Abstract
In analogy with many other proteins, Na+/Ca2+ exchangers (NCX) adapt an inverted twofold symmetry of repeated structural elements, while exhibiting a functional asymmetry by stabilizing an outward-facing conformation. Here, structure-based mutant analyses of the Methanococcus jannaschii Na+/Ca2+ exchanger (NCX_Mj) were performed in conjunction with HDX-MS (hydrogen/deuterium exchange mass spectrometry) to identify the structure-dynamic determinants of functional asymmetry. HDX-MS identified hallmark differences in backbone dynamics at ion-coordinating residues of apo-NCX_Mj, whereas Na+or Ca2+ binding to the respective sites induced relatively small, but specific, changes in backbone dynamics. Mutant analysis identified ion-coordinating residues affecting the catalytic capacity (kcat/Km), but not the stability of the outward-facing conformation. In contrast, distinct “noncatalytic” residues (adjacent to the ion-coordinating residues) control the stability of the outward-facing conformation, but not the catalytic capacity. The helix-breaking signature sequences (GTSLPE) on the α1 and α2 repeats (at the ion-binding core) differ in their folding/unfolding dynamics, while providing asymmetric contributions to transport activities. The present data strongly support the idea that asymmetric preorganization of the ligand-free ion-pocket predefines catalytic reorganization of ion-bound residues, where secondary interactions with adjacent residues couple the alternating access. These findings provide a structure-dynamic basis for ion-coupled alternating access in NCX and similar proteins.
Highlights
Inverted twofold symmetry of repeated structural elements, which evolved via gene duplication and fusion events, represents a common motif among multi-helical proteins including enzymes, receptors, pumps, transporters, and channels
In order to envisage the backbone dynamics within the ion-binding pocket and assign the binding sites occupied by each ion, Hydrogen/Deuterium exchange Mass Spectrometry (HDX-MS) analysis[23,24,25,26,27] was performed on apo and ion-bound NCX_Mj
The present work was undertaken to analyze the structure-dynamic determinants of ion-transport catalysis and the intrinsic asymmetry of bidirectional movements in the NCX_Mj protein as related to the inverted twofold symmetry of ion-coordinating pair residues
Summary
Inverted twofold symmetry of repeated structural elements, which evolved via gene duplication and fusion events, represents a common motif among multi-helical proteins including enzymes, receptors, pumps, transporters, and channels. NCX orthologs vary as much as 104-fold in the turnover rates of the transport cycle[13,14,15,16] to match dynamic swings in cytosolic Ca2+ levels[7,8] Despite these kinetic differences, NCXs share a common ability to stabilize the outward-facing (extracellular) access[13,17]. Molecular dynamics simulations and ion-flux analyses offer an alternative interpretation, suggesting that 3Na+ ions occupy Sext, Sint, and SCa, whereas Ca2+ occupies SCa22 (Fig. 1C,D) According to this point of view, Smid does not bind either Na+ or Ca2+ ions and one water molecule is bound to protonated D240. Since HDX-MS can detect small and slow conformational changes in the presence or absence of ligand[26,27], this technique is well suited for monitoring the folding/refolding dynamics of interest in NCX_Mj
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