New insights into the structure and function of CNNM proteins

Magnesium (Mg2+) is the most abundant divalent cation in cells and plays key roles in almost all biological processes. CBS‐pair domain divalent metal cation transport mediators (CNNMs) are a newly characterized class of Mg2+ transporters present throughout biology. Originally discovered in bacteria, there are four CNNM proteins in humans, which are involved in divalent cation transport, genetic diseases, and cancer. Eukaryotic CNNMs are composed of four domains: an extracellular domain, a transmembrane domain, a cystathionine‐β‐synthase (CBS)‐pair domain, and a cyclic nucleotide‐binding homology domain. The transmembrane and CBS‐pair core are the defining features of CNNM proteins with over 20 000 protein sequences known from over 8000 species. Here, we review the structural and functional studies of eukaryotic and prokaryotic CNNMs that underlie our understanding of their regulation and mechanism of ion transport. Recent structures of prokaryotic CNNMs confirm the transmembrane domain mediates ion transport with the CBS‐pair domain likely playing a regulatory role through binding divalent cations. Studies of mammalian CNNMs have identified new binding partners. These advances are driving progress in understanding this deeply conserved and widespread family of ion transporters.


Introduction
Magnesium (Mg 2+ ) is the most abundant divalent cation in cells and essential for life [1].Mg 2+ plays key roles in the stabilization of cell membranes, neuronal transmission, and muscle contraction; it regulates the permeability of various ion channels and is required for over 600 enzymatic reactions, including topoisomerases, helicases, exonucleases, protein kinases, cyclases, ATPases, DNA and RNA polymerases, and ribosomes [2].Perturbed serum Mg 2+ levels are associated with many diseases, such as osteoporosis, diabetes, hypertension, neurological disorders, and immunodeficiency [3,4].
Human adults contain approximately 25 g of magnesium that is unequally distributed across different tissues [5].The largest amount is found in bones (~60%), where it resides on the surface of hydroxyapatite and in the hydration shell around the crystal, thus contributing to maintaining the integrity of the skeleton [6].Intracellular Mg 2+ concentrations range from 5 to 20 mM; 1-5% (0.2-1 mM) is free, and the remainder is bound to ATP, proteins, and other negatively charged molecules [1].Extracellular Mg 2+ accounts for 1% of total body magnesium, which is primarily found in serum and red blood cells [7].The balance between intestinal absorption and renal excretion is tightly regulated to keep the serum Mg 2+ concentration in its physiological range (0.7-1.1 mM) [8].Mg 2+ is unique among divalent cations in that it has the smallest ionic radius and largest hydrated radius [9].The transport of Mg 2+ across the membrane requires the action of Mg 2+ channels and transporters, such as SLC41A1/3, TRPM6/7, MRS2, and CNNMs [10,11].

CNNM proteins
CBS-pair domain divalent metal cation transport mediator proteins were first identified in Salmonella typhimurium among genes related to cobalt sensitivity [12].The gene first identified was corA, which is unrelated to CNNMs and encodes the major Mg 2+ uptake system in prokaryotes.Subsequently, the authors identified corB, encoding a CNNM ortholog, as playing a role in Mg 2+ to elicit efflux [13].The authors found that although the CorA protein alone is necessary and sufficient for influx, efflux requires the presence of co-effectors, CorB or the related proteins, CorC, or CorD.
Human CNNMs (CBS-pair domain divalent metal cation transport mediators) were discovered in 2003 and comprise four integral membrane proteins: CNNM1, CNNM2, CNNM3, and CNNM4.They were first called ancient conserved domain proteins (ACDP) due to the deep evolutionary of the core domains [14].CNNMs are found in bacteria [13,15,16], archaea [17], yeast [18,19], plants [20,21], and animals [22][23][24] (Fig. 1A).Later studies of the human proteins misidentified them as cyclin M based on the erroneous identification of a cyclin box motif.Although they are unrelated in structure or function to cyclins, the accepted name became CNNM proteins.We use the backronym, CBS-pair domain divalent cation transport mediator, that reflects their role in divalent cation transport and the conserved CBS-pair domain.

CNNMs are implicated in magnesium homeostasis
There is an abundance of evidence indicating eukaryotic CNNM proteins are involved in divalent cation transport.For example, CNNM1-4 alleles have been shown to strongly associate with fluctuations in serum Mg 2+ concentrations in a genome-wide association study [25].There is upregulation of CNNM2 mRNA in mice kept on a low Mg 2+ diet and in kidney cells grown in low Mg 2+ media, suggesting a role in Mg 2+ absorption and retention [26,27].More convincingly, CNNM4 À/À knockout mice exhibit hypomagnesemia, characterized by low magnesium serum levels, indicating these mice have altered magnesium metabolism [28].The mice had elevated levels of magnesium in their feces suggesting a role in magnesium absorption in the gastrointestinal tract [28].In addition, when fed a Mg 2+ -deficient diet, these mice have a higher mortality rate, indicating the importance of CNNM proteins in coping with low Mg 2+ -diets [28].On the contrary, the CNNM2 À/À knockout mice die in utero, and heterozygotic CNNM2 +/À mice have lower Mg 2+ serum levels than wild-type mice [29].Altogether, these results strongly suggest that CNNMs proteins play critical roles in magnesium homeostasis.

