In Silico and In Vitro Analysis of MAP3773c Protein from Mycobacterium avium subsp. Paratuberculosis

SIMPLE SUMMARY: Paratuberculosis is a disease that is caused by Mycobacterium avium subsp. paratuberculosis, a bacterium that survives inside a cell to cause disease. This bacterium therefore needs the nutrients of the cell to survive. Zinc and iron are very important elements in its nutrition and are necessary to carry out many of its survival functions, so the cell develops mechanisms to eliminate these pathogenic bacteria to continue living. One of these mechanisms is the elimination of iron as a strategy to kill the bacteria. In this research, we took on the task of studying one of the proteins of the bacterium called MAP3773c, along with its structure, some of its properties and its particular characteristics. In relation to the affinity for zinc and iron to bind to it, we are interested in discovering and making it known to the scientific community whether MAP3773c is related to the pathology of the disease. ABSTRACT: Paratuberculosis is a disease caused by Mycobacterium avium subsp. paratuberculosis (MAP). It is of great interest to better understand the proteins involved in the pathogenicity of this organism in order to be able to identify potential therapeutic targets and design new vaccines. The protein of interest–MAP3773c–was investigated, and molecular modeling in silico, docking, cloning, expression, purification, and partial characterization of the recombinant protein were achieved. In the in silico study, it was shown that MAP3773c of MAP has 34% sequence similarity with Mycobacterium tuberculosis (MTB) FurB, which is a zinc uptake regulator (Zur) protein. The docking data showed that MAP3773c exhibits two metal-binding sites. The presence of structural Zn(2+) in the purified protein was confirmed by SDS-PAGE PAR staining. The purification showed one band that corresponded to a monomer, which was confirmed by liquid chromatography–mass spectrometry (LC-MS). The presence of a monomer was verified by analyzing the native protein structure through BN-SDS-PAGE (Native Blue (BN) Two-Dimensional Electrophoresis) and BN–Western blotting. The MAP3773c protein contains structural zinc. In conclusion, our results show that MAP3773c displays the features of a Fur-type protein with two metal-binding sites, one of them coordinating structural Zn(2+).