Crystal structure of an archaeal CorB magnesium transporter

Abstract

CNNM/CorB proteins are a broadly conserved family of integral membrane proteins with close to 90,000 protein sequences known. They are associated with Mg²⁺ transport but it is not known if they mediate transport themselves or regulate other transporters. Here, we determine the crystal structure of an archaeal CorB protein in two conformations (apo and Mg²⁺-ATP bound). The transmembrane DUF21 domain exists in an inward-facing conformation with a Mg²⁺ ion coordinated by a conserved π-helix. In the absence of Mg²⁺-ATP, the CBS-pair domain adopts an elongated dimeric configuration with previously unobserved domain-domain contacts. Hydrogen-deuterium exchange mass spectrometry, analytical ultracentrifugation, and molecular dynamics experiments support a role of the structural rearrangements in mediating Mg²⁺-ATP sensing. Lastly, we use an in vitro, liposome-based assay to demonstrate direct Mg²⁺ transport by CorB proteins. These structural and functional insights provide a framework for understanding function of CNNMs in Mg²⁺ transport and associated diseases.

Fig. 1. Overall structure and domain organization of CNNM/CorB Mg²⁺ transporters.

a Phylogenetic analysis of representative CNNM/CorB orthologs generated using the neighbor-joining method. b Domain organization of eukaryotic CNNM and prokaryotic CorB. TMD transmembrane domain, AHB acidic helical bundle, CNBH cyclic nucleotide-binding homology domain, CorC cobalt resistance C domain. c Crystal structure of the Mg²⁺-ATP-bound MtCorB without the C-terminal CorC domain as a homodimer. One chain is colored by domains. d Topology of a MtCorB monomer showing the transmembrane and juxtamembrane helices of the TMD (residues 1–154, cyan), the two helices of the AHB (residues 166–199, yellow), the Mg²⁺-ATP-binding CBS-pair domain (residues 200–324, green), and the CorC domain (residues 325–426, grey).

Fig. 2. TM domain in an inward-facing conformation with a negatively charged cavity.

a TM domain homodimerizes with interface formed by TM2 and TM3 of each protomer. b Electrostatic surface potential representation (±5 kT e⁻¹) of MtCorB TM domain showing a cross-sectional view of the negatively charged cavity. c Close-up view of the polar residues forming the cavity. d A magnesium ion (Mg²⁺) bound in the cavity with F_o–F_c omits map contoured at 5.0σ. e π-helical turn preceding Pro114 in TM3. Highly conserved residues are highlighted in blue. f Conservation of residues in the π-helical turn from archaea to humans.

Fig. 3. Juxtamembrane helix and acidic helical bundle.

a, b UDM detergent molecules bound by the juxtamembrane helix. Simulated annealing 2F_o–F_c composite omit map contoured at 1.0σ. c A sulfate ion bound between JM and helix–turn–helix motif with F_o–F_c omit map contoured at 5.0σ. d Conservation of acidic residues in the AHB domain. e Sequence conservation of conserved glutamates in the AHB.

Fig. 4. Mg²⁺-ATP binding to CBS-pair domain.

a Structural basis of Mg²⁺-ATP binding. Mg²⁺ ions and water molecules are shown in magenta and red, respectively. The Mg²⁺-ATP F_o–F_c omits map was contoured at 3.0σ. b Affinities of MtCorBΔC to adenosine nucleotides with and without 50 mM Mg²⁺ measured by ITC. c Dimerization of the MtCorB CBS-pair domain in the presence of 1 mM adenosine nucleotides as measured by SV-AUC experiments. d Conformational change in the MtCorBΔC in the presence and absence of Mg²⁺-ATP measured by HDX-MS. Regions that showed significant decreases in exchange (defined as >5%, 0.4 Da, and a two-tailed Student’s t test p < 0.01) in the presence of Mg²⁺-ATP are colored blue. Peptides in the CBS-pair domain dimerization interface show the most protection from deuterium exchange upon Mg²⁺-ATP binding.

Fig. 5. Apo conformation of MtCorBΔC captured by R235L mutant.

a Overall structure of MtCorBΔC R235L mutant. One chain is colored by domains. b Novel domain–domain contact between TMD and CBS-pair domain. c AHB binds to CBS-pair domain and competes for Mg²⁺-ATP site. d Large conformational change in the AHB and CBS-pair domains upon Mg²⁺-ATP binding. e The apo and ATP-bound structures are similar with differences limited to the AHB and α8 helix.

Fig. 6. Liposome-based assay shows direct Mg²⁺ transport by CorB proteins.

a Liposomal assay showing direct transport of Mg²⁺ by MtCorB and TtCorB. Proteoliposomes (protein/lipid ratio of 1:30) or empty liposomes containing mag-fura-2 were equilibrated for 1 min before the addition of 5 mM MgCl₂ to initiate Mg²⁺ uptake (arrowhead). The data points represent mean ± SEM (n = 3 independent measurements). b An inward negative membrane potential generated by valinomycin leads to enhanced Mg²⁺ transport by TmCorA (protein/lipid ratio of 1:270) but not TtCorB (protein/lipid ratio of 1:30), suggesting electroneutral transport of Mg²⁺ by TtCorB. The data points represent mean ± SEM (n = 3 independent measurements). c Homology model of TtCorB showing locations of conserved residues in the negatively charged cavity and Mg²⁺ binding site. d, e Liposomal Mg²⁺ transport assays of TtCorB mutants under the same experimental conditions as panel (a). Mutations in the Mg²⁺-binding site increased transport activity while mutations near the π-helical turn reduced transport. The data points represent mean ± SEM (n = 3 independent measurements).

Fig. 7. Molecular modeling.

a Homology model of human CNNM2 (HsCNNM2) color-coded according to identity to CNNM/CorB proteins shown in Supplementary Fig. 2. Conservation is highest around the Mg²⁺ binding site, negatively charged cavity, and the CBS-pair dimerization interface. b Electrostatic surface potential analysis (±5 kT e⁻¹) shows conservation of negatively charged cavity in HsCNNM2 in cross-sectional view. c Conservation of the polar residues in the Mg²⁺ binding site and π-helical turn of HsCNNM2. Asterisk denotes disease-causing mutations. d Mutations in CNNM2 and CNNM4 responsible for hypomagnesemia and Jalili syndrome cluster around the core of the TMD and nucleotide-binding site. For clarity, mutations are only shown on one chain. e Outward-facing conformation generated by MD simulations. f Proposed model of MtCorB transport and regulation. Loss of Mg²⁺-ATP binding, CBS-TMD contact inactivates TMD in the inward-facing conformation. Upon Mg²⁺-ATP binding, CBS-TMD contact is alleviated, thereby activating and allowing TMD to mediate Mg²⁺ transport.