G503 Is Obligatory for Coupling of Regulatory Domains in NCX Proteins

Abstract

In multidomain proteins, interdomain linkers allow an efficient transfer of regulatory information, although it is unclear how the information encoded in the linker structure coins dynamic coupling. Allosteric regulation of NCX proteins involves Ca(2+)-driven tethering of regulatory CBD1 and CBD2 (through a salt bridge network) accompanied by alignment of CBDs and Ca(2+) occlusion at the interface of the two CBDs. Here we investigated "alanine-walk" substitutions in the CBD1-CBD2 linker (501-HAGIFT-506) and found that among all linker residues, only G503 is obligatory for Ca(2+)-induced reorientations of CBDs and slow dissociation of occluded Ca(2+). Moreover, swapping between positions A502 and G503 in the CBD1-CBD2 linker results in a complete loss of slow dissociation of occluded Ca(2+), meaning that dynamic coupling of CBDs requires an exact pose of glycine at position 503. Therefore, accumulating data revealed that position 503 occupied by glycine is absolutely required for Ca(2+)-driven tethering of CBDs, which in turn limits the linker's flexibility and, thus, restricts CBD movements. Because G503 is extremely well conserved in eukaryotic NCX proteins, the information encoded in G503 is essential for dynamic coupling of the two-domain CBD tandem and, thus, for propagation of the allosteric signal.

Population Shift Underlies Ca2+-induced Regulatory Transitions in the Sodium-Calcium Exchanger (NCX)

“…The linker encodes specific information that governs CBD interactions (21). Recently, we identified a Ca 2ϩ -driven regulatory switch at the interface between the two CBDs.…”

“…The amino acids contributing to the interface are highly conserved among NCX orthologs and splice variants (18). Evidently, the interdomain linker governs CBD coupling both in intact NCX (13) or isolated CBD12 proteins (12,21). Moreover, in Gly-503, its dihedral / angles and its precise position in the linker are both essential for slow dissociation of occluded Ca 2ϩ and CBD coupling (21).…”

“…Evidently, the interdomain linker governs CBD coupling both in intact NCX (13) or isolated CBD12 proteins (12,21). Moreover, in Gly-503, its dihedral / angles and its precise position in the linker are both essential for slow dissociation of occluded Ca 2ϩ and CBD coupling (21). Thus, Gly-503 is indispensable for Ca 2ϩ -driven stabilization of interdomain salt bridges that limit the linker flexibility and thus, rigidifying CBD movements (21,29).…”

“…Moreover, in Gly-503, its dihedral / angles and its precise position in the linker are both essential for slow dissociation of occluded Ca 2ϩ and CBD coupling (21). Thus, Gly-503 is indispensable for Ca 2ϩ -driven stabilization of interdomain salt bridges that limit the linker flexibility and thus, rigidifying CBD movements (21,29). Therefore, the linker and perhaps additional components at the interface, e.g.…”

Structure-dynamic determinants governing a mode of regulatory response and propagation of allosteric signal in splice variants of Na+/Ca2+ exchange (NCX) proteins

“…Although these results suggested a common mechanism for signal transduction in NCX proteins, they do not account for the differential physiological responses of different orthologue/splice variants to regulatory Ca 2 + . The short interdomain linker was shown to play a critical role in Ca 2 + -dependent interactions of the CBDs [13,15,24], but the exact pathway by which the allosteric signal propagates from the Ca3-Ca4 sites (on CBD1) to the membrane-embedded ion translocation sites remains elusive.…”