Resolving Conformational Heterogeneity in Intrinsically Disordered Proteins via Experimentally Guided Multi-Replica...

Area of Science:

• Structural Biology
• Computational Biology
• Biophysics

Background:

• Intrinsically disordered proteins (IDPs) possess dynamic conformational landscapes that are challenging to characterize due to ensemble averaging in experimental data.
• Understanding IDP conformational ensembles is crucial for elucidating their diverse biological functions.

Purpose of the Study:

• To present Multi-replica Averaged Restraint Simulation (MARS), a novel data-driven framework for reconstructing IDP conformational landscapes.
• To validate MARS using the estrogen receptor alpha N-terminal domain (ERα-NTD) and integrate diverse biophysical data.

Main Methods:

• MARS enforces ensemble-averaged restraints across multiple simulation replicas without prior structural assumptions.
• Simultaneously integrated small-angle X-ray scattering (SAXS) and six paramagnetic relaxation enhancement (PRE) profiles, providing over 600 pairwise restraints.
• Generated conformational ensembles quantitatively fitting all experimental input data.

Main Results:

• The MARS-derived ensemble for ERα-NTD accurately reproduced experimental data, including backbone relaxation measurements.
• Identified two distinct conformational states: a predominant extended state and a functionally relevant, low-populated compact state.
• Demonstrated that SAXS and PRE data provide orthogonal global and local structural constraints, respectively.

Conclusions:

• MARS successfully reconstructs complex conformational landscapes of IDPs by integrating orthogonal experimental data.
• The framework resolves both dominant and rare functional states, offering insights into IDP mechanisms.
• MARS provides a scalable and powerful approach for structural characterization of dynamic biological molecules.