In Silico and In Vitro Analysis of MAP3773c Protein from Mycobacterium avium subsp. Paratuberculosis
Esteban Hernández-Guevara1, José A Gutiérrez-Pabello2, Kaina Quintero-Chávez3
1Facultad de Odontología, Universidad Autónoma de Baja California, Tijuana 22390, Mexico.
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 Zn2+ 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 Zn2+.
Insights
Researchers investigated the MAP3773c protein in Mycobacterium avium subsp. paratuberculosis (MAP), a pathogen causing paratuberculosis. This protein has two metal-binding sites and contains structural zinc, suggesting potential as a therapeutic target.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Paratuberculosis is caused by Mycobacterium avium subsp. paratuberculosis (MAP).
- Understanding MAP pathogenicity proteins is crucial for developing therapeutics and vaccines.
- MAP3773c was identified as a protein of interest for investigation.
Purpose of the Study:
- To investigate the MAP3773c protein from MAP.
- To characterize its structure and function, particularly its metal-binding capabilities.
- To assess its potential as a therapeutic or vaccine target.
Main Methods:
- In silico molecular modeling and docking studies.
- Cloning, expression, and purification of recombinant MAP3773c.
- Biochemical characterization including SDS-PAGE, PAR staining, LC-MS, and BN-SDS-PAGE/BN-Western blotting.
Main Results:
- MAP3773c shares 34% similarity with Mycobacterium tuberculosis FurB, a zinc uptake regulator.
- Docking analysis revealed two metal-binding sites in MAP3773c.
- Purified MAP3773c exists as a monomer and contains structural Zn2+.
- Structural zinc coordination was confirmed in the purified protein.
Conclusions:
- MAP3773c exhibits characteristics of a Fur-type protein.
- The protein possesses two metal-binding sites, one coordinating structural zinc.
- These findings highlight MAP3773c as a potential target for paratuberculosis intervention strategies.
