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.

Peroxiredoxins (Prxs) are versatile enzymes that demonstrate various cell functions as peroxidases, protein chaperones, functions of signal modulators and binding partners. It is well established that Prxs can interact with multiple proteins in cells, such as ASK1, Cdk5-p35, JNK, MIF, PDGF, TK R4 and others. In this study, we attempted to evaluate a possible association between ubiquitous Prx II and ATP/ADP buffering enzyme - brain-type creatine kinase (CK BB). Our co-immunoprecipitation (Co-IP) results from the A549 and HeLa cell lysates with overexpressed HA-Prx II and Flag-CK BB have demonstrated strong association between two proteins under non-stressed conditions. This protein interaction was enhanced by the heat treatment with further HA-Prx II precipitation to the immobilized Flag-CK BB depending on the temperature increase. Temperature induced oligomerization of Prx II may contribute to the formation of Prx II conglomerates, which in turn, can associate with CK BB and increase signal intensities on the blotted membranes. Thus, such association and oligomerization of Prx II could take part in recovery and protection of the CK BB enzyme activity from inactivation during heat-induced stress.

Peroxiredoxins (Prxs) are a family of peroxidase proteins which, although, exhibit chaperone activity due to the structural transition from dimer to decamer or high‐molecular weight complex. We have identified brain‐type creatine kinase (CKBB) as a Prx I binding protein from the heat‐stressed rat brain extracts. Co‐IP results from the transfected cells demonstrated that the interaction of CKBB with PrxII in addition to PrxI. PrxII showed much higher binding affinity to CKBB than PrxI under normal physiological condition as well as heat‐stressed condition. Prx II effectively protected the kinase activity of CKBB from oxidative inactivation and heat inactivation. This protection activity is not depends on the structural transition but depends on the site‐specific interaction. Thus, we suggest this chaperoning activity of Prx through the sitespecific interaction as “client specific chaperoning”, and that through structural transition as “general chaperoning”. For specific clients, Prx binds to their native state and provides the protection for maintain their native state. The beneficiary of chaperoning through structural transition (general chaperoning) opens to wide variety of clients, however, that through site‐specific interaction (client specific chaperoning) opens to very limited number of clients.

Protein oxidation has detrimental effects on the brain functioning, which involves inhibition of the