Reuben Hiller

Kinetic and equilibrium properties of regulatory calcium sensors of NCX1 protein.

The crystal structures of the CBD1 and CBD2 domains of the Na+/Ca2+ exchanger protein (NCX1) provided a major breakthrough in Ca2+-dependent regulation of NCX1, although the dynamic aspects of the underlying molecular mechanisms are still not clear. Here we provide new experimental approaches for evaluating the kinetic and equilibrium properties of Ca2+ interaction with regulatory sites by using purified preparations of CBD1, CBD2, and CBD12 proteins. CBD12 binds ∼6 Ca2+ ions (mol/mol), whereas the binding of only ∼2 Ca2+ ions is observed (with a Hill coefficient of nH = ∼2) either for CBD1 or CBD2. In the absence of Mg2+, CBD1 has a much higher affinity for Ca2+ (Kd = 0.3 ± 1.2 μm) than CBD2 (Kd = 5.0 ± 1.2 μm). The Ca2+ dissociation from CBD2 (koff = 230 ± 70 s–1) is at least 25 times faster than from CBD1 (koff = 10 ± 3 s–1), whereas the kon values indicate fast kinetics for Ca2+ binding (kon = koff/Kd = 107–108 m–1 s–1) for both CBDs. At 2–5 mm Mg2+, both CBDs bind Ca2+, with a Kd of 1–2 μm (Mg2+ has very little effect on Ca2+ off rates). Mg2+ cannot occupy the primary site of CBD2, whereas the other Ca2+ sites of CBDs interact with Mg2+ as well. There is no competition between Na+ and Ca2+ for any CBD site. The kinetically diverse Ca2+ sensors may sense differentially the dynamic swings in [Ca2+] within specific subcellular compartments (dyadic cleft, submembrane space, bulk cytosol, etc.).

Essential Role of the CBD1-CBD2 Linker in Slow Dissociation of Ca2+ from the Regulatory Two-domain Tandem of NCX1

In NCX proteins CBD1 and CBD2 domains are connected through a short linker (3 or 4 amino acids) forming a regulatory tandem (CBD12). Only three of the six CBD12 Ca2+-binding sites contribute to NCX regulation. Two of them are located on CBD1 (Kd = ∼0.2 μm), and one is on CBD2 (Kd = ∼5 μm). Here we analyze how the intrinsic properties of individual regulatory sites are affected by linker-dependent interactions in CBD12 (AD splice variant). The three sites of CBD12 and CBD1 + CBD2 have comparable Kd values but differ dramatically in their Ca2+ dissociation kinetics. CBD12 exhibits multiphasic kinetics for the dissociation of three Ca2+ ions (kr = 280 s−1, kf = 7 s−1, and ks = 0.4 s−1), whereas the dissociation of two Ca2+ ions from CBD1 (kf = 16 s−1) and one Ca2+ ion from CBD2 (kr = 125 s−1) is monophasic. Insertion of seven alanines into the linker (CBD12–7Ala) abolishes slow dissociation of Ca2+, whereas the kinetic and equilibrium properties of three Ca2+ sites of CBD12–7Ala and CBD1 + CBD2 are similar. Therefore, the linker-dependent interactions in CBD12 decelerate the Ca2+ on/off kinetics at a specific CBD1 site by 50–80-fold, thereby representing Ca2+ “occlusion” at CBD12. Notably, the kinetic and equilibrium properties of the remaining two sites of CBD12 are “linker-independent,” so their intrinsic properties are preserved in CBD12. In conclusion, the dynamic properties of three sites are specifically modified, conserved, diversified, and integrated by the linker in CBD12, thereby generating a wide range dynamic sensor.

