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Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method
Published on: July 27, 2016
An improved expression and purification protocol enables the structural characterization of Mnt1, an antifungal
Patrícia Alves Silva1, Amanda Araújo Souza1, Gideane Mendes de Oliveira1
1Laboratório de Biofísica Molecular, Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, 70910-900, Brazil.
Background:
Candida albicans is one of the most prevalent fungi causing infections in the world. Mnt1 is a mannosyltransferase that participates in both the cell wall biogenesis and biofilm growth of C. albicans. While the cell wall performs crucial functions in pathogenesis, biofilm growth is correlated with sequestration of drugs by the extracellular matrix. Therefore, antifungals targeting CaMnt1 can compromise fungal development and potentially also render Candida susceptible to drug therapy. Despite its importance, CaMnt1 has not yet been purified to high standards and its biophysical properties are lacking.
Results:
We describe a new protocol to obtain high yield of recombinant CaMnt1 in Komagataella phaffii using methanol induction. The purified protein's identity was confirmed by MALDI-TOF/TOF mass spectroscopy. The Far-UV circular dichroism (CD) spectra demonstrate that the secondary structure of CaMnt1 is compatible with a protein formed by α-helices and β-sheets at pH 7.0. The fluorescence spectroscopy results show that the tertiary structure of CaMnt1 is pH-dependent, with a greater intensity of fluorescence emission at pH 7.0. Using our molecular modeling protocol, we depict for the first time the ternary complex of CaMnt1 bound to its two substrates, which has enabled the identification of residues involved in substrate specificity and catalytic reaction. Our results corroborate the hypothesis that Tyr209 stabilizes the formation of an oxocarbenium ion-like intermediate during nucleophilic attack of the acceptor sugar, opposing the double displacement mechanism proposed by other reports.
Conclusions:
The methodology presented here can substantially improve the yield of recombinant CaMnt1 expressed in flask-grown yeasts. In addition, the structural characterization of the fungal mannosyltransferase presents novelties that can be exploited for new antifungal drug's development.
Insights
We developed a new method to produce high yields of recombinant CaMnt1, a key enzyme in Candida albicans. This structural characterization aids in developing novel antifungal drugs against this prevalent fungus.
Area of Science:
- Biochemistry
- Mycology
- Structural Biology
Background:
- Candida albicans is a major fungal pathogen responsible for widespread infections.
- Mnt1, a mannosyltransferase, is crucial for C. albicans cell wall synthesis and biofilm formation.
- Targeting CaMnt1 offers a potential strategy to combat fungal infections and enhance drug susceptibility.
Purpose of the Study:
- To establish a high-yield purification protocol for recombinant CaMnt1.
- To characterize the biophysical properties of CaMnt1.
- To elucidate the structural basis of CaMnt1's enzymatic activity and substrate specificity.
Main Methods:
- Recombinant expression of CaMnt1 in Komagataella phaffii with methanol induction.
- Protein purification and identity confirmation via MALDI-TOF/TOF mass spectrometry.
- Far-UV circular dichroism and fluorescence spectroscopy for structural analysis.
- Molecular modeling to depict the ternary complex of CaMnt1 with substrates.
Main Results:
- A novel protocol achieved high yields of purified recombinant CaMnt1.
- Circular dichroism confirmed a secondary structure composed of alpha-helices and beta-sheets.
- Fluorescence spectroscopy revealed a pH-dependent tertiary structure.
- Molecular modeling identified key residues involved in substrate binding and catalysis, supporting a specific reaction mechanism.
Conclusions:
- The presented methodology significantly enhances recombinant CaMnt1 yield in yeast.
- The structural insights into CaMnt1 provide a foundation for developing new antifungal therapies targeting Candida albicans.
