Nucleotide binding triggers a conformational change of the CBS module of the magnesium transporter CNNM2 from a twisted towards a flat structure

Abstract

Recent studies suggest CNNM2 (cyclin M2) to be part of the long-sought basolateral Mg2+ extruder at the renal distal convoluted tubule, or its regulator. In the present study, we explore structural features and ligand-binding capacities of the Bateman module of CNNM2 (residues 429-584), an intracellular domain structurally equivalent to the region involved in Mg2+ handling by the bacterial Mg2+ transporter MgtE, and AMP binding by the Mg2+ efflux protein CorC. Additionally, we studied the structural impact of the pathogenic mutation T568I located in this region. Our crystal structures reveal that nucleotides such as AMP, ADP or ATP bind at only one of the two cavities present in CNNM2429-584. Mg2+ favours ATP binding by alleviating the otherwise negative charge repulsion existing between acidic residues and the polyphosphate group of ATP. In crystals CNNM2429-584 forms parallel dimers, commonly referred to as CBS (cystathionine β-synthase) modules. Interestingly, nucleotide binding triggers a conformational change in the CBS module from a twisted towards a flat disc-like structure that mostly affects the structural elements connecting the Bateman module with the transmembrane region. We furthermore show that the T568I mutation, which causes dominant hypomagnesaemia, mimics the structural effect induced by nucleotide binding. The results of the present study suggest that the T568I mutation exerts its pathogenic effect in humans by constraining the conformational equilibrium of the CBS module of CNNM2, which becomes 'locked' in its flat form.

PRL2 links magnesium flux and sex-dependent circadian metabolic rhythms

“…CBS domains act as energy-sensing modules; for instance, the γ subunit of AMPK possesses 4 CBS domains with allosteric binding sites for AMP as well as ATP, allowing AMPK activity to vary according to the intracellular ATP:AMP ratio (50). Such energy-sensing mechanisms could modulate PRL2/CNNM functions, as ATP binds to CBS domains of CNNM2 in a Mg 2+ -dependent manner (51) and adenosine nucleotides trigger a conformational change in the CBS domains of CNNM2 (52). Taking into account that PRLs directly interact with CBS domains of CNNMs (8, 53), these findings imply that the function of the PRL2/CNNMs complex must be tightly regulated according to cellular metabolic status.…”

Structural Basis of the Oncogenic Interaction of Phosphatase PRL-1 with the Magnesium Transporter CNNM2

“…Upon binding of phospho-nucleotides (AMP, ADP, ATP, and MgATP), a network of polar interactions centered around Thr-568 is disrupted, thus causing a coordinated reorientation of helices H0 and H4 (supplemental Fig. S2), concomitant to a relative rotation of the two CBS motifs (37). This apparently smooth displacement of secondary elements within the subunit induces a dramatic structural change in the dimeric disc that evolves toward a "flat" arrangement (PDB codes 4P1G, 4IY0, and 4P1O) (supplemental Movie S2) in which both CBS1 domains interact symmetrically (37).…”

“…S2), concomitant to a relative rotation of the two CBS motifs (37). This apparently smooth displacement of secondary elements within the subunit induces a dramatic structural change in the dimeric disc that evolves toward a "flat" arrangement (PDB codes 4P1G, 4IY0, and 4P1O) (supplemental Movie S2) in which both CBS1 domains interact symmetrically (37). Importantly, the conformational change in the CBS module is believed to be transmitted to the transmembrane region through the C-terminal ␣-helix H0 connecting both Bateman modules with the transmembrane domains, thus putatively modulating the activity of CNNM2 and its ability to regulate Mg 2ϩ homeostasis (37).…”

“…We recently found that the Bateman module of CNNM2, consisting of two consecutive CBS domains, associates itself with disc-like dimers commonly referred to as "CBS modules" (supplemental Fig. S2) that accommodate nucleotides and metal ions (37,38). In the absence of bound nucleotide, the CBS module adopts a Y-shaped or "twisted" conformation (PDB code 4IYS) (supplemental Movie S1), where the interfacial helices H3 and H4 maintain the CBS2 domains from complementary subunits in close contact with each other, although the CBS1 domains are separated (37).…”

“…S2) that accommodate nucleotides and metal ions (37,38). In the absence of bound nucleotide, the CBS module adopts a Y-shaped or "twisted" conformation (PDB code 4IYS) (supplemental Movie S1), where the interfacial helices H3 and H4 maintain the CBS2 domains from complementary subunits in close contact with each other, although the CBS1 domains are separated (37). Upon binding of phospho-nucleotides (AMP, ADP, ATP, and MgATP), a network of polar interactions centered around Thr-568 is disrupted, thus causing a coordinated reorientation of helices H0 and H4 (supplemental Fig.…”