Proton-sensing Ca2+ Binding Domains Regulate the Cardiac Na+/Ca2+ Exchanger

The cardiac Na+/Ca2+ exchanger (NCX) regulates cellular [Ca2+]i and plays a central role in health and disease, but its molecular regulation is poorly understood. Here we report on how protons affect this electrogenic transporter by modulating two critically important NCX C2 regulatory domains, Ca2+ binding domain-1 (CBD1) and CBD2. The NCX transport rate in intact cardiac ventricular myocytes was measured as a membrane current, INCX, whereas [H+]i was varied using an ammonium chloride “rebound” method at constant extracellular pH 7.4. At pHi = 7.2 and [Ca2+]i < 120 nm, INCX was less than 4% that of its maximally Ca2+-activated value. INCX increases steeply at [Ca2+]i between 130–150 nm with a Hill coefficient (nH) of 8.0 ± 0.7 and K0.5 = 310 ± 5 nm. At pHi = 6.87, the threshold of Ca2+-dependent activation of INCX was shifted to much higher [Ca2+]i (600–700 nm), and the relationship was similarly steep (nH = 8.0±0.8) with K0.5 = 1042 ± 15 nm. The Vmax of Ca2+-dependent activation of INCX was not significantly altered by low pHi. The Ca2+ affinities for CBD1 (0.39 ± 0.06 μm) and CBD2 (Kd = 18.4 ± 6 μm) were exquisitely sensitive to [H+], decreasing 1.3–2.3-fold as pHi decreased from 7.2 to 6.9. This work reveals for the first time that NCX can be switched off by physiologically relevant intracellular acidification and that this depends on the competitive binding of protons to its C2 regulatory domains CBD1 and CBD2.

Sodium recognition by the Na+/Ca2+ exchanger in the outward-facing conformation.

Significance Na+/Ca2+ exchangers (NCXs) have a key role in the homeostasis of cellular Ca2+ and consequently are implicated in diverse human-health disorders, including neurodegenerative and cardiovascular diseases. A detailed understanding of the molecular mechanisms of these membrane proteins is therefore of interest from fundamental and biomedical standpoints. Here, we establish the structural mechanism of Na+ recognition in a prokaryotic NCX homolog, using atomistic molecular-dynamics simulations based on recently reported crystallographic data, as well as experimental transport assays of wild-type and mutagenized exchangers. The results have general implications pertaining to the ion exchange stoichiometry and electrogenicity of the Na+/Ca2+ transport cycle across the NCX family, and provide the basis for future investigations of the conformational mechanism of these important transporters. Na+/Ca2+ exchangers (NCXs) are ubiquitous membrane transporters with a key role in Ca2+ homeostasis and signaling. NCXs mediate the bidirectional translocation of either Na+ or Ca2+, and thus can catalyze uphill Ca2+ transport driven by a Na+ gradient, or vice versa. In a major breakthrough, a prokaryotic NCX homolog (NCX_Mj) was recently isolated and its crystal structure determined at atomic resolution. The structure revealed an intriguing architecture consisting of two inverted-topology repeats, each comprising five transmembrane helices. These repeats adopt asymmetric conformations, yielding an outward-facing occluded state. The crystal structure also revealed four putative ion-binding sites, but the occupancy and specificity thereof could not be conclusively established. Here, we use molecular-dynamics simulations and free-energy calculations to identify the ion configuration that best corresponds to the crystallographic data and that is also thermodynamically optimal. In this most probable configuration, three Na+ ions occupy the so-called Sext, SCa, and Sint sites, whereas the Smid site is occupied by one water molecule and one H+, which protonates an adjacent aspartate side chain (D240). Experimental measurements of Na+/Ca2+ and Ca2+/Ca2+ exchange by wild-type and mutagenized NCX_Mj confirm that transport of both Na+ and Ca2+ requires protonation of D240, and that this side chain does not coordinate either ion at Smid. These results imply that the ion exchange stoichiometry of NCX_Mj is 3:1 and that translocation of Na+ across the membrane is electrogenic, whereas transport of Ca2+ is not. Altogether, these findings provide the basis for further experimental and computational studies of the conformational mechanism of this exchanger.

