Crystal Structures of Progressive Ca2+ Binding States of the Ca2+ Sensor Ca2+ Binding Domain 1 (CBD1) from the CALX Na+/Ca2+ Exchanger Reveal Incremental Conformational Transitions

Wu, Mingqin; Le, Huy; Wang, Meitian; Yurkov, Vladimir; Omelchenko, Alexander; Hnatowich, Mark; Nix, Jay C.; Hryshko, Larry V.; Zheng, Lei

doi:10.1074/jbc.m109.059162

Cited by 37 publications

(70 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…6). Our findings are consistent with recent structural studies indicating that Ca 2ϩ binding enhances the stability of the Ca 2ϩ binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected (28). The underlying biological relevance is that the linker-dependent modification of Ca 2ϩ on/off kinetics at a specific regulatory site can diversify and widen the dynamic range of Ca 2ϩ sensing of CBD12 by nearly 2 orders of magnitude (while retaining a submicromolar range of Ca 2ϩ affinity).…”

Section: Discussionsupporting

confidence: 82%

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

Giladi

Boyman

Mikhasenko

et al. 2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

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 Ca , whereas the kinetic and equilibrium properties of three Ca 2؉ sites of CBD12-7Ala and CBD1 ؉ CBD2 are similar. Therefore, the linker-dependent interactions in CBD12 decelerate the Ca 2؉ on/off kinetics at a specific CBD1 site by 50 -80-fold, thereby representing Ca 2؉ "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.The Na ϩ /Ca 2ϩ exchanger proteins (NCX1-3) and their splice variants are expressed in a tissue-specific manner (1-3) and mediate Ca 2ϩ entry/extrusion depending on various factors (4, 5). NCX is "autoregulated" by cytosolic Ca 2ϩ at sites that do not directly participate in the ion translocation (6, 7). For example, the cardiac/neuronal NCX1 variants respond to 0.3-10 M cytosolic Ca 2ϩ , whereas the kidney variant does not (8, 9). Allosteric regulation of NCX involves the interaction of cytosolic Ca 2ϩ with the regulatory loop-f of NCX (10 -14). Although the high affinity regulatory site of NCX1 was discovered 16 years ago (15), only recent studies identified two Ca 2ϩ regulatory domains, CBD1 and CBD2, which form a tandem (CBD12) through a short linker (amino acids 501-504) (16). High resolution x-ray and NMR revealed C 2 -type protein folding in both CBDs (16 -20) that display four Ca 2ϩ sites (Ca1-Ca4) on CBD1 (17,18,20) and two Ca 2ϩ sites (CaI and CaII) on CBD2 (16, 19). The NCX splice variants arise from a combination of 6 small exons (A, B, C, D, E, F), while the splice variant region is exclusively restricted to the CBD2 domain (2-4, 16, 19, 24). Excitable tissues, such as those of the brain (AD splice variant) and heart (ACDEF splice variant) contain exon A, whereas kidney, stomach, and skeletal muscle tissues comprise NCX with exon B (2-5).Equilibrium binding studies have shown that isolated CBD1 protein contains two sites with high affinity for Ca ]-dependent regulation by cytosolic Ca 2ϩ in cellular systems, whereas the primary site of CBD2 (CaI) may contribute to the Ca 2ϩ -dependent relief of Na ϩ -dependent inactivation of intact NCX (23-25). Importantly, the segment that undergoes alternative splicing is located solely within the CBD2 domain, meaning that specific domain-domain interactions may shape the regulatory properties of primary Ca 2ϩ sites at CBD1 (16 -20, 24). Fluorescence resonance energy transfer studies demonstrated that CBD domains of NCX can undergo significant conformational changes in the cell within the excitation-contraction coupling (22,27), although dynamic aspects of relevant conformational transitions, as well as the contribution of each specific Ca 2ϩ site to regulatory even...

show abstract

Section: Discussionsupporting

confidence: 82%

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

Giladi

Boyman

Mikhasenko

et al. 2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…However, this interpretation is unlikely, because although CBD1 was crystallized without G503 and upstream residues, the structure shows ion coordination identical to that of CBD12 for all CBD1's sites. 30,32 Moreover, G503 participates in the β-strand interactions of CBD2. 31 Finally, the G555P mutation in CALX-CBD12 (corresponding to G503P in this study) resulted in intact capacity but reduced affinity.…”

Section: ■ Resultsmentioning

confidence: 99%

G503 Is Obligatory for Coupling of Regulatory Domains in NCX Proteins

et al. 2012

View full text Add to dashboard Cite

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.

show abstract

“…In spite of high sequence homology within the intracellular regions (Figure 1B), CALX exhibits a unique negative Ca 2+ regulatory property in contrast to the positive effect of other characterized NCXs (Hryshko et al, 1996). Our recent X-ray crystallographic analyses of individual CBD1 and CBD2 domains from the CALX 1.1 isoform showed that only CBD1 is a functional Ca 2+ binding domain ([Wu et al, 2010] and [Wu et al, 2009]). It has a nearly identical 4-Ca 2+ binding conformation to that of canine NCX1 (Nicoll et al, 2006), further supporting the importance of CBD1 in the Ca 2+ regulatory mechanism of NCXs.…”

Section: Introductionmentioning

confidence: 99%

“…However, Ca 2+ interaction with CBD1 from CALX or NCX1 results in a protein conformational change limited to the local Ca 2+ binding site, mainly the 1E-1F loop of CBD1 (the loop between the β strands 1E and 1F), arguing overall protein conformational change is not required for the Ca 2+ regulation ([Wu et al, 2010] and [Johnson et al, 2006]). NCXs have a very short linker predicted between CBD1 and CBD2 (Figure 1B).…”

Section: Introductionmentioning

confidence: 99%

Structural Basis of the Ca2+ Inhibitory Mechanism of Drosophila Na+/Ca2+ Exchanger CALX and Its Modification by Alternative Splicing

Wu¹,

Shu²,

González³

et al. 2011

Structure

Self Cite

View full text Add to dashboard Cite

Summary The Na+/Ca2+ exchanger CALX promotes Ca2+ efflux in Drosophila sensory neuronal cells to facilitate light-mediated Ca2+ homeostasis. CALX activity is negatively regulated by specific Ca2+ interaction within its two intracellular Ca2+ regulatory domains CBD1 and CBD2, yet how the Ca2+ binding is converted to molecular motion to operate the exchanger is unknown. Here we report crystal structures of the entire Ca2+ regulatory domain CBD12 from two alternative splicing isoforms, CALX, 1.1 and 1.2, exhibiting distinct regulatory Ca2+-dependency. The structures show an open V-shaped conformation with four Ca2+ ions bound on the CBD domain interface, confirmed by LRET analysis. The structures together with Ca2+ binding analysis support that the Ca2+ inhibition of CALX is achieved by interdomain conformational changes induced by Ca2+ binding at CBD1. The conformational difference between the two isoforms also indicates that alternative splicing adjusts the interdomain orientation angle to modify the Ca2+ regulatory property of the exchangers.

show abstract

Crystal Structures of Progressive Ca2+ Binding States of the Ca2+ Sensor Ca2+ Binding Domain 1 (CBD1) from the CALX Na+/Ca2+ Exchanger Reveal Incremental Conformational Transitions

Cited by 37 publications

References 27 publications

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

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

G503 Is Obligatory for Coupling of Regulatory Domains in NCX Proteins

Structural Basis of the Ca2+ Inhibitory Mechanism of Drosophila Na+/Ca2+ Exchanger CALX and Its Modification by Alternative Splicing

Contact Info

Product

Resources

About