Probing Charge-Controlled Inter-Domain Flexibility: Integrating Experimental and Coarse-Grained Approaches

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• The Na+/Ca2+ exchanger (NCX) is crucial for calcium homeostasis in excitable cells.
• Its intracellular loop (IL) contains a two-domain Ca2+-sensor (CBD12) that regulates exchanger activity.
• Ca2+ binding to CBD12 activates the NCX and reduces Na+-dependent inactivation, but the mechanism remains unclear.

Purpose of the Study:

• To investigate the atomistic mechanism by which Ca2+ binding to CBD12 induces NCX activation.
• To elucidate the role of interdomain flexibility and Ca2+ binding in stabilizing the active conformation of CBD12.

Main Methods:

• Coarse-grained molecular dynamics simulations using a dual-basin structure-based model (SBM) were performed on the Drosophila CALX exchanger.
• Simulations sampled large-scale conformational transitions between open and closed states of CBD12.
• Free energy profiles of CBD12 were calculated in both Ca2+-bound and Ca2+-free states.

Main Results:

• Ca2+ binding was found to reshape the CBD12 free energy landscape, stabilizing a rigid, open interdomain conformation.
• This stabilization aligns with experimental observations of increased rigidity upon Ca2+ binding.
• Significant differences in the free energy landscape were observed between two CALX CBD12 isoforms, correlating with experimental NMR data.

Conclusions:

• Ca2+ binding to CBD12 stabilizes an open conformation, providing atomistic insights into NCX activation.
• Interdomain flexibility and Ca2+ sensing by CBD12 are critical for regulating NCX function.
• Isoform-specific differences in CBD12 energy landscapes may influence exchanger activity and Ca2+ sensitivity.