1.A.112. The Cyclin M Mg2+ Exporter (CNNM) Family
The hemolysin C of Brachyspira hyodysenteriae (268 aas; ter Huurne et al., 1994) and the Co2+-resistance protein, CorC of Salmonella typhimurium (273 aas with one putative TMS (residues 163-181)) are homologous throughout most of their lengths to each other. They are also similar to the C-terminal hydrophilic portions of 5 close paralogues in Bacillus subtilis, all of which are about 440 aas long, have an N-terminal 4 TMS domain, and may be Mg2+ exporters (Ulens 2018). One representative B. subtilis paralogue is YrkA (434 aas; spP54428). The CorC protein was believed to function as an auxiliary protein to the CorA Co2+/Mg2+ channel (TC #1.A.35) of S. typhimurium (Gibson et al., 1991). The bacterial CorB/CorC/CNNM sub-family of proteins is involved in resistance to antibiotic exposure and the survival of pathogenic microorganisms in their host environment. CorC proteins possess a cytoplasmic region containing a (regulatory?) ATP-binding site (Huang et al. 2021).
Mouse ACDP2 (CNNM2) was cloned from mouse distal convoluted tubule cells of the kidney, expressed in Xenopus laevis oocytes, and studied with two-electrode voltage-clamp techniques. When expressed in oocytes, ACDP2 was reported to mediate saturable Mg2+ uptake with a Michaelis constant of 0.56 mM. Transport of Mg2+ by ACDP2 was rheogenic and voltage-dependent, but was not coupled to Na+ or Cl- ions. ACDP2 transports a range of divalent cations: Mg2+ , Co2+, Mn2+, Sr2+, Ba2+, Cu2+, and Fe2+, thus exhibiting wide substrate selectivity. The cations Ca2+, Cd2+, Zn2+, and Ni2+ did not induce currents, and only Zn2+ effectively inhibited transport. The ACDP2 transcript is abundantly present in kidney, brain, and heart with lower amounts in liver, small intestine, and colon. Moreover, ACDP2 mRNA is upregulated with magnesium deficiency, particularly in the distal convoluted tubule cells, kidney, heart, and brain. Thus, ACDP2 may be a regulated transporter for Mg2+ and other divalent cations in epithelial cells (Goytain et al., 2005).
Mutations in the cyclin M2 (CNNM2) gene were identified to be causative for severe hypomagnesemia. In kidney, CNNM2 is a basolaterally expressed protein with predominant expression in the distal convoluted tubule. Transcellular magnesium (Mg2+) reabsorption in the distal convoluted tubule represents the final step before Mg2+ is excreted into the urine, thus fine-tuning its final excretion via a tightly regulated mechanism. Membrane topology studies showed that CNNM2 has an extracellular N terminus and an intracellular C terminus (de Baaij et al., 2012). The loss-of-function mutation found in patients disturbs ATP binding by the intracellular cystathionine β-synthase domains. In the endoplasmic reticulum, the signal peptidase complex cleaves off a large N-terminal signal peptide of about 64 amino acids. Mutagenesis screening showed that CNNM2 is glycosylated at residue Asn-112, stabilizing CNNM2 on the plasma membrane. CNNM2a forms heterodimers with the smaller isoform CNNM2b.
The bacterial proteins, YrkA and YhdP, have three recognized domains: the 4-TMS DUF21 domain (residues 1-170), a nucleotide binding CBS domain (residues 225-335) and a CorC/HlyC hydrophilic domain (residues 360-430). The mammalian homologues have at least the first two of these domains which are preceded by an N-terminal TMS and an unidentified hydrophilic domain. The bacterial HlyC and CorC proteins (1.C.126) lack the 4 TMS DUF21 domain, but have the CBS and CorC/HlyC domains. Only the proteins with the DUF21 domain are likely to be transporters. All of the evidence is consistent with the conclusion that these homologues form divalent-cation-specific porters or channels.
There is controversy as to (1) whether the CNNM (cyclin) proteins are transporters or regulators, and (2) if they are transporters, whether they are channels or carriers. The CBS domains bind ATP (Hirata et al. 2014). In humans, the CNNM family is encoded by four genes: CNNM1-4. CNNM1 is thought to act as a cytosolic copper chaperone, whereas CNNM2 and CNNM4 have been associated with magnesium handling. Mutations in the CNNM2 gene cause familial dominant hypomagnesaemia (MIM:607803), a rare human disorder characterized by renal and intestinal magnesium (Mg2+) wasting, which may lead to symptoms of Mg2+ depletion such as tetany, seizures and cardiac arrhythmias (Gómez-García et al. 2012). In marine fish, there are 6 CNNM paralogues, each with a different location and function, probably catayzing Mg2+ efflux (Islam et al. 2014). Three conserved dileucine motifs in CNNM4 are necessary for both basolateral sorting and interaction with the μ1As (AP1A) and μ1B (AP1B) proteins (Hirata et al. 2014). Ishii et al. 2016 also concluded that CNNM proteins are efflux permeases.
