Structure-dynamic determinants governing a mode of regulatory response and propagation of allosteric signal in splice variants of Na+/Ca2+ exchange (NCX) proteins
Abstract:The Ca(2+)-dependent allosteric regulation of Na(+)/Ca(2+) exchanger (NCX) proteins represents Ca(2+) interaction with the cytosolic domains, CBD1 (calcium-binding domain 1) and CBD2, which is associated either with activation, inhibition or no response to regulatory Ca(2+) in a given splice variant. CBD1 contains a high affinity Ca(2+)-sensor (which is highly conserved among splice variants), whereas primary information upon Ca(2+) binding to CBD1 is modified by alternative splicing of CBD2, yielding the dive… Show more
“…This result reveals that the alternative splicing segment (Fig. 1, located on CBD2) affects the folding stability of CBD12 in the apo state and thus, may predefine distinct conformational responses of CBDs to Ca 2+ -binding in a given splice variant 6, 29, 31 .Figure 4Effect of alternative splicing on the conformational dynamics of NCX3-CBD12. Deuterium uptake differences between NCX3-AC and NCX3-B in the apo form ( A ) or in the Ca 2+ -bound form ( B ) are color-coded onto the model of NCX3-AC.…”
Section: Resultsmentioning
confidence: 91%
“…Therefore, it remains unclear how Ca 2+ -dependent NCX regulation is diversified between the different isoforms and splice variants. To better understand the differential conformational dynamics of NCX-CBD12 isoforms and splice variants, the isolated preparations of NCX2-CBD12 and NCX3-CBD12 proteins were analyzed in the apo and Ca 2+ -bound states by HDX-MS 29, 31 .…”
Section: Resultsmentioning
confidence: 99%
“…This population shift of conformational states is essential for regulation, thereby representing a common mechanism for NCX1 splice variants regardless of their regulatory specificity 28 . Finally, hydrogen-deuterium exchange mass-spectrometry (HDX-MS) analyses of NCX1-CBD12 splice variants have shown that Ca 2+ binding to all tested variants mainly rigidifies the backbone dynamics of CBD2 (and not that of CBD1), where the alternative splicing of CBD2 secondarily modifies the strength and expansion of rigidification throughout CBD2 29, 31 . Importantly, the Ca 2+ -induced effects on CBD2 backbone dynamics correlate well with the regulatory specificity found in the matching splice variants of full-size NCX1s 29, 31 .…”
Section: Introductionmentioning
confidence: 99%
“…Thus far, NCX1 splice variants share a common mechanism for the initial decoding of the regulatory signal upon Ca 2+ occlusion and tethering of CBDs (i.e., through the population shift mechanism), whereas alternative splicing of CBD2 controls the propagation and strength of Ca 2+ -dependent rigidification and thereby, diversifies the regulatory response 28, 29, 31 . Currently it is unclear to what extent (if at all) the above-described mechanisms are also valid for NCX2 and NCX3 splice variants and how the exons control the CBD dynamics as related to shaping the regulatory specificity in a given isoform/splice variant.…”
Section: Introductionmentioning
confidence: 99%
“…Currently it is unclear to what extent (if at all) the above-described mechanisms are also valid for NCX2 and NCX3 splice variants and how the exons control the CBD dynamics as related to shaping the regulatory specificity in a given isoform/splice variant. To overcome this gap, here we analyzed the apo and Ca 2+ -bound forms of isolated NCX2-CBD12, NCX3-CBD12-AC, and NCX3-CBD12-B proteins with SAXS 19, 28, 29 and HDX-MS 29, 31 in order to identify their conformational dynamics and the effect of alternative splicing. The present analysis reveals a general mechanism for regulatory coupling associated with Ca 2+ interactions with regulatory CBD domains of NCX1, NCX2, and NCX3 isoforms and their splice variants, where the dynamic effects of alternative splicing on conformational dynamics of the two-domain regulatory tandem (CBD12) control the contributions of a given isoform/splice variant to dynamic handling of cytosolic Ca 2+ oscillations in distinct cell types.…”
Mammalian Na+/Ca2+ exchangers, NCX1 and NCX3, generate splice variants, whereas NCX2 does not. The CBD1 and CBD2 domains form a regulatory tandem (CBD12), where Ca2+ binding to CBD1 activates and Ca2+ binding to CBD2 (bearing the splicing segment) alleviates the Na+-induced inactivation. Here, the NCX2-CBD12, NCX3-CBD12-B, and NCX3-CBD12-AC proteins were analyzed by small-angle X-ray scattering (SAXS) and hydrogen-deuterium exchange mass-spectrometry (HDX-MS) to resolve regulatory variances in the NCX2 and NCX3 variants. SAXS revealed the unified model, according to which the Ca2+ binding to CBD12 shifts a dynamic equilibrium without generating new conformational states, and where more rigid conformational states become more populated without any global conformational changes. HDX-MS revealed the differential effects of the B and AC exons on the folding stability of apo CBD1 in NCX3-CBD12, where the dynamic differences become less noticeable in the Ca2+-bound state. Therefore, the apo forms predefine incremental changes in backbone dynamics upon Ca2+ binding. These observations may account for slower inactivation (caused by slower dissociation of occluded Ca2+ from CBD12) in the skeletal vs the brain-expressed NCX2 and NCX3 variants. This may have physiological relevance, since NCX must extrude much higher amounts of Ca2+ from the skeletal cell than from the neuron.
