Large-Scale Conformational Changes of FhaC Provide Insights Into the Two-Partner Secretion Mechanism

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• The Two-Partner secretion (TPS) pathway is crucial for protein transport across the Gram-negative bacterial outer membrane.
• TpsB transporters, part of the Omp85 superfamily, facilitate membrane protein translocation without external energy.
• These transporters feature a transmembrane β-barrel and periplasmic POTRA domains for substrate binding.

Purpose of the Study:

• To investigate the transient conformational changes of the TpsB transporter FhaC.
• To understand the mechanism of protein secretion mediated by TpsB transporters.
• To elucidate how TpsB transporters function without cofactors.

Main Methods:

• Integrative approach combining in vivo assays, mass spectrometry, nuclear magnetic resonance (NMR), and electron paramagnetic resonance (EPR).
• Techniques were optimized to detect minor conformational states in heterogeneous protein populations.
• Focus on exploring transient conformers of the FhaC transporter.

Main Results:

• Identified substantial, spontaneous conformational changes in FhaC occurring on a slow timescale.
• Observed parts of the POTRA2 domain approaching the lipid bilayer and surface loops.
• Evidence suggests an amphipathic POTRA2 β-hairpin can insert into the β-barrel.

Conclusions:

• Proposed that conformational motions enlarge the transporter channel, initiating substrate secretion.
• Concluded that intrinsic protein dynamics, rather than cofactors, enable TpsB-mediated protein secretion.
• Highlighted the role of dynamic conformational changes in bacterial protein transport mechanisms.