Tissue and cellular localization
CNNM1 is only expressed in brain and testis, while other three are ubiquitously expressed with higher expressions in certain tissues [14,30].CNNM2 is highly expressed in kidney, brain, and heart [26,29,31].Immunohistochemistry on human kidney sections showed that CNNM2 is predominantly expressed in both distal convoluted tubule (DCT) and thick ascending limb (TAL) of Henle's loop, the two consecutive nephron segments primarily responsible for Mg 2+ reabsorption.CNNM3 is highly expressed in kidney, brain, lung, spleen, and heart [32].CNNM4 shows highest expression in small intestine and colon, where dietary Mg 2+ absorption occurs [32].
In terms of cellular localization, immunostaining studies of kidney cells have shown that CNNM2 localizes at the basolateral membrane of DCT epithelial cells [27,29,32].Likewise, CNNM4 has been shown to localize at the basolateral membrane of intestinal epithelia [28], and the localization is dependent on AP-1 clathrin adaptor proteins, which recognizes multiple dileucine motifs in the cytosolic domains of CNNM4 [33].In both intestinal absorption and renal reabsorption, Mg 2+ entry into the epithelial cells is mediated by apically localized Mg 2+ -permeable channels, TRPM6/7 [34,35].However, the identities of Mg 2+ transporters involved in Mg 2+ efflux at the basolateral membrane have remained unknown.Due to their basolateral localization, CNNMs have been proposed to be the long-sought Mg 2+ -efflux proteins [28].

Disease relevance
CBS-pair domain divalent metal cation transport mediator are associated with a myriad of genetic diseases linked to abnormal Mg 2+ handling, such as hypomagnesemia, Jalili syndrome, hypertension, infertility, schizophrenia, nonalcoholic steatohepatitis, and cancer (Fig. 1B).

Hypomagnesemia
In 2011, mutations in CNNM2 were first linked to dominant renal hypomagnesemia (OMIM# 613882) (Table 1), in which patients from two unrelated families suffer from muscle weakness, tremor, and headaches accompanied by low Mg 2+ serum concentrations (0.3-0.5 mM) [27].Analysis of the heterozygous missense mutation T568I showed that the mutant protein was properly localized to the plasma membrane but caused a significant decrease in CNNM2 activity in Mg 2+ -sensitive Na + currents [27] and Mg 2+ efflux activity [36].In 2014, another study reported four CNNM2 mutations in five unrelated families suffering from hypomagnesemia, seizures, and mental retardations (OMIM# 616418) [37].In the first family, the patients had a homozygous missense mutation of E122K inherited recessively [37].The four remaining cases consisted of dominant, de novo heterozygous mutations: S269W, L330F, or E357K [37].All these mutants except for L330F exhibited severely reduced 25 Mg 2+ uptake, possibly due to destabilization of CNNM2.The E112K and S269W mutants showed reduced plasma membrane expression [37].Subsequently, many more mutations in CNNM2 were identified (Table 1).Together with the finding that CNNM2 +/À knockout mice showed mild hypomagnesemia and developmental defects [38], all these mutations likely alter CNNM2 normal function in Mg 2+ uptake and brain development.Mutations in CNNM2 have also been observed in patients suffering from mild intellectual disability and refractory epilepsy, but without hypomagnesemia symptoms [39,40] (Table 1).Interestingly, these mutations (S743F and R746Q) reside in the cytosolic CNBH domain, which may not participate directly in Mg 2+ transport, but more in a regulatory function.

Hypertension
Several genome-wide association studies have identified CNNM2 as a candidate gene associated with blood pressure variation and hypertension [45][46][47][48][49]. Studies of CNNM2 and CNNM4 knockdown mice suggested opposing roles of CNNM proteins on blood pressure: CNNM2 +/À mice had lower blood pressure, while CNNM4 À/À mice had increased blood pressure [29].The authors proposed the following explanation: CNNM2 is involved in renal absorption of Mg 2+ , and when CNNM2 expression is knocked down, it leads to impaired activity of renal reabsorption, thus leading to lower blood pressure [29].On the contrary, knockdown of CNNM4 results in lower intestinal Mg 2+ absorption and lower serum magnesium level.This in turn upregulates renal Mg 2+ reabsorption by CNNM2, and the increased renal activity leads to increased blood pressure [29].More recently, Cnnm2 knockdown in zebrafish has also resulted in hypertension-related phenotypes: higher blood flow, increased arterial pulse, and elevated linear velocity [50].

Infertility
Evidence of CNNM involvement in infertility comes from CNNM knockout studies in C. elegans and mice.In C. elegans, CNNM-1 and CNNM-3 double mutant worms were sterile due to gonadogenesis defect that severely attenuated the proliferation of germ cells [23].In mice, CNNM4 À/À males are almost infertile because of loss of sperm mobility and fertility [51].Detailed analyses of these sperm revealed that their lack of hyperactivation motility is due to defects in the activation of CatSper, a sperm-specific Ca 2+ channel required for hyperactivation [51].These sperm also showed higher levels of magnesium, in which excessive Mg 2+ accumulation was considered to impair the Cat-Sper Ca 2+ channel function, suggesting a functional relationship between Mg 2+ homeostasis and Ca 2+ signaling [51].In addition, CNNM2 +/À CNNM4 À/À mice were infertile, and their sperm showed a more severe motility-defective phenotype than those from CNNM4 À/À mice, thus also implicating the role of CNNM2 in sperm motility [52].

