Intersubunit interactions in Plasmodium falciparum thioredoxin reductase

Area of Science:

• Biochemistry
• Molecular Biology
• Redox Biology

Background:

• The thioredoxin redox system, comprising thioredoxin reductase (TrxR) and thioredoxin, is vital for cellular processes including disulfide bond reduction and oxidative stress defense.
• High molecular weight TrxRs (high M(r) TrxRs), found in mammals, C. elegans, and P. falciparum, possess unique C-terminal cysteine or selenenylsulfide motifs crucial for thioredoxin reduction.
• The precise contribution of each subunit to the active site of P. falciparum TrxR (PfTrxR) remains to be fully elucidated.

Purpose of the Study:

• To investigate whether the active site residues of P. falciparum TrxR are contributed by one or both subunits.
• To determine if PfTrxR utilizes intersubunit active sites for its enzymatic function.

Main Methods:

• Co-expression of differentially tagged wild-type and mutant P. falciparum TrxR proteins in E. coli.
• Purification of recombinant protein species using affinity chromatography specific to the tags.
• Analysis of homodimeric and heterodimeric protein species formed during co-expression.

Main Results:

• Co-expression yielded three protein species: wild-type homodimers, mutant homodimers, and wild-type/mutant heterodimers.
• Co-expression of wild-type PfTrxR with a double mutant (PfTrxRC88AC535A) resulted in an inactive heterodimer.
• The formation of an inactive heterodimer indicates that PfTrxR requires contributions from both subunits for activity.

Conclusions:

• The results strongly suggest that P. falciparum TrxR possesses intersubunit active sites.
• The findings imply a potential cooperative interaction between the active sites of the two PfTrxR subunits.
• This highlights the importance of the dimeric structure for PfTrxR enzymatic function.