Structurally distinct unfolding intermediates formed from a staphylococcal capsule-producing enzyme retained NADPH...

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• CapF is a capsule-producing enzyme from Staphylococcus aureus, crucial for virulence.
• CapF orthologs are present in many virulent bacteria, highlighting their importance.
• Understanding CapF's folding-unfolding mechanism is key to its function and potential inhibition.

Purpose of the Study:

• To investigate the folding-unfolding mechanism of recombinant CapF (rCapF) from Staphylococcus aureus.
• To characterize the intermediates formed during denaturation by urea and guanidine hydrochloride (GdnCl).
• To assess the impact of unfolding on NADPH binding activity and protein structure.

Main Methods:

• Recombinant CapF (rCapF) was subjected to denaturation using urea and guanidine hydrochloride (GdnCl).
• Protein unfolding intermediates were analyzed for structural changes (shape, secondary and tertiary structure) and NADPH binding activity.
• Sequence alignment and tertiary structural modeling were employed to identify sensitive regions.

Main Results:

• Unfolding of rCapF by both denaturants was reversible, forming distinct dimeric intermediates.
• Urea produced three intermediates (rCapF1-3), while GdnCl produced two (rCapF4-5).
• Intermediate rCapF5 showed reduced NADPH binding; others retained activity. Intermediates exhibited altered shapes, with rCapF4 showing maximum shape loss and rCapF3 resembling native protein.

Conclusions:

• The folding-unfolding pathway of S. aureus CapF is complex, yielding unique intermediates with varied structural and functional properties.
• The region around Trp137 appears most susceptible to unfolding, while the N-terminal NADPH binding motif is more resistant, especially at low denaturant concentrations.
• These findings provide insights into the structural dynamics of CapF and its interaction with NADPH.