Schizophrenia
Many genome-wide association studies have identified CNNM2 as a risk locus for schizophrenia [53][54][55][56][57].A study found that a CNNM2 SNP variant (rs7914558; located within an intron) is associated with gray matter morphological vulnerability of the bilateral inferior frontal gyri, which may represent the mechanism by which CNNM2 increases the risk for schizophrenia [58].Rose et al. [59] also investigated the effect of CNNM2 rs7914558 variant and showed that this variant has an effect on both social cognition (i.e., attributional style) and gray matter volume in regions previously implicated in the processing of social stimuli.Large-scale phenotypic landscape characterization of schizophreniaassociation genes in zebrafish also identified CNNM2 as a promising candidate for the disease; CNNM2 mutants caused decreased brain activity in retinal arborization field, tectum, and hypothalamus [60].In addition, genome sequencing of individuals affected with schizophrenia has identified missense mutations in CNNM1, CNNM3, and CNNM4, thereby linking all four members to schizophrenia (Table 1) [61].
Recently, two studies in flies have implicated the CNNM ortholog, UEX, in brain development and function [24,62].Normal and enhanced fly memory requires the expression of UEX, and regulated neuronal Mg 2+ efflux is critical for normal and Mg 2+enhanced memory [62].Phosphatase of regenerating liver (PRL)-1, which acts upstream of UEX, was shown to be required for proper synapse formation and fly movement [63].PRL-1 and UEX were also found to play neuroprotective roles.Loss of either UEX or PRL-1 led to neural dysfunction characterized by a wing-up phenotype upon CO 2 exposure [24].The physical basis of the interaction between PRL-1 and UEX is conserved between flies and mammals [64].

Nonalcoholic steatohepatitis
CNNM4 overexpression is found in patients with nonalcoholic steatohepatitis (NASH), a condition in which fat builds up on the liver and causes inflammation [65].Examination of hepatic mRNA levels shows a significant overexpression of CNNM4 in both clinical samples and NASH rodent models.These symptoms were alleviated through silencing of CNNM4.Mechanistically, CNNM4 knockdown in hepatocytes is said to induce cellular Mg 2+ accumulation, thereby reducing ER stress and increasing microsomal triglyceride transfer activity, which promote hepatic lipid clearance by increasing the secretion of very-low-density lipoprotein and causes NASH.Decreasing CNNM4 was also observed to protect against acetaminophen-induced liver damage [66].CNNM4 was upregulated in hepatocytes and rodent models of acetaminophen poisoning and led to disturbances in mitochondrial magnesium reservoirs, ATP production, and endoplasmic reticulum homeostasis.

Cancer
CNNM4 has been shown to act as a tumor suppressor in a mouse model of colon cancer.Knockdown of CNNM4 significantly augmented the number of tumor nodules on the mice lungs, confirming the tumorsuppressing role of endogenous CNNM4 [67].In addition, in Apc D/14+ mice, which spontaneously form benign polyps in the intestine, deletion of CNNM4 promoted malignant progression of intestinal polyps to adenocarcinomas [67].CNNM4 mRNA levels in colon cancer were significantly reduced, and this reduction was more evident in metastatic colon cancer than nonmetastatic ones [68], implicating CNNM4 downregulation in human colon cancer development.
Two groups independently identified an interaction between CNNM proteins and phosphatases of regenerating liver (PRLs), a family of potent oncogenes frequently overexpressed in malignant human cancers [67,69].Both groups found that CNNM-PRL interaction promoted oncogenesis and disruption of interaction abolished tumor progression [67,70].Mechanistically, the two groups proposed different mechanisms but with the same downstream effect.Funato et al. [67] postulated that CNNM4 mediates Mg 2+ efflux with PRL3 inhibiting CNNM4 activity and increasing intracellular Mg 2+ .In contrast, Hardy et al. [69] postulated that CNNM3 mediates Mg 2+ influx with PRL2 enhancing influx activity to again increase intracellular Mg 2+ levels.Both groups suggested that increased binding of Mg 2+ to ATP would promote the activity of enzymes associated with energy metabolism and protein synthesis, thereby promoting cellular proliferation and tumor invasiveness [71][72][73][74].It has also been proposed that CNNM4 deficiency promotes cell proliferation in the colon epithelia through suppressing Ca 2+ signaling [75].

CNNM-Domain organization and structure
CBS-pair domain divalent metal cation transport mediators are multidomain proteins.Eukaryotic CNNMs contain four domains: the extracellular domain (ECD), transmembrane domain (TMD), cystathionine-b-synthase (CBS)-pair domain, and cyclic nucleotide-binding homology (CNBH) domain (Fig. 2).The transmembrane and CBS-pair domains are the most conserved domains and found associated together in essentially all organisms from humans to bacteria.Prokaryotic CNNMs lack the ECD and have a smaller C-terminal domain, termed CorC, that is unrelated to the eukaryotic CNBH domain.

Extracellular domain
Based on sequence analysis, all four human CNNMs contain a signal peptide prior to the N-terminal ECD [76].In CNNM2, the signal peptide has been experimentally determined to be 64 amino acid long and is cleaved by signal peptidase complex (SPC) in the ER [32].A glycosylation site at Asn-112 in CNNM2 is required for proper localization and membrane stability as N112A mutation resulted in 90% reduction in plasma membrane expression [32].Several mutations in this domain have been linked to both hypomagnesemia and Jalili syndrome (Table 1).Currently, there are no experimental structures for this domain.Alpha-Fold prediction model suggests this domain forms a broll structure with two large, disordered loops (Fig. 2B) [77].

