Structure-Based Function and Regulation of NCX Variants: Updates and Challenges

Figures

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  Figure 1. Structure-topological arrangements of prokaryotic and eukaryotic NCX proteins. Eukaryotic (A) and prokaryotic (B) NCX proteins consist of 10 trans-membrane helices (TM1–10), where highly conserved α-repeat regions form a four-helix ion passageway with TM2/TM3 (α1) and TM7/TM8 (α2) bands (shown boxed). Eukaryotic NCXs contain a very long (~520 residues) cytosolic 5L6 loop (f-loop) between TM5 and TM6 with the Ca2+ binding regulatory domains, CBD1 and CBD2. In the lack of regulatory domains, the 5L6 loop of prokaryotic NCXs is very short (12–16 residues). (C) The crystal structure of the archaeal NCX_Mj protein (PDB 3V5U) in the OF orientation describes a tightly packed core of eight helices (TM2-TM5 and TM7–TM10), where two long and loosely packed helices (TM1 and TM6) form a two-helix slanted bundle (TM1/TM6) in front of a rigid eight-helix hub (TM2–TM5 and TM7–TM10). (D) The α1 and α2 repeats, covering the ion-transporting four-helix structure (TM2/TM3 and TM7/TM8), are shown from the extracellular side of the NCX_Mj (PDB 3V5U) crystal structure. The combined data, attained from X-ray crystallography, MD simulations, and mutational effects on ion fluxes, support a model…

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  Figure 2. Isoform/splice variants of eukaryotic NCX variants. (A) The eukaryotic NCX variants share a mode of Ca2+-dependent activation (due to the Ca2+ binding to CBD1). NCX1 and NCX3 display a mode of Na+-induced inactivation (due to the Na+ binding to some unknown site). NCX2 lacks the Na+-induced inactivation mode. The Ca2+ binding to CBD2 can alleviate the Na+-induced inactivation only in the splice variants of NCX1 and NCX3 that have the Ca2+ binding sites at CBD2. Exon A generates the Ca2+ binding sites in CBD2 of NCX1 (but not of NCX3). In contrast, exon B breads the Ca2+ sites in CBD2 of NCX3 (but not of NCX1). (B) The protein products of NCX1, NCX2, and NCX3 genes (isoforms) are expressed in a tissue-specific manner, thereby underscoring diverse regulatory responses of NCX variants to cell-specific Ca2+ signaling/homeostasis. (C) The Ca2+-binding regulatory domains, CBD1 and CBD2, form a head-to-tail tandem of two regulatory domains (CBD12). The alternatively spliced region is located exclusively with CBD2. The splice variants of NCX1 and NCX3 arise from a combination of six small exons (A, B, C, D, E and F), where a mutually exclusive exon (either A or B) appears in every splice…

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  Figure 3. Structural organization and sequence alignment of ion-coordinating residues in NCXs and similar proteins. The sliding mechanism is a distinct variant of the alternating access mechanism while describing specific conformational changes associated with the OF/IF swapping. The open (X-ray structure) (A) and closed (computed) (B) structures of NCX_Mj at the extracellular side. Upon ion binding, the two-helix sliding bundle, TM1/TM6 (presented in red), might relocates in front of other helixes to swap the OF and IF states. (C) Sequence alignment of NCX, NCKX, NCLX, and CAX antiporters (belonging to the superfamily of Ca2+/CA antiporters). Twelve ion-coordinating residues (in red boxes), attached to four helixes (TM2, TM3, TM7, and TM8) form an ion-passageway entity. The ion-coordinating residues are highly conserved in Ca2+/CA proteins—e.g., the NCX_Mj and mammalian NCXs have three (out of twelve) different ion-coordinating residues (T50S, E213D and D240N) although their transport rates differ by several orders of magnitude.

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  Figure 4. The Ca2+- and Na+-bound intermediates during the transport cycle of Na+/Ca2+ exchange. (A) The Na+/Ca2+ exchange cycle involves separate steps of 3Na+ or 1Ca2+ translocation across the membrane where the transition state (either for Na+ or Ca2+ transport step) is between the occluded states at the extracellular and cytosolic sides. According to this model, the TM1/TM6 undergoes a sliding movement toward performing the OF/IF swapping. Ion interactions with respective sites generate the occluded states either at the extracellular or cytosolic side (green and red spheres represent the Na+ and Ca2+ ions, respectively). Dashed lines refer to the two-helix sliding bundle, TM1/TM6). (B) The emerging working hypothesis is that the occupation of the SCa site plays a critical role in closing the hydrophilic gap between the TM2C (P53) and TM7B (P212) segments, thereby forming a transient hydrophobic patch. The transient hydrophobic environment between TM2 and TM7 can facilitate the sliding movement of the TM1/TM6 bundle on the protein surface, thereby accomplishing the OF/IF swapping.

