKBB) and peroxiredoxin II (Prx II).By the same example of Prx II overexpression in Alzhiemer's disease and protection of neuron cell from amyloid-β-peptide toxicity (Yao et al., 2007), we can speculate on importance of Prx II to prevent deterioration effects of hydrogen peroxide on CKBB.

Target Sequences
The protein structures of Peroxiredoxin II and Brain-type creatine kinase (CKBB) were publicly available.The complete aminoacid sequences of human Peroxiredoxin II 22 kDa molecule, which consist of 198 amino acids, and human Creatine kinase b-type42 kDa molecule with 381 amino acids, were retrieved from RCSB Protein Databank (Prx II -1QMV; CKBB -3B6R).Protein structure analyzing was performed with the Expasy'sProtPram Proteomic server (Gasteiger et al., 2005)

Assay conditions
The creatine kinase b-type was heat inactivated at 42 C in a solution contained 30 nM CKBB and different molar concentrations (1:1, 1:5, 1:10, 1:20) of Prx II protein.Everything was diluted in a 5 mM Glycine-NaOH (pH 9.0) buffer.Aliquots (60 µl) were removed at specific time intervals to determine CK activity.The activity measurements were made after a brief preincubation of heat treated samples on ice.Measurements of creatine kinase activity were conducted in accordance with (Yao et al., 1982) with minor modifications (Rakhmetov et al., 2015).Hydrogen peroxide inactivation was established by adding H 2 O 2 at final concentration of 1 mM to the samples containing the same assay mixture used for thermal inactivation of CK enzyme.Residual activity was measured at the 597 nm by adding sample aliquots (60 µl) to the reaction mixture with final volume of 600 µl.

Molecular docking
To investigate the molecular interaction between human peroxiredoxin II and the brain-type creatine kinase rigid protein docking was performed using ClusPro 2.0.Within this server enclosed three computational steps that include: rigid body docking using the FFT (Fast Fourier Transform) correlation approach, RMSD (Root Mean Square Deviation) based clustering of the structures generated to find the largest cluster that will represent the likely models of the complex, and refinement of selected structures (Kozakov et al., 2013).By default server settings ClusPro 2.0 simultaneously generates four types of models using the scoring algorithms called designated as balanced, electrostatic-favored, hydrophobic-favored, and van der Waals + electrostatic.We selected the first ten docking structureswith the relative low energies that were scored by the server.According to (Kozakov et al., 2013) the only refinement currently applied in CluPro 2.0 is minimizing the Charm energy.
PyMol software was used for visual representation and assessing of the complex interaction, measuring of the distances between the interacting aminoacid residues.By using the multifaceted molecular software were rendered cartoons and surfaces of the docking Prx II decamer and CKBB dimer.

RESULTS AND DISCUSSIONS
Peroxidase enzymes peroxiredoxins exhibit negligible catalytic activity toward neutralizing metabolic hydrogen peroxide compare to the major H 2 O 2 scavengers like catalyze, and superoxide dismutase (SOD) (Hanschmann et al., 2013).Therefore, they may play a major role as chaperone molecules.Chaperone function of ubiquitous peroxiredoxin enzymes is well recognized (Moon et al., 2005;Saccoccia et al., 2012), however some functional aspects of the structural organization remain to be elucidated.
It is established that protein structures consist of specific geometric arrangements of the secondary structure elements (Tsai et al., 1997).Some of the motifs of the α-helixes and of the βsheets have specific associated biological functions.The solvent exposed parts of these motifs are typically hydrophilic and the interiors are hydrophobic with the limited number of special arrangements.Peroxiredoxin II inactivated by hyperoxidation of its cysteine active sites is reversibly capable of transformation from a peroxidase to a high molecular weight chaperone (Lowtherand Haynes, 2011).The substantial structural change of Prx II is associated with disulfide bond formation within the dimeric structure of the enzyme, therefore the peroxiredoxin molecule predominantly cycles between dimeric and decamericoligomeric states.
Here we have utilized a dataset of stable interfaces of human peroxiredoxin II and creatine kinase b-type derived from the PDB database.ClusPro2.0 is aprotein docking server that comparable to GRAMM-X, ZDOCK, RossetaDock, SymmDockand PatchDock.Models built by ClusPro2.0have been reported in over 200 publications (Kozakov et al., 2013).Several interaction models of Prx II and CKBB were yielded during a simulation run on the ClusPro 2.0 protein docking webserver, with the first model presented (Fig 1.).Processing of these generated models by the molecular graphic software PyMolhas allowed us to outline the most important aminoacid residues that participated in the interaction of two proteinsPrx II and CKBB.As a result, we showed that the C-terminal residue Lys 196 of Prx II, involved in stabilization of the COOH terminal domain, has formed a hydrogen bong interaction with the Ala 204 aminoacid residue of creatine kinase (Fig. 2