Transmembrane domain
The TMD is the defining feature of CNNM proteins.The domain is very broadly conserved and constitutes the largest family of protein domains of unknown function (DUFs) on the Pfam database (PF01595) [78].Called domain of unknown function 21 (DUF21), there are close to 20 000 protein sequences from over 8000 species ranging from bacteria to plants and animals.Several hypomagnesemia and Jalili syndromeassociated mutations reside in this domain of CNNM2 and CNNM4, respectively (Table 1).In 2021, our group and the group of Motoyuki Hattori independently determined the crystal structures of the TMDs of two prokaryotic CNNMs: MtCorB from a thermophilic archaeon (Methanoculleus thermophilus) and TpCorB from a Gram-negative bacterium (Thermus parvatiensis) [17,79] (Table 2).Structurally, the TMD forms a homodimer with each chain composed of three transmembrane helices (TM1-3), a pair of short, intracellular helices, and a belt-like juxtamembrane (JM) helix (Fig. 3A).The dimerization is predominantly formed by hydrophobic contacts of TM2 and TM3 of each protomer.
The TMD resembles a transporter fold in an inward-facing conformation as it contains a solventexposed cavity open toward the cytosol (Fig. 3B).With a maximum diameter of 10 A, the cavity is lined with several polar residues making it highly electronegative.A well-resolved electron density was observed deep within the cavity and assigned as a Mg 2+ ion based on coordination distance (Fig. 3C).In MtCorB, the Mg 2+ ion is coordinated by hydroxyl groups of Ser21, Ser25, and Ser71; carboxyl group of Glu111; and main-chain carbonyl groups of Ser21 and Gly110 [17].All these coordinating residues are highly conserved across species, suggesting the protein has a conserved function across species.Another feature of the TMD is the p-helical turn in TM3, in which the helix is composed of (i + 5) ?i instead of (i + 4) ?i hydrogen bonding, resulting in a bulge in the helix (Fig. 3C).The residues forming the p-helical turn (Glu111, Ile112, and Pro114 in MtCorB) are completely invariant from archaea to humans (Fig. 3D).Glu111 points toward the negative cavity and directly participates in Mg 2+ coordination, whereas Pro114 acts as a helix breaker that allows the shift in hydrogen bonding of the p-helical turn.The positively charged Lys115 is also highly conserved across species.
As previously predicted, the TMD contains a reentrant helix as the fourth helix unlike the previously suggested second helix [32].The JM helix wraps around TM1 and TM2 like a belt through several hydrophobic contacts.The curvature is imposed by a conserved proline, causing a kink in the helix (Fig. 3A).The JM helix is amphipathic and rich in aromatic residues, and it is nearly parallel to the plane of membrane.In the bacterial structure, the JM helix is longer and forms extensive contacts with TM3 of other protomer [79] (Fig. 3E).In the archaeal structure, several UDM detergent molecules were observed to lie across the JM helix, mimicking the binding mode with phospholipids [17] (Fig. 3F).The helix-turn-helix motif underneath the JM helix forms a basic patch that binds a sulfate molecule from crystallization buffer (Fig. 3F).This may serve to bind the negatively charged phospho-headgroups.

Acidic helical bundle
A structural region consisting of two ɑ-helices resides between the TMD and cytosolic domains (Fig. 4).In the Mg 2+ -ATP-bound MtCorB structure, the two helices can be seen to dimerize and form a four-helical bundle that sits on top of the CBS-pair domain [17] (Fig. 4E).Because both helices are concentrated in acidic residues (Glu/Asp), they were termed the acidic helical bundle (AHB).In the apo conformation, the AHB dissociates into a two-helical bundle and binds to the Mg 2+ -ATP binding site of CBS-pair domain (Fig. 4D).

CBS-pair domain
Following the AHB is a cytosolic domain called CBSpair domain, also known as a Bateman module [80] (Fig. 4).CBS-pair domains are found in many proteins: AMP-activated protein kinase (AMPK), inosine-5 0 -monophosphate dehydrogenase (IMPDH), chloride channel (ClC), and bacterial Mg 2+ transporter MgtE [81].In AMPK, this domain acts as an energy sensor as it is able to bind different adenine nucleotides, such as AMP, ADP, and ATP [82].In CNNM, this domain binds ATP [36] and is also the site of PRL binding [83].Several disease-associated mutations are found in this domain (Table 1).