“…This result reveals that the alternative splicing segment (Fig. 1, located on CBD2) affects the folding stability of CBD12 in the apo state and thus, may predefine distinct conformational responses of CBDs to Ca 2+ -binding in a given splice variant 6, 29, 31 .Figure 4Effect of alternative splicing on the conformational dynamics of NCX3-CBD12. Deuterium uptake differences between NCX3-AC and NCX3-B in the apo form ( A ) or in the Ca 2+ -bound form ( B ) are color-coded onto the model of NCX3-AC.…”
Section: Resultsmentioning
confidence: 91%
“…Therefore, it remains unclear how Ca 2+ -dependent NCX regulation is diversified between the different isoforms and splice variants. To better understand the differential conformational dynamics of NCX-CBD12 isoforms and splice variants, the isolated preparations of NCX2-CBD12 and NCX3-CBD12 proteins were analyzed in the apo and Ca 2+ -bound states by HDX-MS 29, 31 .…”
Section: Resultsmentioning
confidence: 99%
“…This population shift of conformational states is essential for regulation, thereby representing a common mechanism for NCX1 splice variants regardless of their regulatory specificity 28 . Finally, hydrogen-deuterium exchange mass-spectrometry (HDX-MS) analyses of NCX1-CBD12 splice variants have shown that Ca 2+ binding to all tested variants mainly rigidifies the backbone dynamics of CBD2 (and not that of CBD1), where the alternative splicing of CBD2 secondarily modifies the strength and expansion of rigidification throughout CBD2 29, 31 . Importantly, the Ca 2+ -induced effects on CBD2 backbone dynamics correlate well with the regulatory specificity found in the matching splice variants of full-size NCX1s 29, 31 .…”
Section: Introductionmentioning
confidence: 99%
“…Thus far, NCX1 splice variants share a common mechanism for the initial decoding of the regulatory signal upon Ca 2+ occlusion and tethering of CBDs (i.e., through the population shift mechanism), whereas alternative splicing of CBD2 controls the propagation and strength of Ca 2+ -dependent rigidification and thereby, diversifies the regulatory response 28, 29, 31 . Currently it is unclear to what extent (if at all) the above-described mechanisms are also valid for NCX2 and NCX3 splice variants and how the exons control the CBD dynamics as related to shaping the regulatory specificity in a given isoform/splice variant.…”
Section: Introductionmentioning
confidence: 99%
“…Currently it is unclear to what extent (if at all) the above-described mechanisms are also valid for NCX2 and NCX3 splice variants and how the exons control the CBD dynamics as related to shaping the regulatory specificity in a given isoform/splice variant. To overcome this gap, here we analyzed the apo and Ca 2+ -bound forms of isolated NCX2-CBD12, NCX3-CBD12-AC, and NCX3-CBD12-B proteins with SAXS 19, 28, 29 and HDX-MS 29, 31 in order to identify their conformational dynamics and the effect of alternative splicing. The present analysis reveals a general mechanism for regulatory coupling associated with Ca 2+ interactions with regulatory CBD domains of NCX1, NCX2, and NCX3 isoforms and their splice variants, where the dynamic effects of alternative splicing on conformational dynamics of the two-domain regulatory tandem (CBD12) control the contributions of a given isoform/splice variant to dynamic handling of cytosolic Ca 2+ oscillations in distinct cell types.…”