PRL phosphatases and CNNM proteins form complexes, regulated by the formation of phosphocysteine. Gulerez et al. 2016 showed that cysteine in the PRL phosphatase catalytic site is endogenously phosphorylated as part of the catalytic cycle and that phosphocysteine levels change in response to Mg2+ levels. Phosphorylation blocks PRL binding to the CNNM Mg2+ transporters, and mutations that block the PRL-CNNM interaction prevent regulation of Mg2+ efflux in cultured cells. The crystal structure of the complex of PRL2 and the CBS-pair domain of the Mg2+ transporter CNNM3 revealed the molecular basis for the interaction (Gulerez et al. 2016). The structural basis underlying the interaction between PRL phosphatases and CNNM transporters has been further studied (Giménez-Mascarell et al. 2017). Renal function of CNNM2 is necessary for maintenance of blood pressure (Funato et al. 2017), and Funato et al. 2018. have concluded that CNNM porters are Na+:Mg2+ antiporters. However Arjona and de Baaij 2018 suggested that they function as regulators of Mg2+ transport, although Funato et al. 2018 rebutted this suggestion. Chen et al. 2018 concluded that the CBS domains in CNNM proteins mediate dimerization and are important for Mg2+ transport activity. Most of the evnidence therefore suggests that CNNM proteins are Mg2+ exporters (Ulens 2018).
The cyclin M family (CNNMs; also called ancient conserved domain proteins, or ACDPs) may be Mg2+ transporters (Chen et al. 2018). CNNMs are associated with a number of genetic diseases affecting ion movement and cancer via their association with highly oncogenic phosphatases of regenerating liver (PRLs). Structurally, CNNMs contain an N-terminal extracellular domain, a transmembrane domain (DUF21), and a large cytosolic region containing a cystathionine-β-synthase (CBS) domain and a putative cyclic nucleotide-binding homology (CNBH) domain. Chen et al. 2018 determined the crystal structures of the CNBH domains of CNNM2 and CNNM3 at 2.6 and 1.9 Å resolutions. These domains did not bind cyclic nucleotides, but mediated dimerization both in crystals and in solution. An inverse correlation between the propensity of the CNBH domains to dimerize and the ability of CNNMs to mediate Mg2+ efflux was noted. CNBH domains from active family members were observed as both dimers and monomers, whereas the inactive member, CNNM3, was observed only as a dimer. Mutational analysis revealed that the CNBH domain was required for Mg2+ efflux activity of CNNM4.
CNNM/CorB proteins are a broadly conserved and are associated with Mg2+ transport but it is not known if they mediate transport themselves or regulate other transporters. Chen et al. 2021 determined 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 pi-helix. In the absence of Mg2+-ATP, the CBS-pair domain adopts an elongated dimeric configuration with previously unobserved domain-domain contacts. A role of the structural rearrangements in mediating Mg2+-ATP sensing was suggested. An in vitro, liposome-based assay was used to demonstrate direct Mg2+ transport by CorB proteins (Chen et al. 2021).
In prokaryotes, cellular Mg2+ homeostasis is orchestrated via the CorA, MgtA/B, MgtE, and CorB/C Mg2+ transporters (Franken et al. 2022). For CorA, MgtE, and CorB/C, the motifs that form the selectivity pore, are conserved during evolution. Thus, CNNM proteins, the vertebrate orthologues of CorB/C, also have Mg2+ transport capacity. Whereas CorA and CorB/C proteins share the gross quaternary structure and functional properties with their respective orthologues, the MgtE channel only shares the selectivity pore with SLC41 Na+/Mg2+ transporters (Franken et al. 2022).
The generalized reaction that may be catalyzed by CNNM porters is:
Mg2+ (in) → Mg2+ (out)
Brandao K., Deason-Towne F., Perraud AL. and Schmitz C. (2013). The role of Mg2+ in immune cells. Immunol Res. 55(1-3):261-9.