CBS-pair domain homodimerizes in various dimeric configurations
Several crystal structures of CBS-pair domain of CNNMs have been determined in the presence and absence of various adenine nucleotides (Table 2).The CBS-pair domain is made of two consecutive CBS motifs (CBS1 and CBS2) that fold together (Fig. 4A) [84].Both CBS motifs adopt babba folds and contact each other via three-stranded b-sheets (where the first two b strands run parallel and the third one runs antiparallel) [84].The two CBS-pair domains associate to form a homodimer, referred to as a CBS module.Depending on the presence of bound nucleotides, four dimeric configurations have been observed: one liganded state and three unliganded states (Fig. 4A-D).
In the liganded state with bound adenine nucleotides and metal ions, the CBS module exists in a flat, disklike dimeric configuration, in which CBS2 and CBS1 motifs of each protomer closely associate with that of the other protomer (Fig. 4A).The dimerization interface is mediated by hydrophobic contacts of both CBS2 and CBS1 motifs.The CBS2 motif contact is ).(D) The CBS-pair domain in a disease-mimicking (R235L) mutant of MtCorB adopts a split dimeric configuration (PDB: 7M1U [17]).The dimeric AHB separates and buries into the Mg 2+ -ATP binding site.(E) Acidic helical bundle in MtCorB is decorated with many acidic residues (PDB: 7M1T [17]).(F) Structural basis of Mg 2+ -ATP binding to CBS-pair domain.Crystal structure of CNNM2 CBS-pair domain bound to Mg 2+ -ATP-analog (PDB: 6N7E [85]).ATP binds to the canonical nucleotide-binding site.Mg 2+ ions are shown as magenta spheres.(G) Structural basis of CNNM-PRL interaction.Crystal structure of CNNM3 CBS-pair domain in complex with PRL2 (PDB: 5K22 [83]).The interaction is mediated by the extended loop structure from CNNM3 (green) into the catalytic pocket of PRL2 (magenta).All structural figures were created with PYMOL, Version 2.5.0 (www.pymol.org).mediated through a4 and a8 helices of each protomer, while CBS1 motif contact is mediated by a5 and a6 helices.This conformation is stabilized by the bound adenine nucleotides and metal ions, in which the metal ions are proposed to neutralize the charge repulsion between the nucleotide phosphate groups and nearby acidic residues.In the whole cytosolic fragment structure of CNNM2 in complex with Mg 2+ -AMP-PNP, there are two potential Mg 2+ binding sites observed near the base of CBS1 motifs [85].Simultaneous mutation of all three aspartate residues to alanine resulted in a slight decrease in ATP binding affinity.
On the contrary, three unliganded states have been observed so far: twisted, half-open, and split (Fig. 4B-D).The twisted state was obtained with isolated CBSpair domain of CNNM2 in absence of adenine nucleotides (Fig. 4B) [84].In this state, the CBS2 motifs remain in contact while the CBS1 motifs have separated, retaining only scarce hydrophobic contacts between a6 helices of each protomer.The half-open state was observed in the crystal structure of whole cytosolic fragment of CNNM3 (Fig. 4C) [85].Here, the dimerization contact is only observed in the CBS2 motif, more specifically by the a8 helices of both protomers.The CBS1 motif is completely swung open making a 90°rotation with respect to the flat, nucleotide-bound dimer.Lastly, the split dimeric configuration was observed in the crystal structure of a disease-mimicking mutant in an archaeal CNNM (Fig. 4D) [17].In this case, the CBS1 motif has separated even further and undergoes a complete 180°r otation with respect to the flat, nucleotide-bound dimer.The dimeric AHB has dissociated and bound to the CBS2 motif of the neighboring molecule, competing for the Mg 2+ -ATP binding site.
In all three unliganded states, the CBS2 motifs remain in contact while the CBS1 motifs separate, and the relative orientation of helix a4 that connects to TMD is markedly different in comparison with the liganded state.The a4 helix goes from a parallel dimeric configuration (flat) to a cross-shaped configuration (twisted) and then ultimately to a nontouching configuration (split).These changes suggest that the movements induced by nucleotide binding could be translated to the TMD, affecting ion transport.

CBS-pair domain binds adenine nucleotides in the central cavity
The CBS-pair domain of CNNMs is known to bind adenine nucleotides in a Mg 2+ -dependent manner.Using [ 32 P]ATP filter-binding assay, in the absence of Mg 2+ , the CBS-pair domains of all four CNNM members showed negligible binding [36].On the contrary, in the presence of 10 mM MgCl 2 , that of CNNM2 and CNNM4 binds ATP with a low micromolar affinity (159 AE 28 and 43.4 AE 8.9 lM, respectively), while that of CNNM1 and CNNM3 showed negligible binding.While they possess ATP binding ability, they do not possess ATPase activity [85].
The binding mode of adenine nucleotide was elucidated through various crystal structures of CBS-pair domains of human CNNM2 (Table 2).The CBS-pair domain of CNNM2 has been extensively studied in complex with various adenine nucleotides (AMP, ADP, Mg 2+ -ATP, or Mg 2+ -AMP-PNP).The binding mode for each adenine nucleotide is very similar.The adenine ring is sandwiched between Tyr478 and Ile566 in a hydrophobic pocket comprising Pro482, Ile481, Cys456, and Phe457, while the ribose ring forms hydrogen bonds with the side chains of Thr451 and Asp571 (Fig. 4F) [84].For the phosphate groups, the ɑ-phosphate is stabilized by Thr568.When present, the b-phosphate and/or c-phosphate are hydrogen-bonded by Thr479 and Arg480 [84].In hypomagnesemia patients, this Thr568 residue is mutated to a bulkier isoleucine residue, consequently causing steric clashing with the ribose moiety, thus inhibiting adenine nucleotide binding [27].In the Mg 2+ -ATP-bound structure, the Mg 2+ is coordinated by all three phosphates and is considered an allosteric activator for ATP binding as Mg 2+ binding alleviates the otherwise negative charge repulsion existing between acidic residues and phosphate groups of ATP [84].
In the whole cytosolic domain structure of CNNM2, the interdomain linker connecting to CNBH domain can be seen to participate in Mg 2+ -ATP binding (Fig. 4F) [85].The acidic residues, Glu584 and Asp586, in the linker make interactions with the ATPcoordinated Mg 2+ ion.Additionally, acidic aspartate residue Asp583 from the linker interacts with R480 from another protomer, strengthening the dimerization contact.The importance of these linker residues was confirmed by ITC binding studies of mutants that lacking these acidic residues, in which the affinity for Mg 2+ -ATP was impaired by 100-fold [85].

