A finely balanced order-disorder equilibrium sculpts the folding-binding landscape of an antibiotic sequestering...

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• TipA is a MerR family transcription factor from *Streptomyces lividans* that confers antibiotic resistance by sequestering thiopeptide antibiotics.
• TipAS, an isoform of TipA, contains a partially disordered N-terminal subdomain that folds upon antibiotic binding, suggesting a role in promiscuous binding and function.
• Understanding the molecular heterogeneity of TipAS is critical for elucidating antibiotic sequestration mechanisms.

Purpose of the Study:

• To investigate the molecular heterogeneity and conformational dynamics of the TipAS protein.
• To elucidate the mechanism of antibiotic binding and sequestration by TipAS.
• To determine the functional and evolutionary significance of partial disorder in MerR family antibiotic-sequestering proteins.

Main Methods:

• Equilibrium and time-resolved experiments.
• Statistical modeling.
• Molecular simulations.

Main Results:

• The native TipAS ensemble exists in a pre-equilibrium between binding-incompetent and binding-competent substates.
• The binding-competent state, with a partially structured N-terminus, loses structure with increasing temperature and exhibits slow conformational exchange.
• Antibiotic binding, such as to thiostrepton, occurs via a hybrid induced-fit/conformational selection mechanism, stabilizing the binding-competent substate.
• These ensemble features are conserved in orthologs from human-pathogenic bacteria.

Conclusions:

• Partial intrinsic disorder in the native ensemble of TipAS is essential for its antibiotic-sequestering function.
• The dynamic conformational landscape of TipAS facilitates its interaction with multiple antibiotics.
• Conserved ensemble features highlight the functional importance of partial disorder in MerR family antibiotic resistance proteins.