Calcium regulation of the human mitochondrial ATP-Mg/Pi carrier SLC25A24 uses a locking pin mechanism

Abstract

Mitochondrial ATP-Mg/Pi carriers import adenine nucleotides into the mitochondrial matrix and export phosphate to the cytosol. They are calcium-regulated to control the size of the matrix adenine nucleotide pool in response to cellular energetic demands. They consist of three domains: an N-terminal regulatory domain containing four calcium-binding EF-hands, a linker loop domain with an amphipathic α-helix and a C-terminal mitochondrial carrier domain for the transport of substrates. Here, we use thermostability assays to demonstrate that the carrier is regulated by calcium via a locking pin mechanism involving the amphipathic α-helix. When calcium levels in the intermembrane space are high, the N-terminus of the amphipathic α-helix is bound to a cleft in the regulatory domain, leading to substrate transport by the carrier domain. When calcium levels drop, the cleft closes, and the amphipathic α-helix is released to bind to the carrier domain via its C-terminus, locking the carrier in an inhibited state.

Calcium‐regulated mitochondrial ATP‐Mg/Pi carriers evolved from a fusion of an EF‐hand regulatory domain with a mitochondrial ADP/ATP carrier‐like domain

“…There could be other interactions in the cytoplasmic network that have not yet been appreciated. However, in the current assessment the strength of the cytoplasmic and matrix salt‐bridge networks in APC are roughly equal, implying that APCs are strict exchangers of substrates, agreeing with experimental evidence that APC does not catalyse a significant uniport activity .…”

“…Therefore, calcium concentrations required to stimulate adenosine nucleotide transport are different in each case, because the affinity of EF‐hands for calcium binding has been adapted through specific mutations. The calcium concentration for half‐maximal activity has been estimated to be 15–30 μM calcium for yeast APC 180 μM calcium for human APC and 0.2–0.8 μM calcium for Arabidopsis thaliana APC . Calcium affects the V max but not the K m for substrate transport in APC, suggesting a non‐competitive mechanism of calcium regulation .…”

“…Electroneutral exchange implies that ATP is transported together with magnesium or protons in exchange for phosphate, as both exchanged species would then carry the same charge . The affinity of APC for ATP has been observed in the range of 30–300 μM for a number of different orthologues . This affinity is 10 to 100‐fold less than the affinity of AAC for ATP, which is in the range of 2–20 μM .…”

“…To understand how the AAC‐like carrier domain and the EF‐hand‐containing regulatory domain function together to provide calcium‐regulated adenosine nucleotide transport, an understanding of the overall position of the three APC domains relative to one another is required. In the absence of an experimental structure, a model of the protein has been pieced together using evolution coupling and structural data . This model, coupled with all available biophysical characterisation data, suggests that the carrier and regulatory domains move independently of each other and interact only via the linker‐loop region .…”

“…In the absence of an experimental structure, a model of the protein has been pieced together using evolution coupling and structural data . This model, coupled with all available biophysical characterisation data, suggests that the carrier and regulatory domains move independently of each other and interact only via the linker‐loop region . In the proposed locking pin mechanism , the amphipathic α‐helix is released from the regulatory domain when the calcium levels are low, leading to closure of the hydrophobic pockets and to its binding with residues of the cavity in the carrier domain, inhibiting the transporter domain.…”