Molecular Basis of MgATP Selectivity of the Mitochondrial SCaMC Carrier

Abstract

SUMMARY The mitochondrial matrix is the supplier of cellular adenosine triphosphate (ATP). The short Ca2+-binding mitochondrial carrier (SCaMC) is one of the two mitochondrial carriers responsible for transporting ATP across the mitochondrial inner membrane. While the ADP/ATP carrier (AAC) accounts for the bulk ADP/ATP recycling in the matrix, the function of SCaMC is important for mitochondrial activities that depend on adenine nucleotides, such as gluconeogenesis and mitochondrial biogenesis. A key difference between SCaMC and AAC is that SCaMC selectively transport MgATP whereas AAC only transport free nucleotides. Here we use a combination of nuclear magnetic resonance (NMR) experiments and functional mutagenesis to investigate the structural basis of the MgATP selectivity in SCaMC. Our data revealed an MgATP binding site inside the transporter cavity, while identifying an aspartic acid residue that plays an important role in the higher selectivity for MgATP over free ATP.

In vitro reconstitution, functional dissection, and mutational analysis of metal ion transport by mitoferrin-1

“…Moreover, eukaryotic membrane proteins are fragile molecules once extracted from the membrane and the harsh detergents used for solubilization and purification of the carriers using this method cast doubt about the integrity of the proteins throughout such preparations. Accordingly, biophysical data on interactions between the carriers and their respective substrates exist for only a few MCs (24)(25)(26)(27).…”

A functional NMR for membrane proteins: dynamics, ligand binding, and allosteric modulation

“…In another study of substrate binding to carriers, Run et al . used paramagnetic ATP‐Mn 2+ to probe ATP‐Mg 2+ binding site in SCaMC, a calcium regulated carrier that selectively transports ATP‐Mg 2+ . The SCaMC is one of the two carriers responsible for transporting ATP across the mitochondrial inner membrane.…”

“…Unlike AAC that selectively transports free ATP, SCaMC has strong selectivity for ATP‐Mg 2+ over free ATP, and the structural determinant of this selectivity remained elusive. Since the endogenous substrate of SCaMC is ATP‐Mg 2+ , it was convenient to substitute Mg 2+ with paramagnetic Mn 2+ . Addition of ATP‐Mn 2+ to the carrier domain of SCaMC caused strong broadening of a subset of NMR peaks [Fig.…”

“…(D)]. Functional mutagenesis showed that, indeed, mutating Asp361 to tyrosine completely removed selectivity for ATP‐Mg 2+ over ATP in SCaMC …”

“…53 In another study of substrate binding to carriers, Run et al used paramagnetic ATP-Mn 21 to probe ATP-Mg 21 binding site in SCaMC, a calcium regulated carrier that selectively transports ATP-Mg 21 . 55 The SCaMC is one of the two carriers responsible for transporting ATP across the mitochondrial inner membrane. While the AAC accounts for the bulk ADP/ATP recycling in the matrix, the function of SCaMC is important for mitochondrial activities that depend on adenine nucleotides, such as gluconeogenesis and mitochondrial biogenesis.…”