Structure and function of the human mitochondrial MRS2 channel

Abstract

The human Mitochondrial RNA Splicing 2 protein (MRS2) has been implicated in Mg²⁺ transport across mitochondrial inner membranes, thus playing an important role in Mg²⁺ homeostasis critical for mitochondrial integrity and function. However, the molecular mechanisms underlying its fundamental channel properties such as ion selectivity and regulation remain unclear. Here, we present structural and functional investigation of MRS2. Cryo-electron microscopy structures in various ionic conditions reveal a pentameric channel architecture and the molecular basis of ion permeation and potential regulation mechanisms. Electrophysiological analyses demonstrate that MRS2 is a Ca²⁺-regulated, non-selective channel permeable to Mg²⁺, Ca²⁺, Na⁺ and K⁺, which contrasts with its prokaryotic ortholog, CorA, operating as a Mg²⁺-gated Mg²⁺ channel. Moreover, a conserved arginine ring within the pore of MRS2 functions to restrict cation movements, likely preventing the channel from collapsing the proton motive force that drives mitochondrial ATP synthesis. Together, our results provide a molecular framework for further understanding MRS2 in mitochondrial function and disease.

Extended Data Fig. 1 |. Cryo-EM data processing and validation.

a-c, Image processing and map validation of MRS2_Mg (a), MRS2_EDTA (b), and MRS2_Ca (c). Representative micrographs and 2D classes are shown. The final 3D reconstructions are colored by local resolution. Also shown are Fourier shell correlations (FSC) and orientation distribution of particles used in the final reconstruction.

Extended Data Fig. 2 |. Cryo-EM density.

a, Ribbon representation of a single subunit of MRS2_Mg and cryo-EM density. b, Segments of the final refined model of MRS2_Mg and the corresponding cryo-EM densities.

Extended Data Fig. 3 |. Structural comparison of human MRS2 and TmCorA.

a-b, A single subunit of human MRS2 (a) and TmCorA (b, PDB ID: 4I0U). The N-terminal α/β domain is also highlighted for comparison. c, Divalent ion binding sites in human MRS2 (left panels) and TmCorA (right panels).

Extended Data Fig. 4 |. Sequence alignment of MRS2 channels.

Multiple protein sequences, including Homo sapiens MRS2 (hMRS2, NCBI sequence: NP_065713.1), Mus musculus MRS2 (mMRS2, NCBI sequence: NP_001013407.2), Rattus norvegicus MRS2 (rMRS2, NCBI sequence: NP_076491.1), Danio rerio MRS2 (zMRS2, NCBI sequence: XP_693621.5), Saccharomyces cerevisiae MRS2 (ScMRS2, NCBI sequence: NP_014979.1), Schizosaccharomyces pombe MRS2 (SpMRS2, NCBI sequence: NP_596358.1), Arabidopsis thaliana MRS2 (AtMRS2, NCBI sequence: AAM62917.1),and Thermotoga maritima CorA (TmCorA, NCBI sequence: WP_004081315.1). Secondary structure elements on the basis of hMRS2 are indicated above the sequences. Critical amino acids are highlighted.

Extended Data Fig. 5 |. Ion densities.

a-b, Cryo-EM densities in the three ion binding sites for Mg²⁺ (a) and Ca²⁺ (b). Mg²⁺ and Ca²⁺ are shown as magenta and yellow spheres, respectively.

Fig. 1 |. Structure of human MRS2.

a, Cryo-EM reconstruction of human MRS2. Each subunit is uniquely colored and the contour indicates membrane boundaries. b, The overall structure of human MRS2. c, The central ion pathway, estimated using the HOLE program and represented as colored dots (pore radius: 1.15 Å < green < 2.3 Å < blue). Two opposite subunits are shown for clarity. d, Cutaway view of the channel surface, colored by surface electrostatic potential (red, −5 kT/e; white, neutral; blue, +5 kT/e). e, Details of the pore-lining residues and the dimension of the central pore.

Fig. 2 |. Mg²⁺ and Ca²⁺ recognition.

a, Mg²⁺ ions (purple spheres) identified in the structure of MRS2 in the presence of Mg²⁺. Also shown are the details of the three Mg²⁺ ions (site 1 and 2) coordinated within the channel. Only two adjacent subunits are shown for clarity. b, Orthogonal view as in (a). c, Ca²⁺ ions (yellow spheres) in the MRS2_Ca structure.

Fig. 3 |. MRS2 currents.

a, Mg²⁺ transport by TmCorA or MRS2. The western blot image compares the expression of TmCorA, MRS2_FL, and MRS_EM, all tagged with a C-terminal 1D4 sequence and detected with an anti-1D4 antibody. b, MRS2 Mg²⁺ currents induced by the R332S mutation. MRS2_RS exhibits ~60% expression level of MRS2_WT. c, Inhibition of MRS2 activity by cobalt hexammine. Na⁺ currents were presented here, but similar inhibition was obtained when recording Mg²⁺ currents. d, Dose response of cobalt hexammine inhibition of MRS2 Na⁺ currents. Data points were fit with a standard saturation binding equation (black curve). Maximal inhibition achieved is ~50%, comparable with that obtained with TmCorA.

Fig. 4 |. Ion permeation properties.

a, Monovalent and divalent cation currents through MRS2_WT or MRS2_RS. Peak-level amplitudes of Ca²⁺ currents were presented in the bar chart. **P < 0.01; ***p < 0.001. b, Ca²⁺ currents conducted by TmCorA or MRS2_RS. c, Recovery of MRS2_RS from inactivation. d, The effect of Mg²⁺ on Na⁺ currents. e, Lack of Ca²⁺ inhibition of MRS2 Na⁺ currents. 500 µM of EGTA was added later in the recording to test if the trace amount of Ca²⁺ in the NaCl buffer can inhibit Na⁺ currents.

Fig. 5 |. Divalent cation-binding sites.

a-b, Functional impacts of site 1 or site 2 mutations on MRS2_RS. The Western-blot image shows that these mutations do not affect MRS2_RS expression. RS: MRS2_RS. NA: N362A; DS: D329S; DR: D329R. c, Mg²⁺ inactivation of TmCorA. d, The effect of site 3 mutations on MRS2_RS function (quad: E138A-E243A-D247A-E312A mutation). Western blot shows that these mutations do not reduce surface expression.