A self-sequestered calmodulin-like Ca²⁺ sensor of mitochondrial SCaMC carrier and its implication to Ca²⁺-dependent ATP-Mg/P(i) transport

Abstract

The mitochondrial carriers play essential roles in energy metabolism. The short Ca²⁺-binding mitochondrial carrier (SCaMC) transports ATP-Mg in exchange for Pi and is important for activities that depend on adenine nucleotides. SCaMC adopts, in addition to the transmembrane domain (TMD) that transports solutes, an extramembrane N-terminal domain (NTD) that regulates solute transport in a Ca²⁺-dependent manner. Crystal structure of the Ca²⁺-bound NTD reveals a compact architecture in which the functional EF hands are sequestered by an endogenous helical segment. Nuclear magnetic resonance (NMR) relaxation rates indicated that removal of Ca²⁺ from NTD results in a major conformational switch from the rigid and compact Ca²⁺-bound state to the dynamic and loose apo state. Finally, we showed using surface plasmon resonance and NMR titration experiments that free apo NTDs could specifically interact with liposome-incorporated TMD, but that Ca²⁺ binding drastically weakened the interaction. Our results together provide a molecular explanation for Ca²⁺-dependent ATP-Mg flux in mitochondria.

Figure 1. Structure of the Ca²⁺-bound NTD of human SCaMC-1 shows a self-sequestered CaM-like architecture

(A) Cartoon and surface representations of the 2.1 Å crystal structure of the Ca²⁺-bound NTD. The NTD construct used for structure determination consists of residues 1–193, but electron density was observed only for residues 22–173. EF-hands are colored in orange, yellow, cyan, and blue. The extra helix (H9) after the CaM-like domain is colored in magenta. Ca²⁺ ions are represented by green sphere, and their simulated omit map are shown as blue mesh (contoured at 4 σ). See also Figure S2. (B) Structures of the Ca²⁺-CaM with and without the bound peptide, shown for comparison with the NTD. Ca²⁺-CaM and Ca²⁺-CaM-peptide complexes are adapted from PDB: 4CLN and 2BBN, respectively. The N- and C-lobes are colored in yellow and cyan, respectively. The central flexible loop is in gray and the target peptide is in green. (C) Correlation of ¹D_NH measured for Ca²⁺-bound NTD in solution to ¹D_NH values calculated based on the 2.1 Å crystal structure. The goodness of fit was accessed by Pearson correlation coefficient (R) and the quality factor (Q). The outliers with discrepancy between measured and calculated ¹D_NH larger than 10 Hz are mapped onto the structure of the Ca²⁺-bound NTD (Insert).

Figure 2. Helix-9 appears to be an intramolecular target peptide for the SCaMC EF hands

(A) Detailed view of H9 interacting with EF-hands III & IV, showing the residues involved in the interaction. The color scheme used is the same as in Figure 1. (B) Sequence alignment of H9 from SCaMC paralogs and homologs. Residues with more than 70% conservation are colored in red. The five hydrophobic residues involved in binding to the EF hands are highlighted in yellow. A more complete alignment is shown in Figure S3. (C) Structure alignment between Ca²⁺-NTD and CaM-peptide complex. The N- and C-lobes of NTD are colored in orange and blue, respectively, whereas the CaM N- and C-lobes are in pink and cyan, respectively. The SCaMC-1 H9 (magenta) aligns well with the target peptide of CaM (green) in the CaM-peptide complex.

Figure 3. The apo NTD is more dynamic and less structured than Ca²⁺-bound NTD

A, C, and E show the residue-specific heteronuclear ¹⁵N(¹H) NOE, R₂, and R₁ values of the Ca²⁺-bound NTD, measured at 30 °C and ¹H frequency of 750 MHz. The corresponding NOE and relaxation rates for the apo NTD are shown in B, D, and F. Secondary structure elements of the Ca²⁺-bound NTD are indicated above the plots. The color scheme used here is the same as in Figure 1A. See also Figure S1. (G) The flexible regions (¹⁵N(¹H) NOE < 0.75) found in the apo NTD (salmon) are mapped onto the structure of the Ca²⁺-bound NTD, for showing regions of the Ca²⁺-bound NTD that become largely disordered in the absence of Ca²⁺. The unassigned residues (139–155) in the apo state are colored in blue.

Figure 4. The apo, but not the Ca²⁺-bound NTD binds specifically to TMD

(A) SPR sensorgrams of the binding of NTD to immobilized TMD proteoliposomes at different NTD concentrations in the absence of Ca²⁺ (left) and in the presence of 5 mM Ca²⁺ (right). (B) Binding isotherms derived from data in (A) determined K_D of 250 ± 50 μM for the apo NTD and 5600 ± 1000 μM for the Ca²⁺-bound NTD. In this analysis, equilibrium signals at various NTD concentrations were plotted and fit with a single binding isotherm to determine K_D. The K_D values presented are mean ± SD, calculated from 3 independent measurements. (C) SPR sensorgrams of the binding of 33 μM NTD, which does not contain EDTA, to the immobilized TMD proteoliposomes in the presence of 0, 3, 10, 30, 100, 300 and 1000 μM of Ca²⁺. (D) Binding isotherms derived from data in (C) determined apparent K_D of 64.7 ± 18.8 μM for Ca²⁺. In this analysis, the RU at 0 μMCa ²⁺ was used as a reference point and the change in RU (ΔRU) as compared to 0 μM Ca²⁺ for the rest of the data points were plotted and fit with a single binding isotherm to determine apparent K_D. The K_D values presented are mean ± SD, calculated from 2 independent measurements. See also Figure S5 (E) The ¹H-¹⁵N TROSY-HSQC spectra of 0.1 mM apo NTD in the presence of empty liposomes (left) or 0.1 mM TMD incorporated proteoliposomes (right), both containing 20 mM total lipid. The majority of the peaks disappeared with the exception of the dynamic regions, residues 53–72 and 172–193. (F) The same spectra as in (C) recorded for Ca²⁺-bound NTD, showing essentially no change in the presence of the TMD. See also Figure S4

Figure 5. A proposed model for Ca²⁺-regulated ATP-Mg/Pi transport by SCaMC

(A) In the absence of Ca²⁺, the NTD has a dynamic and loose conformation; it can specifically interact with and probably undergo conformational change and “cap” the intermembrane space side of the TMD. Apo form NTD is represented by the ellipsoid. (B) In the presence of Ca²⁺, the NTD is in a compact and self-sequestered form that no longer interacts with the TMD. The “uncapping” allows solutes to be transported. The TMD structure was modeled after the crystal structure of the inhibited state of the ADP/ATP carrier (PDB ID: 1OKC).