Gibson, M.M., Bagga, D.A., Miller, C.G., and Maguire, M.E. (1991). Magnesium transport in Salmonella typhimurium: the influence of new mutations conferring Co2+ resistance on the CorA Mg2+ transport system. Mol Microbiol. 5: 2753-2762.
Quamme GA. (2010). Molecular identification of ancient and modern mammalian magnesium transporters. Am J Physiol Cell Physiol. 298(3):C407-29.
Kidney metal (Mg2+) transporter, Cyclin (CNN) M2 isoform CRA_b (CNNM2). Defects cause hypomagnesemia. It has an extracellular N-terminus, an N-terminal TMS, a hydrophilic domain followed by 4 TMSs, another hydrophilic domain, and an intracellular C-terminus (de Baaij et al., 2012). CNNM2a forms heterodimers with the smaller isoform CNNM2b. The human splice variant 1 of CNNM2 (ACDP2; Q9H8M5) is a Mg2+ transporter (Brandao et al. 2012). The Bateman module is involved in AMP binding and Mg2+ sensing, and their binding causes a conformational change in the CBS module, transmitted to the transmembrane domain (Corral-Rodríguez et al. 2014). It may be able to transport divalent metal cations, Mg2+, Co2+, Mn2+, Sr2+, Ba2+, Cu2+, Fe2+ and monvalent cation, Na+. In prokaryotes, homologs are CorB/C.
Cyclin M2, CNNM2, of Mus musculus (Q3TWN3)
Metal transporter CNNM3 (Ancient conserved domain-containing protein 3) (mACDP3) (Cyclin-M3) of 713 aas and probably 5 TMSs with one N-terminal, and four together in the first half of the protein (Chen et al. 2018). See the family description for the domain order of the CNNM proteins.
CNNM3 of Mus musculus
Metal transporter CNNM3 (Ancient conserved domain-containing protein 3) (Cyclin-M3). As of 2018, the function of this protein as a Mg2+ transporter is under debate (Schäffers et al. 2018).
CNNM3 of Homo sapiens
Metal transporter CNNM4 (Ancient conserved domain-containing protein 4) (Cyclin-M4). As of 2018, the function of this protein as a Mg2+ transporter was under debate (Schäffers et al. 2018).
CNNM4 of Homo sapiens
Mg2+ exporter of 951 aas and 5 TMSs in a 1 + 4 TMS arrangement, CNNM1 (Chen et al. 2018).
CNNM1 of Homo sapiens
Putative Mg2+ exporter of 875 aas and 5 TMSs, CNNM2 or ACDP2 (Chen et al. 2018). The bacterial CorC is involved in resistance to antibiotic exposure and to the survival of pathogenic microorganisms in their host environments. CorC possesses a cytoplasmic region containing the (regulatory ?) ATP-binding site (Huang et al. 2021). An inhibitor, IGN95a, targets the ATP-binding site and blocks both ATP binding and Mg2+ export. The cytoplasmic domain structure in complex with IGN95a was determined (Huang et al. 2021). With ATP bound to the cytoplasmic domain, the conformational equilibrium of CorC shifts toward the inward-facing state of the transmembrane domain (Huang et al. 2021). These considerations suggest that CorC may be an ATP-driven Mg2+ efflux porter, and if so, the family belongs in TC sub-class, 3.A. CorC homologs may be able to export Mg2+, Co2+, Mn2+, Sr2+, Ba2+, Cu2+ and Fe2+.
CNNM2 of Homo sapiens
Uncharacterized protein of 734 aas and 5 N-terminal TMSs.
UP of Trypanosoma cruzi
CNNM Mg2+ transport protein of 494 aas and 5 TMSs in a 1 + 4 TMS arrangement. It has a CNNM domain (residues 1 - 220) and three CBS domains, CBS1, 2, and 3 (residues 230 - 425).
CNNM Mg2+ transporter of Arabidopsis thaliana
CNNM putative Mg2+ transport channel of 527 aas with 5 TMSs. The hydrophobic CNNM domain is N-terminal followed by 3 CBS domains.
CNNM of Arabidopsis thaliana
Uncharacterized protein of 384 aas and 4 TMSs. The region of sequence similarity with established CorC proteins is in a hydrophilic region following the TMSs, putting into question the assignment of this protein to family 9.A.40. The N-terminal domain of 100 aas and 2 TMSs does not show sequence similarity with anything outside of the Candidatus Saccharibacteria bacteria.
