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

The Na⁺/Ca²⁺ exchanger (NCX) is a membrane protein that couples the downhill transport of Na⁺ across the plasma membrane to the uphill movement of Ca²⁺ in the opposite direction. The NCX is a key Ca²⁺ extrusion mechanism in excitable cells. It contains a transmembrane domain that catalyzes the counter-transport of Na⁺ and Ca²⁺, and a large intracellular loop (IL) that is responsible for the allosteric regulation of the exchanger by its substrates. The NCX intracellular loop contains a two-domain Ca²⁺-sensor, CBD12, which harbors Ca²⁺ regulatory sites. Ca²⁺-binding to CBD12 triggers NCX activation and alleviates Na⁺-dependent inactivation. An outstanding question in this field is how Ca²⁺-binding to CBD12 activates the exchanger? Previous experimental studies showed that CBD12 displays considerable interdomain flexibility in the unbound state, while Ca²⁺ binding near the linker between the two domains stabilizes a rigid and widely opened interdomain conformation. This phenomenon could be an important step in the Ca²⁺ regulation mechanism. Using the Drosophila exchanger, CALX, as a model system, we carried out coarse-grained molecular dynamics simulations using a dual-basin structure-based model (SBM) to sample large-scale conformational transitions between open and closed states. In addition, we calculated the CBD12 free energy profile along the open-closed transition coordinate, in the Ca²⁺-bound and in the free states. We found that Ca²⁺ binding reshapes the CBD12 free energy landscape, stabilizing a widely opened interdomain conformation in agreement with previously published experimental data. Notably, the energy landscape of the two CALX CBD12 isoforms, which differ by only five amino acids near the interdomain linker, is substantially different. These results provide atomistic insights into the open-closed conformational transition experienced by this two-domain construct, and are consistent with the considerable broadening of the solution NMR resonances of CBD12 1.2 in comparison with the 1.1 isoform.