A Common Ca2+-Driven Interdomain Module Governs Eukaryotic NCX Regulation

Na+/Ca2+ exchanger (NCX) proteins mediate Ca2+-fluxes across the cell membrane to maintain Ca2+ homeostasis in many cell types. Eukaryotic NCX contains Ca2+-binding regulatory domains, CBD1 and CBD2. Ca2+ binding to a primary sensor (Ca3-Ca4 sites) on CBD1 activates mammalian NCXs, whereas CALX, a Drosophila NCX ortholog, displays an inhibitory response to regulatory Ca2+. To further elucidate the underlying regulatory mechanisms, we determined the 2.7 Å crystal structure of mammalian CBD12-E454K, a two-domain construct that retains wild-type properties. In conjunction with stopped-flow kinetics and SAXS (small-angle X-ray scattering) analyses of CBD12 mutants, we show that Ca2+ binding to Ca3-Ca4 sites tethers the domains via a network of interdomain salt-bridges. This Ca2+-driven interdomain switch controls slow dissociation of “occluded” Ca2+ from the primary sensor and thus dictates Ca2+ sensing dynamics. In the Ca2+-bound conformation, the interdomain angle of CBD12 is very similar in NCX and CALX, meaning that the interdomain distances cannot account for regulatory diversity in NCX and CALX. Since the two-domain interface is nearly identical among eukaryotic NCXs, including CALX, we suggest that the Ca2+-driven interdomain switch described here represents a general mechanism for initial conduction of regulatory signals in NCX variants.

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

Background: Ca2+ binding to the regulatory two-domain tandem (CBD12) activates ion transport in NCX. Results: Ca2+ binding to high affinity sites of CBD12 results in a population shift, where more constraint conformational states become highly populated. Conclusion: Ca2+-induced population shift governs NCX activation with no significant contribution of global conformational changes in CBD alignment. Significance: Population shift may represent a general mechanism for regulating NCX. In eukaryotic Na+/Ca2+ exchangers (NCX) the Ca2+ binding CBD1 and CBD2 domains form a two-domain regulatory tandem (CBD12). An allosteric Ca2+ sensor (Ca3–Ca4 sites) is located on CBD1, whereas CBD2 contains a splice-variant segment. Recently, a Ca2+-driven interdomain switch has been described, albeit how it couples Ca2+ binding with signal propagation remains unclear. To resolve the dynamic features of Ca2+-induced conformational transitions we analyze here distinct splice variants and mutants of isolated CBD12 at varying temperatures by using small angle x-ray scattering (SAXS) and equilibrium 45Ca2+ binding assays. The ensemble optimization method SAXS analysis demonstrates that the apo and Mg2+-bound forms of CBD12 are highly flexible, whereas Ca2+ binding to the Ca3–Ca4 sites results in a population shift of conformational landscape to more rigidified states. Population shift occurs even under conditions in which no effect of Ca2+ is observed on the globally derived Dmax (maximal interatomic distance), although under comparable conditions a normal [Ca2+]-dependent allosteric regulation occurs. Low affinity sites (Ca1–Ca2) of CBD1 do not contribute to Ca2+-induced population shift, but the occupancy of these sites by 1 mm Mg2+ shifts the Ca2+ affinity (Kd) at the neighboring Ca3–Ca4 sites from ∼ 50 nm to ∼ 200 nm and thus, keeps the primary Ca2+ sensor (Ca3–Ca4 sites) within a physiological range. Thus, Ca2+ binding to the Ca3–Ca4 sites results in a population shift, where more constraint conformational states become highly populated at dynamic equilibrium in the absence of global conformational transitions in CBD alignment.

Asymmetric Preorganization of Inverted Pair Residues in the Sodium-Calcium Exchanger

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.

Functional asymmetry of bidirectional Ca2+-movements in an archaeal sodium–calcium exchanger (NCX_Mj)