UP of Candidatus Saccharibacteria bacterium
Magnesium and cobalt efflux protein, CorC or MpfA (Magnesium Protection Factor A) of 449 aas and 4 N-terminal TMSs, apparently in a 2 + 2 TMS arrangement. Evidence has been presented that this protein catalyzes active Mg2+ extrusion from the cell (Armitano et al. 2016). If so, It must be an active transporter, either a secondary carrier (TC subclass 2.A) or an ATP hydrolysis-driven exporter (TC subclass 3.A).
MpfA of Staphylococcus aureus
PaeA, YtfL, UPF0053 inner membrane protein, Duf21 domain containing protein, HlyC/CorC family transporter, hemolysin homolog of 447 aas and 4 N-terminal TMSs (residues 1 - 200) followed by a large hydrophilic domain (cystathionine beta-synthase, CBS, residues 201 - 447), possibly with a single TMS at about residue 320. It transports cadaverine and putrescine. In fact, Salmonella, Klebsiella pneumoniae (TC# 1.A.112.1.12) and E. coli synthesize, import, and export cadaverine, putrescine, and spermidine to maintain physiological levels of polyamines and provide pH homeostasis. Both low and high intracellular levels of polyamines confer pleiotropic phenotypes or lethality. Iwadate et al. 2021 demonstrated that PaeA (YtfL) is required for reducing cytoplasmic cadaverine and putrescine concentrations. PaeA is involved in stationary phase survival when cells are grown in acidic medium in which they produce cadaverine. The paeA mutant is sensitive to putrescine, but not spermidine or spermine. Sensitivity to external cadaverine in stationary phase is only observed at pH > 8, suggesting that the polyamines need to be deprotonated to passively diffuse into the cell. In the absence of PaeA, intracellular polyamine levels increase and the cells lose viability. Ectopic expression of the known cadaverine exporter, CadB, in stationary phase partially suppresses the paeA mutant phenotype, and overexpression of paeA in exponential phase partially complements a cadB mutant grown in acidic medium. Thus, PaeA is a cadaverine/putrescine exporter, reducing potentially toxic levels under certain stress conditions (Iwadate et al. 2021).
PaeA of E. coli
YtfA, DUF21 domain-containing protein, HlyC/CorC family transporter, magnesium and cobalt efflux protein CorC_2 or CorC_3, of 445 aas and 4 N-terminal TMSs plus a large hydrophilic domain as the C-terminal 250 residues. Based on the E. coli ortholog, it probably transports putrescine and canavanine (Iwadate et al. 2021). Klebsiella pneumoniae is a source of widespread contamination of medical equipment, causing pneumonia as well as other multiorgan metastatic infections. During K. pneumoniae infections of lung epithelia, microtubules are severed and then eliminated, and YtfA plays a role, probably by secreting a relevant compound (Chua et al. 2019).
YtfL of Klebsiella pneumoniae
Uncharacterized protein of 434 aas with an N-terminal 4 TMSs, YrkA.
YrkA of Bacillus subtilis (Q45494)
Probable Mg2+ exporter of 452 aas. It does not exhibit hemolysin activity (Sałamaszyńska-Guz and Klimuszko 2008).
TylC-like protein of Campylobacter jejuni
Uncharacterized protein of 444 aas and 4 TMSs, YhdP. Mutations in yhdP increase the activity of sigmaW (Turner and Helmann 2000).
YhdP of Bacillus subtilis
CorC homologue, YfjD or YpjE, of 428 aas and 4 TMSs in a 1 + 3 TMS arrangement at the N-terminus of the protein. This hydrophobic region is followed by a larger hydrophilic domain. It is encoded within a two gene operon with YpjD (CorE), a putative cytochrome c assembly protein of 8 or 9 TMSs (P64432; TC# 9.B.14.3.6) (Huang et al. 2021).
YfjD of E. coli
Uncharacterized CorC protein of 327 aas and 3 N-terminal TMSs. The region of sequence similarity with HlyC proteins (TC# 1.C.126) is in a hydrophilic C-terminal region following the TMSs.
UP of Candidatus Saccharibacteria bacterium
Mg2+ and Co2+ transporter, CorB, of 413 aas and 3 TMSs. It contains DUF21, CBS pair, and CorC-HlyC domainsin succession.
CorB of Pseudomonas bauzanensis
HlyC/CorC family transporter of 354 aas and 4 TMSs.
CorC domain protein of Micromonospora peucetia
Uncharacterized protein of 329 aas and 4 TMSs.
UP of Verrucomicrobia bacterium