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  Figure 5. Structural basis of exon-dependent regulatory diversity in eukaryotic NCX variants. (A) Experimentally obtained NMR structures of CBD1 (2FWS), CBD2-AD (2FWU), and CBD2-BD (2KLT) of NCX1 were superimposed on the template of NCX1-CBD12 crystal structure (PBD 3US9) to demonstrate the structural positions of mutually exclusive (A or B) and cassette (CDEF) exons at CBD2. (B) The crystal structure of NCX1-CBD2-AD (PDB 2QVM) shows that K585 forms a salt bridge with D552 and E648 in the absence of Ca2+ (thereby stabilizing apo-CBD2), whereas in the presence of Ca2+, two Ca2+ binding sites (CaI and CaII) can be occupied by Ca2+. Different residues at the three key positions (522, 578, and 585) can predefine the number of Ca2+ binding sites at CBD2 in an exon-dependent manner. For example, in NCX1, exon B variants contain Arg (instead of Asp or Glu) and Cys (instead of Lys) at positions 578 and 585, respectively, which presents Ca2+ binding to CaI of NCX1-CBD2, thereby destabilizing the CBD2 folding. In contrast, the A-exon containing variants of NCX1 retain its structural integrity even in the absence of Ca2+ since K585 can form salt bridges with neighboring negatively charged residues…

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  Figure 6. The interface structures of the two-domain CBD12 tandem in the NCX and CALX orthologs. Carton presentation of NCX1-CBD12-AD (PDB 3US9) and CALX1.1-CBD12 (PDB 3RB5) and CALX1.2 (PDB 3RB7) crystal structures depict highly conserved residues participating in the network of interdomain salt bridges. Green balls indicate the Ca2+ binding sites at the interface of NCX1-CBD12-AD, CALX1.1-CBD12, and CALX1.2-CBD12. The blue ball in NCX1-CBD12 refers to the Ca2+ binding site that cannot be resolved in the PDB 3US9 crystal structure, although the position of this Ca2+ binding site is nearly identical to the Ca2+ binding site of CALX1.1-CBD12 (PDB 3RB5) and CALX1.2 (PDB 3RB7) as revealed by these crystal structures. The Ca2+ occlusion occurs at the high-affinity binding sites (Ca3-Ca4) of CBD1 in all three proteins (shown in the red-dotted cycle). Although the interdomain salt-bridge structures are highly conserved among NCX and CALX variants, a striking difference between the NCX and CALX variants occurs in the structural organization of the FG-loop α-helix at CBD2. More specifically, the CALX1.1-CBD12 and CALX1.2-CBD12 variants have a two-headed (H1 and H2) short-helix structure, which…

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  Figure 7. Structural elements that contribute to the propagation and integration of allosteric signals. (A) The occupation of the Ca3-Ca4 sites by Ca2+ rigidifies the backbone dynamics at the two-domain interface of CBDs. The allosteric signal propagation from the C-terminal tip of CBD1 to the C-terminal end of CBD2 (shown in a solid red arrow) is under the control of the splicing segment at CBD2 and the two-domain interface. (B) The positively charged XIP (autoinhibitory) domain can anchor the negatively charged helix (PAL) at the palmitoylation site. Since the PAL domain links to TM6, the XIP/PAL unit may affect the sliding of the TM1/TM6 bundle, thereby modulating the transport rates. The interactions of inhibitory (e.g., Na+) and activating (e.g., PIP2) ligands with respective sites (located of the XIP/PAL unit) can shift a steady-state equilibrium between the active and inactive states. Blue and red cylinders represent the XIP and PAL domains, respectively. The interdomain movements of the CBD1 and CBD2 domains might involve translational (C) and rotational (D) movements. Specific structural elements (e.g., the two-domain interface, XIP/PAL unit, and varying exon compositions) may…