CBS-pair domain binds PRL using an extended loop
Our group reported the first co-crystal structure of a CBS-pair domain (CNNM3) and phosphatase (PRL2; Table 2) [83].Additional structures of CNNM2 and CNNM3 with different PRL proteins were subsequently reported [86][87][88].In all the CNNM-PRL structures, the CBS-pair domain is always present as a flat disk-like dimer, similar to the nucleotide-bound conformation (Fig. 4A).The CBS-pair domain interacts with PRL using the extended loop that connects b5 and b6 (Fig. 4G).The extended size of the loop is unique to CNNMs and absent in other CBS domaincontaining proteins.PRL binding is mainly mediated by an aspartic acid (Asp558 in CNNM2; Asp426 in CNNM3) in the CBS-pair domain that inserts into the catalytic pocket of PRL mimicking a phosphorylated substrate.

Cyclic nucleotide-binding homology domain
Eukaryotic CNNMs contain a CNBH domain after the CBS-pair domain (Fig. 2).This domain shares sequence similarity to cyclic nucleotide-binding (CNB) domains [14].The CNBH domain does not interact with CBS-pair domain, nor does it interact with PRLs [86].Several mutations in the domain have been linked to various diseases (Table 1).
Our group characterized structurally and functionally the CNBH domains from two human CNNM proteins.Structurally, the CNBH domains resemble a CNB domain fold with an eight-stranded antiparallel b-roll capped by an a-helical bundle on the side (Fig. 5A).However, they have several differences.The first difference lies in the loop connecting ɑ10 and b13.In CNB domain, the loop is 5 residues long while in CNNMs, the loop varies between 30 to 70 residues in length.Although disordered, the loop likely plays a regulatory role through interaction with regulatory proteins.CNNM2 missense mutations in this loop are linked to intellectual disability and epilepsy (Table 1).Second, it is missing the signature Cterminal helix of CNB domains.In CNBs, this Cterminal helix (aC) is involved in binding the cyclic nucleotide through enclosing the bound nucleotide (Fig. 5B) [89].This region is unstructured in CNNM crystal structures despite being in the crystallized construct.Secondary structure prediction shows low sequence conservation.In AlphaFold-predicted structures, this portion is also unstructured [77].These strongly suggest the region does not form a helix in CNNMs.Third, the putative cyclic nucleotide-binding site has a bulkier tyrosine residue that would sterically clash with any bound nucleotides (Fig. 5B), suggesting CNBH domain has no cyclic nucleotide-binding capacity.Indeed, several attempts have been made to measure the affinity of cyclic nucleotides to CNBH domain, but no cyclic nucleotide-binding activities have been detected [90,91].Instead, this domain has been shown to function as a dimerization domain [90].The domain dimerizes using the putative cyclic nucleotide-binding surface in the b-roll, forming an elongated homodimer (Fig. 5C).This dimeric configuration is associated with the Mg 2+ -ATP-bound CBS-pair domain [85].In a later structure of the whole cytosolic region of CNNM3, a different dimeric configuration for the CNBH domain was observed showing a compact homodimer (Fig. 5D) [85].The new configuration was coupled to the unliganded state of CBS-pair domain, suggesting an interplay between the two domains.

CorC domain
In prokaryotes, instead of CNBH domain, the CBSpair domain is followed by a smaller C-terminal CorC domain (Fig. 2).Structures from several prokaryotic CNNMs have been determined (Table 2).The domain is made up of a five-stranded antiparallel b-roll and two a-helices (Fig. 5E).Whether CorC domain has the ability to dimerize like the other domains is unclear.In crystal structures of CorC domains (Table 2), divalent cations (e.g., Mg 2+ , Ca 2+ , and Mn 2+ ) have been consistently observed near a cluster of acidic residues in the b-roll of two symmetry-related molecules (Fig. 5F).Whether these are crystallographic artefacts or of functional importance remains to be elucidated.

Mechanism of Mg 2+ transport
Despite the clear association with Mg 2+ transport, over the years, the molecular mechanism of Mg 2+ transport by CNNM proteins has been debated.Some groups suggest that CNNMs transport ions directly and other that CNNMs act through other proteins [92][93][94].Structures of the TMDs of prokaryotic CNNMs (CorB proteins) support a direct role but the question is not completely resolved.
In 2010, Sponder et al. [95] overexpressed human CNNM2 in a Salmonella strain MM281 missing three major Mg 2+ influx systems (CorA, MgtA, and MgtB), which requires high Mg 2+ -media to proliferate.They reported that CNNM2 was able to partly restore the Mg 2+ -deficient growth phenotype.Using mag-fura-2 fast filter spectroscopy, they detected a large inwardoriented Mg 2+ influx in CNNM2-expressing cells and concluded that CNNM2 is a functional Mg 2+ transporting entity by itself [95].Later work by the same group reversed that conclusion and claimed that CNNM2 is not a Mg 2+ transporter by itself, but a Mg 2+ homeostatic factor.In 2016, Sponder et al. [96] performed patch-clamp analyses on HEK293 cells overexpressing CNNM2.They were not able to detect constitutive membrane currents above the control, concluding CNNM2 is unable to mediate Na + or Mg 2+ currents.
In 2011, Stuiver et al. [27] performed patch-clamp analysis of HEK293 cells transiently transfected with mouse CNNM2, and they detected Mg 2+ -sensitive Na + currents rather than Mg 2+ -induced currents.Additionally, these Na + currents were blocked by increased extracellular Mg 2+ concentrations (20 mM) as well as addition of 20 mM ZnSO 4 [27].Therefore, the authors suggested CNNM2 might contribute to a Mg 2+ -sensing mechanism rather than act as a transporter itself [27].
In 2013, Yamazaki et al. [28] performed a series of experiments that proposed CNNMs as Na + /Mg 2+ exchangers.They performed elemental analyses of CNNM4-overexpressing HEK293 cells and detected a decrease of Mg 2+ and an increase of Na + levels, without effects on other major metals.Imaging analyses with Magnesium Green, a fluorescent indicator for Mg 2+ , showed that the fluorescent signal in cells expressing CNNM4 rapidly decreased after exchanging bathing solution from 40 to 0 mM Mg 2+ (Fig. 6A).They concluded CNNM4 is able to stimulate Mg 2+ extrusion [28].Performing the same assay with Na + replaced by N-methyl-D-glucamine (NMDG) abolished the Mg 2+ efflux, suggesting the efflux is Na + -dependent [28].They also performed electrophysiological analyses on CNNM4 or CNNM2-expressed HEK293 cells and found that CNNM4 expression induced no significant electronic currents while CNNM2 expression generated an inward current of Na + [28].Using ratiometric fluorescent probes, sodium-binding benzofuran isophthalate (SBFI) and mag-fura-2, the molar ratio of changes in Na + and Mg 2+ was close to 2 : 1, suggesting electroneutral exchange of Na + and Mg 2+ by CNNM4 [28].
In 2014, Arjona et al. [37] performed 25 Mg 2+ uptake in HEK293 cells transiently transfected with mouse CNNM2 and found that CNNM2-transfected cells displayed a higher 25 Mg 2+ uptake compared with control cells.They also found that the CNNM2-dependent 25 Mg 2+ uptake was significantly inhibited by the addition of 2-APB, an inhibitor of TRPM7 [37].Additionally, uptake was found to be independent of Na + and Cl À availability and present in assays performed with NMDG or gluconate buffers [37].These results suggest that CNNM2 is not a Mg 2+ transporter itself but a regulator of TRPM7 [37].