Dynamic features of Ca(2+) interactions with transport and regulatory sites control the Ca(2+)-fluxes in mammalian Na(+)/Ca(2+)(NCX) exchangers bearing the Ca(2+)-binding regulatory domains on the cytosolic 5L6 loop. The crystal structure of Methanococcus jannaschii NCX (NCX_Mj) may serve as a template for studying ion-transport mechanisms since NCX_Mj does not contain the regulatory domains. The turnover rate of Na(+)/Ca(2+) exchange (kcat=0.5±0.2 s(-1)) in WT-NCX_Mj is 10(3)-10(4) times slower than in mammalian NCX. In NCX_Mj, the intrinsic equilibrium (Kint) for bidirectional Ca(2+) movements (defined as the ratio between the cytosolic and extracellular Km of Ca(2+)/Ca(2+) exchange) is asymmetric, Kint=0.15±0.5. Therefore, the Ca(2+) movement from the cytosol to the extracellular side is ∼7-times faster than in the opposite direction, thereby representing a stabilization of outward-facing (extracellular) access. This intrinsic asymmetry accounts for observed differences in the cytosolic and extracellulr Km values having a physiological relevance. Bidirectional Ca(2+) movements are also asymmetric in mammalian NCX. Thus, the stabilization of the outward-facing access along the transport cycle is a common feature among NCX orthologs despite huge differences in the ion-transport kinetics. Elongation of the cytosolic 5L6 loop in NCX_Mj by 8 or 14 residues accelerates the ion transport rates (kcat) ∼10 fold, while increasing the Kint values 100-250-fold (Kint=15-35). Therefore, 5L6 controls both the intrinsic equilibrium and rates of bidirectional Ca(2+) movements in NCX proteins. Some additional structural elements may shape the kinetic variances among phylogenetically distant NCX variants, although the intrinsic asymmetry (Kint) of bidirectional Ca(2+) movements seems to be comparable among evolutionary diverged NCX variants.

Structure-dynamic basis of splicing-dependent regulation in tissue-specific variants of the sodium-calcium exchanger

Tissue‐specific splice variants of Na+/Ca2+ exchangers contain 2 Ca2+‐binding regulatory domains (CBDs), CBD1 and CBD2. Ca2+ interaction with CBD1 activates sodium‐calcium exchangers (NCXs), and Ca2+ binding to CBD2 alleviates Na+‐dependent inactivation. A combination of mutually exclusive (A, B) and cassette (C‐F) exons in CBD2 raises functionally diverse splice variants through unknown mechanisms. Here, the effect of exons on CBDs backbone dynamics were investigated in the 2‐domain tandem (CBD12) of the brain, kidney, and cardiac splice variants by using hydrogen‐deuterium exchange mass spectrometry and stopped‐flow techniques. Mutually exclusive exons stabilize interdomain interactions in the apoprotein, which primarily predefines the extent of responses to Ca2+ binding. Deuterium uptake levels were up to 20% lower in the cardiac vs. the brain CBD12, reveling that elongation of the CBD2 FG loop by cassette exons rigidifies the interdomain Ca2+ salt bridge at the 2‐domain interface, which secondarily modulates the Ca2+‐bound states. In matching splice variants, the extent of Ca2+‐induced rigidification correlates with decreased (up to 10‐fold) Ca2+ off rates, where the cardiac CBD12 exhibits the slowest Ca2+ off rates. Collectively, structurally disordered/dynamic segments at mutually exclusive and cassette exons have local and distant effects on the folded structures nearby the Ca2+ binding sites, which may serve as a structure‐dynamic basis for splicing‐dependent regulation of NCX.—Lee, S. Y., Giladi, M., Bohbot, H., Hiller, R., Chung, K. Y., Khananshvili, D., Structure‐dynamic basis of splicing‐dependent regulation in tissue‐specific variants of the sodium‐calcium exchanger. FASEB J. 30, 1356–1366 (2016). www.fasebj.org

Structure-Based Engineering of Lithium-Transport Capacity in an Archaeal Sodium–Calcium Exchanger

Members of the Ca(2+)/cation exchanger superfamily (Ca(2+)/CA) share structural similarities (including highly conserved ion-coordinating residues) while exhibiting differential selectivity for Ca(2+), Na(+), H(+), K(+), and Li(+). The archaeal Na(+)/Ca(2+) exchanger (NCX_Mj) and its mammalian orthologs are highly selective for Na(+), whereas the mitochondrial ortholog (NCLX) can transport either Li(+) or Na(+) in exchange with Ca(2+). Here, structure-based replacement of ion-coordinating residues in NCX_Mj resulted in a capacity for transporting either Na(+) or Li(+), similar to the case for NCLX. This engineered protein may serve as a model for elucidating the mechanisms underlying ion selectivity and ion-coupled alternating access in NCX and similar proteins.