Mammalian Na+/Ca2+ exchangers, NCX1 and NCX3, generate splice variants, whereas NCX2 does not. The CBD1 and CBD2 domains form a regulatory tandem (CBD12), where Ca2+ binding to CBD1 activates and Ca2+ binding to CBD2 (bearing the splicing segment) alleviates the Na+-induced inactivation. Here, the NCX2-CBD12, NCX3-CBD12-B, and NCX3-CBD12-AC proteins were analyzed by small-angle X-ray scattering (SAXS) and hydrogen-deuterium exchange mass-spectrometry (HDX-MS) to resolve regulatory variances in the NCX2 and NCX3 variants. SAXS revealed the unified model, according to which the Ca2+ binding to CBD12 shifts a dynamic equilibrium without generating new conformational states, and where more rigid conformational states become more populated without any global conformational changes. HDX-MS revealed the differential effects of the B and AC exons on the folding stability of apo CBD1 in NCX3-CBD12, where the dynamic differences become less noticeable in the Ca2+-bound state. Therefore, the apo forms predefine incremental changes in backbone dynamics upon Ca2+ binding. These observations may account for slower inactivation (caused by slower dissociation of occluded Ca2+ from CBD12) in the skeletal vs the brain-expressed NCX2 and NCX3 variants. This may have physiological relevance, since NCX must extrude much higher amounts of Ca2+ from the skeletal cell than from the neuron.
Sodium–calcium exchanger (NCX) proteins are ubiquitously expressed and play a pivotal role in cellular calcium homeostasis by mediating uphill calcium efflux across the cell membrane. Intracellular calcium allosterically regulates the exchange activity by binding to two cytoplasmic calcium‐binding domains, CBD1 and CBD2. However, the calcium‐binding affinities of these domains are seemingly inadequate to sense physiological calcium oscillations. Previously, magnesium binding to either domain was shown to tune their affinity for calcium, bringing it into the physiological range. However, while the magnesium‐binding site of CBD2 was identified, the identity of the CBD1 magnesium site remains elusive. Here, using molecular dynamics in combination with differential scanning fluorimetry and mutational analysis, we pinpoint the magnesium‐binding site in CBD1. Specifically, among four calcium‐binding sites (Ca1–Ca4) in this domain, only Ca1 can accommodate magnesium with an affinity similar to its free intracellular concentration. Moreover, our results provide mechanistic insights into the modulation of the regulatory calcium affinity by magnesium, which allows an adequate NCX activity level throughout varying physiological needs.
The Na(+) /Ca(2+) exchanger provides a major Ca(2+) extrusion pathway in excitable cells and plays a key role in the control of intracellular Ca(2+) concentrations. In Canis familiaris, Na(+) /Ca(2+) exchanger (NCX) activity is regulated by the binding of Ca(2+) to two cytosolic Ca(2+) -binding domains, CBD1 and CBD2, such that Ca(2+) -binding activates the exchanger. Despite its physiological importance, little is known about the exchanger's global structure, and the mechanism of allosteric Ca(2+) -regulation remains unclear. It was found previously that for NCX in the absence of Ca(2+) the two domains CBD1 and CBD2 of the cytosolic loop are flexibly linked, while after Ca(2+) -binding they adopt a rigid arrangement that is slightly tilted. A realistic model for the mechanism of the exchanger's allosteric regulation should not only address this property, but also it should explain the distinctive behavior of Drosophila melanogaster's sodium/calcium exchanger, CALX, for which Ca(2+) -binding to CBD1 inhibits Ca(2+) exchange. Here, NMR spin relaxation and residual dipolar couplings were used to show that Ca(2+) modulates CBD1 and CBD2 interdomain flexibility of CALX in an analogous way as for NCX. A mechanistic model for the allosteric Ca(2+) regulation of the Na(+) /Ca(2+) exchanger is proposed. In this model, the intracellular loop acts as an entropic spring whose strength is modulated by Ca(2+) -binding to CBD1 controlling ion transport across the plasma membrane.
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