Prokaryotic CNNMs
Studies in Staphylococcus aureus identified a CNNM ortholog called MpfA (magnesium protection factor A). MpfA deletion mutants are unable to grow in the presence of high concentrations of magnesium [15].Complementation by MpfA-containing plasmids can reverse these phenotypes, indicating perturbation of magnesium homeostasis [15].Using a Mg 2+ -sensing riboswitch, the authors showed that loss of MpfA results in an increase of internal magnesium concentrations consistent with MpfA acting as a Mg 2+ exporter itself or promoting Mg 2+ export via another protein [98].The DMpfA mutants also showed moderate resistance to cobalt and manganese which the authors attributed to increased competition for Co 2+ and Mn 2+ binding sites by intracellular Mg 2+ [15].
In 2021, Huang et al. [79] carried out Mg 2+ efflux assays with TpCorB, a bacterial CNNM from Thermus parvatiensis.To facilitate cell surface expression of TpCorB in HEK293 cells, they created a stable cell line expressing chimeric CNNM4/TpCorB protein, consisting of ECD of human CNNM4 and full-length TpCorB.Removal of Mg 2+ in the bath solution resulted in loss of fluorescence in Magnesium Greenloaded cells, confirming Mg 2+ efflux function of TpCorB [79].Similar to eukaryotic CNNMs, TpCorBdependent Mg 2+ transport depends on the presence of Na + .Mutational studies of critical residues involved in Mg 2+ -coordination confirmed their functional importance in Mg 2+ efflux [79].
All the transport assays performed with eukaryotic/ prokaryotic CNNMs were done through overexpression of CNNMs in cells, which preclude the differentiation of direct or indirect Mg 2+ transport.In 2021, our group carried out an in vitro, liposome-based Mg 2+ transport assay with TtCorB, a bacterial CNNM from Tepidiphilus thermophilus, demonstrating direct Mg 2+ transport function of CNNMs [17].Purified TtCorB was reconstituted into liposomes encapsulated with ratiometric Mg 2+ indicator, mag-fura-2.Upon addition of Mg 2+ , we observed a time-dependent Mg 2+ uptake into the liposome (Fig. 6B).With an inward negative membrane potential generated with valinomycin, TmCorA showed increased Mg 2+ uptake, while that of TtCorB remains unchanged, suggesting TtCorB mediates transport through an electroneutral antiporter mechanism, similar to that proposed previously for CNNM4 [28].

Regulation
CBS-pair domain divalent metal cation transport mediator function as a signaling hub through which cellular Mg 2+ homeostasis is maintained through simultaneous regulation by intracellular Mg 2+ -ATP levels, PRL, and ARL15.

Mg 2+ -ATP
The CBS-pair domain is important for the function of CNNM as it is conserved across species.In both prokaryotic and eukaryotic CNNMs, the CBS-pair domain binds Mg 2+ -ATP in the central cavity, forming a disk-like dimer [17,84].This binding is essential for Mg 2+ transport.Mutations that affect the Mg 2+ -ATP binding site can impair Mg 2+ efflux activity [36,85].Factors that reduce Mg 2+ -ATP affinity can also negatively impact Mg 2+ efflux function.For instance, mutations to the CBS1 dimerization interface can reduce the formation of the disk-like dimer required for Mg 2+ -ATP binding, leading to reduced Mg 2+ efflux activity [85].Mutations to the dimerization interface of the CNBH domain can also reduce Mg 2+ -ATP affinity and Mg 2+ transport function [85,90].
The exact mechanism by which Mg 2+ -ATP binding regulates Mg 2+ transport function remains unclear.The CBS-pair domain likely serves as a regulatory domain that senses intracellular Mg 2+ -ATP levels and regulates Mg 2+ efflux function.When the intracellular [Mg 2+ ] is high, CNNM actively effluxes Mg 2+ .However, when the intracellular [Mg 2+ ] is low, Mg 2+ -ATP unbinds, and Mg 2+ efflux function is impaired.