. A).
Reversible phosphorylation of Prx II induces temporal inactivation of the peroxidase activity, therefore allowing the transient accumulation of H 2 O 2 for receptor signaling.According to our results the Asp 181 residue that forms possible hydrogen bond with His 197 to prevent phosphorylation of Prx II (Rhee and Woo, 2011), also participates in the interaction with Lys 11 residue of CKBB (Fig. 2. C).Thus, adding the second hydrogen bond to strengthen thePrx II-CKBB protein association.
The salt bridge network that includes Asp 181 and Lys 196 with interatomic distance at 2.64 Å and that between Asp 181 and His 197 at 2.69 Å might stabilize the COOH terminal domain of Prx II (Rhee and Woo, 2011).Arg 95 is the key residue for guanidine substrate recognition for both AK and CK enzymes (Uda and Suzuki, 2004;Edmiston et al., 2001).
The monomer of creatine kinase includes an essential Nterminal region with 1-100 aminoacid residues and a C-terminal α/β domain, residues 125-381 both connected with a long linker, residues 101-124 (Bong et al., 2008).According to our simulation results, the top model that possesses the lowest RMSD (Root Mean Square Deviation is the square root of the mean of the square of the distance between the matched atoms) has aminoacid residues that positioned at the beginning of N-terminal region (Arg 13 , Asp 18 ) and in the middle of C-terminal (Arg 209 , Ala 204 ).Hyperoxidizedhuman Prx II (monomer ~22 kDa, decamer ~ 220 kDa), presumably forming dodecameric structural units with oxidized cysteine to the sulfinic form (Wood et al., 2003), interacts with the dimeric creatine kinase (84 kDa), thus recovering the enzyme activity.Potential interaction suggested by the model include electrostatic hydrogen bond, CKBB Ala 204 with Prx II Lys 196 , as well as electrostatic interaction, CKBB Arg 209 with Prx II Glu 192 (Fig. 2, A, B) Protein folding accompanied by a conformational freedom of the aminoacid chain allows almost any possible protein configuration.This is not the case for protein binding.The rotational and translational packing available to the chains includes only 6 degrees of freedom (Chen and Xu, 2003).The individual contribution of amino acids that compose the side chain of interacting proteins can be investigated experimentally by mutating the amino acid of interest with implementation of the site-direct mutagenesis technique (Tsai et al., 1997).
Prominent residue that we registered throughout examination of 20 docked poses was Arg 13 of creatine kinase.Unequivocal role for arginine residues from rabbit muscle CK in complete inactivation accompanied by the loss of nucleotide binding was previously established (Wood et al., 1998).In addition, the chemical modification of two arginine residue per mitochondrial creatine kinase monomer showed strong negative co-operatively and complete inactivation of Mi-CK (Belousovaand Muizhnek, 2004).
In our experimental docking the Arg 13 residue of CKBB demonstrated prominent interaction with aminoacid residues of peroxiredoxin II moleculeat attained the stable distances of 1.70, 1.90 Å in the entire simulation period (Fig. 3).Our simulation docking approach has yielded the following aminoacid residues of Prx II molecule that interacted with Arg 13 of CKBB: Glu 167 , Gln 163 , Lys 67 , Asp 64 , Asn 60 .These hydrogen bond interactions with attained intermolecular distances along with the salt bridge stabilized the protein-protein association the most.Pictures created using PyMol molecular viewer software.
Due to the remarkable structural rearrangements of Prx molecule, it is quite difficult to predict the exact interacting cluster of aminoacids with a partner protein.Although, a decameric state of peroxiredoxinis characterized as active, with reduced conserved cysteine residues, the equilibrium shifts right after the molecule of Prx has suffered the H 2 O 2 triggered oxidation to a dimeric form with partial decameric presence (Barranco-Medina et al., 2008).Keeping in mind that under a severe hyperoxidationPrxs undergo a unique functional transformation from a low molecular weight species to a high molecular weight conglomerates.This conglomerates, that shaped as a long filament tubes, exhibit chaperonic activity by somehow attracting partner proteins into their custody.In our experiment we have seen, probably, a similar event, where the inactivated creatine kinase BB isoenzyme, which was identified by the coimmunoprecipitation assay as PrxII binding partner, interacted with hyperoxidized PrxII molecule at the specific aminoacid clusters.In this study, we could not verify the correctness of our results in vitro; it can be done by the site direct mutagenesis of interacting residues.Therefore, more studies are needed.

CONCLUSION
In this study, we have speculated that human Prx II and CKBB are the binding partners with definite aminoacid cluster of interaction.This fact was supported by the interaction simulation results using the online docking server ClusPro 2.0.By analyzing all ten generated models, we have come to conclusion that aminoacid residues of pivotal role are located in the N-terminal region of CKBB (Lys 11, Arg 13) and the C-terminal region of Prx II (Asp 181,Glu 192,Lys 196).Therefore, discovered connection of human antioxidant enzyme, effective molecular chaperone Prx II and a key regulator of ATP level in neural cells brain-type creatine kinase could contribute to more comprehensive understanding of the development of neurodegenerative processes in the human brain.

Fig. 1 .
Fig. 1.Ribbon diagram of human decamericPrx II (green) and dimeric CKBB (cyan).DecamericPrx II composed of five dimeric molecules.Outline black square indicates on the interacting region.

Fig. 2 :
Fig. 2:The set of Prx II and CKBB interacting residues.Hydrogen bond and electrostatic interactions between two aminoacid residues is shown with its distances (Å).Where: greyhydrogen, dark bluenitrogen, and redoxygen atoms.Distance between two aminoacids residue indicated by a dashed red line.Pictures created using PyMol molecular viewer software.