Modeling the native ensemble of PhuS using enhanced sampling MD and HDX-ensemble reweighting

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• Pseudomonas aeruginosa utilizes the cytoplasmic heme binding protein PhuS for heme uptake and iron homeostasis.
• PhuS shuttles heme to heme oxygenase (HemO) and binds DNA to regulate small RNAs (sRNAs) involved in iron homeostasis.
• Heme binding to PhuS modulates its dual DNA-binding and heme-shuttling functions.

Purpose of the Study:

• To reconcile discrepancies between static crystal structures and dynamic solution-based data regarding PhuS conformational changes upon heme binding.
• To elucidate the structural basis for how heme binding alters PhuS function.
• To investigate the conformational landscape of apo-PhuS and its implications for heme transfer.

Main Methods:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe protein dynamics.
• Molecular dynamics (MD) simulations and enhanced sampling MD.
• HDXer computational approach to integrate HDX-MS data with structural ensembles.
• Circular dichroism (CD) spectroscopy.

Main Results:

• HDX-MS revealed significant conformational rearrangements in apo-PhuS, particularly in helices α6, 7, and 8, contrasting with crystal structures.
• MD simulations alone did not fully capture these dynamics, but the HDXer approach successfully integrated HDX-MS data.
• A revised conformational landscape for apo-PhuS was proposed, showing altered secondary structure and helix displacement, consistent with functional observations.

Conclusions:

• Heme binding induces substantial conformational changes in PhuS, not evident from crystal structures alone.
• The dynamic nature of PhuS is critical for its dual role in heme uptake and iron regulation.
• Computational integration of experimental data provides a more accurate picture of protein conformational dynamics and function.