Phosphatase of regenerating liver
Eukaryotic CNNMs have an extra layer of regulation as it contains an extra loop structure that allows them to interact with oncogenic PRL [83].PRL is an inhibitor of CNNM Mg 2+ efflux function as their interaction leads to an increase in intracellular [Mg 2+ ] [67].The interaction between PRL and CNNM is regulated by various factors.For example, phosphorylation and oxidation of the catalytic cysteine in PRL affects its ability to bind CNNM.Oxidized PRL has a weaker binding to CNNM, while phosphorylated PRL cannot bind to CNNM at all [83].Changes in extracellular [Mg 2+ ] can also affect protein level and phosphorylation status of PRL, which indirectly affects the function of CNNM.PRL expression is controlled by external Mg 2+ availability with increases in PRL mRNA levels when external Mg 2+ levels are decreased [99].High extracellular [Mg 2+ ] increases PRL phosphorylation while low extracellular [Mg 2+ ] decreases PRL phosphorylation [88].

ARL15
Four groups have characterized the interaction between CNNM and ADP-ribosylation factor-like GTPase 15 (ARL15) [97,[100][101][102].ARL15 locus was associated with urinary Mg 2+ excretion in a GWAS [103].Structurally, ARL15 is similar to Ras-related GTP-binding proteins, which regulate intracellular vesicle trafficking [104].Using co-immunoprecipitation of truncated CNNM2 constructs, ARL15 binding was mapped to the CBS-pair domain of CNNM2 [100].A crystal structure of the complex showed the binding site overlaps with the PRL binding site so that the ARL15 and PRL compete for binding CNNMs.Several groups observed that ARL15 inhibits divalent ion uptake in cells [97,101].This likely occurs through CNNM-binding as an ARL15 mutant that is unable to bind CNNMs was unable to inhibit Zn 2+ uptake by TRPM7, a bifunctional protein with an ion channel and protein kinase domain involved in Mg 2+ homeostasis [35,101,102,105].Knockdown and overexpression of ARL15 were also shown to affect Nglycosylation of CNNMs [100].

Conclusion
Current structural and functional knowledge of the CNNM proteins was laid out in this review.Compared with other Mg 2+ transporters and channels (e.g., MgtE, CorA, and TRPM7) [10,11], studies of CNNMs still have a long way to go.Currently, the structure of TMD domain is only known in one conformation, representing a single snapshot of the transport cycle.Assuming transport by CNNMs occurs through a rocker-switch mechanism, multiple structures of different conformations will be needed to fully understand the mechanism of transport.The location of Na + binding site also remains to be identified.
Functionally, CNNMs have been shown to be key players in maintaining cellular Mg 2+ homeostasis.Their involvement in various diseases testifies to their essential role in proper organismal function.While a role in Mg 2+ homeostasis has been unequivocally established, the exact mechanism by which PRL and other ligands affect CNNM transport activity remains to be elucidated.Despite the challenges, the recent progress in functional and structural studies of these ancient and ubiquitous proteins is evidence of a transformation in our understanding of their role in health and disease.
. C-terminal domains: CNBH and CorC domain.(A) CNBH domain resembles a CNB domain fold with an eight-stranded antiparallel bfold capped by an a-helical bundle (PDB: 6DFD [90]).(B) Structural comparison of CNNM CNBH domain (PDB: 6DFD [90]) and CNB domain from bacterial potassium channel MlotiK1 (PDB: 1VP6 [140]).Putative cyclic nucleotide-binding site in CNNM has bulky aromatic residues that would sterically clash with any bound nucleotides.(C, D) CNBH domain dimerizes using the b-roll region in two different modes depending on liganded state of CBS-pair domain: an elongated dimer (PDB: 6DFD [90]) when Mg 2+ -ATP bound or a compact dimer (PDB: 6MN6 [85]) in the absence of bound ligands.(E) CorC domain resembles a simplified version of CNBH domain consisting of a five-stranded b-roll capped by two a-helices (PDB: 2PLS).(F) CorC domain binds divalent cations using acidic residues in the b-roll of two symmetryrelated molecules (PDB: 2PLS).All structural figures were created with PYMOL, Version 2.5.0 (www.pymol.org).

Fig. 6 .
Fig. 6.Mg 2+ transport properties of CNNMs.(A) Mg 2+ efflux assay showing time-dependent Mg 2+ efflux by CNNM4.HEK293T cells transfected with either empty vector or human CNNM4 were loaded with Magnesium Green and then subjected to Mg 2+ depletion at the indicated time point (arrowhead).The mean relative fluorescence intensities of 10 cells are shown in the graph.Immunofluorescence images of HEK293T cells with anti-FLAG (green) and rhodamine-phalloidin (red) showing CNNM4 are properly co-localized with F-actin adjacent to the cell membrane.Bar, 10 lm.Adapted from [85].(B) Liposome-based Mg 2+ transport assay showing direct Mg 2+ transport by TtCorB.Proteoliposomes or empty liposomes containing mag-fura-2 were equilibrated for 1 min before the addition of 5 mM MgCl 2 to initiate Mg 2+ uptake (arrowhead).An inward negative membrane potential generated by valinomycin leads to enhanced Mg 2+ transport by TmCorA but not TtCorB suggesting electroneutral transport of Mg 2+ by TtCorB.The data points represent mean AE SEM (n = 3 independent measurements).Adapted from[17].