Current Structural Knowledge on the CNNM Family of Magnesium Transport Mediators

Abstract

The cyclin and cystathionine β-synthase (CBS) domain magnesium transport mediators, CNNMs, are key players in maintaining the homeostasis of magnesium in different organs. The human family includes four members, whose impaired activity causes diseases such as Jalili Syndrome or Familial Hypomagnesemia, but is also linked to neuropathologic disorders, altered blood pressure, and infertility. Recent findings demonstrated that CNNMs are associated with the highly oncogenic phosphatases of the regenerating liver to promote tumor growth and metastasis, which has attracted renewed focus on their potential exploitation as targets for cancer treatment. However, the exact function of CNNMs remains unclear and is subject to debate, proposed as either direct transporters, sensors, or homeostatic factors. This review gathers the current structural knowledge on the CNNM family, highlighting similarities and differences with the closely related structural partners such as the bacterial Mg²⁺/Co²⁺ efflux protein CorC and the Mg²⁺ channel MgtE.

Keywords: ACDP; CBS domain; CNBH domain; CNNM; Jalili syndrome; cNMP domain; cancer; hypomagnesemia; magnesium homeostasis; magnesium transport.

Figure 1

Structure of CNNMs. (A) Domain distribution in a related cystathionine β-synthase CBS domain containing metal ion transporters, including CNNM2. The Bateman module (in blue) plays a regulatory role in all the proteins shown. The DUF21 domain represents a major part of the transmembrane domain (TMD) (orange) and is thought to contain from three to four α-helices. CNBH domain is in green. The N-terminal extracellular region of CNNM2 is predicted to be β-strand-enriched (deep purple). The bacterial MgtE channel shares the presence of a CorC-HlyC domain (purple) with CorC and with other proteins such as the bacterial SA0657 protein [57]. (B) The panel shows in ribbons the three-dimensional structure of the isolated domains. The structures of the Bateman module and the CNBH domain were extracted from crystallographic data [43,45,58,59]. The representation of the DUF21 domain corresponds to an in silico model [22]. The CNNM representation is as follows: The N-terminal extracellular domain (dark blue) shows the glycosylation in red; the transmembrane α-helixes are represented in orange. In the intracellular region, the Bateman module (PDB code: 4P1O) is represented in blue; bound MgATP is in pink. The CNBH domain (PDB code: 6DJ3) is in green. The unstructured terminal C-tail is in purple. The locations of all known pathological mutations reported for CNNMs are indicated by arrows and in different colors depending on the variant protein: CNNM2 (red), CNNM3 (cyan), and CNNM4 (black). Helix H0 connects the Bateman module with the DUF21 domain.

Figure 2

Structure of the Bateman domain of CNNM2. (left) Two views of the structural elements configuring the Bateman module of CNNM2 (in ribbons). The main symmetric cavities S1 and S2 are indicated. S2 hosts MgATP (right) CBS module: parallel (head-to-head) dimeric association of Bateman modules. The S2 cavities from the complementary subunits are located on opposite sides of the disc. (bottom) Amino acid sequence of human CNNM2. The secondary elements are indicated.

Figure 3

Structure of the CNNM-PRL complex. The picture shows the crystal structure of the CBS module of CNNM2 (PDB code: 5LXQ) (the two Bateman modules are in yellow and orange) in complex with PRL-1 (in green and blue). As shown, the CBS module adopts a flat conformation. MgATP (in sticks) occupies the complementary S2 cavities. (A) Detail of the main interactions between CNNM2 and PRL-1. D558 from CNNM2 (orange) enters the catalytic cavity of PRL-1 (green); (B) MgATP-binding site (cavity S2) in CNNM2. The main residues involved in the interaction with ATP are represented in sticks. (Bottom) Sequence alignment of the three main blocks forming the walls of the S2 site. The conserved motif h y y′ h′ P (where h is hydrophobic, y is whatever, and P is proline) stabilizes the adenine ring and favors the presence of adenine-derived nucleotides. Conserved T568 and D571 interact with the hydroxyl group of the ribose ring and belong to the conserved motif G h h′ T/S X X D/N (where h is hydrophobic, and X is any residue). The electrostatic repulsion exerted by residues E570, D571, and E572 is partially neutralized by Mg²⁺. Nucleotides bind to Site 2 (S2) in CNNMs, CorC, and ClCs, whereas MgtE binds ATP in Site 1 (S1). However, as shown in the alignment, conserved motifs are also present in S1 of MgTE. (C) Nucleotide-binding sites in MgtE and CorC (adapted from Tomita et al., 2017). (left) Site S1 of MgtE (represented in green), ATP is represented by sticks (PDB code: 5X9G). The conserved threonine of the G h h′ T/S X X D/N motif is substituted by a serine, and the conserved glutamate (E570 in CNNM2) is substituted by an arginine that interacts with the triphosphate chain of the ATP. The hydrophobic environment that stabilizes the position of the adenine ring is also present (motif h y y′ h′ P). (right) Site S2 of CorC (blue) bound to AMP (PDB code: 5YZ2) [69].

Figure 4

Crystal Structure of the CNBH of CNNM2. (Figure prepared using atom coordinates from PDB code 6DJ3 [59]. (A) The structure shows a central eight-stranded β roll preceded by two α-helixes in the N-terminal part (αA′ and αA), and is followed by a C-terminal α helix called αB. The small αB helix is inserted in the beta roll. Residue F705 and some other bulky residues autoinhibit the domain and impair nucleotide binding [59]; (B) The CNBH domain associates in dimers. The complementary subunits are depicted in pink and green. Residues F705 and M703 are relevant in the subunits association.

Figure 5

Structural comparison of CNNM2 and MgtE. (A) Structure of the CBS module of MgtE in its two different conformations: (top) closed and (bottom) open. Mg²⁺ ions are represented by grey spheres. The acidic clusters are represented by sticks. Upon binding of Mg²⁺ ions, the CBS module evolves from its open state (bottom) to its closed Mg²⁺-bound conformation (top). (B) CNNM2 may adopt two different conformations: Flat (top right) when bound to MgATP, and twisted (bottom right) in the absence of a nucleotide. The main residues involved in ATP and Mg²⁺ binding are represented by sticks.