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.

Figure 1. Domain distribution of CNNM2, MgtE and CorC

In CNNM2, the Bateman module (white), consisting of a pair of CBS motifs [–48], connects with the transmembrane region (DUF21 domain, in black) through its N-terminal α-helix. CorC is structurally equivalent in this regard. The location of the hypomagnesaemia-causing mutation T568I [16,17] is indicated. In contrast, in MgtE the Bateman module connects with the transmembrane region through its C-terminal α-helix.

Figure 2. Effect of Mg²+ and/or nucleotides on the Bateman module of CNNM2

The Figure shows five representative groups of signals (columns 1–5) selected from the ¹⁵N-HSQC CNNM2^429–584 protein spectrum and affected upon addition of Mg²+ and/or nucleotides to the protein solution. The signals obtained in the absence of Mg²+ (black) are overlapped with those obtained after addition of: (A) 2.5 mM MgCl₂ (green); (B) 0.5 mM ADPNP (red) and 10 mM ADPNP (magenta); (C) 2.5 mM ADPNP+0.5 mM MgCl₂ (green), 2.5 mM ADPNP+1 mM MgCl₂ (orange), and 2.5 mM ADPNP+2.5 mM MgCl₂ (red); (D) 10 mM MgCl2 +2.5 mM ADPNP (red) or 2.5 mM ADPNP+10 mM MgCl2 (blue); and (E) 0.25 mM AMP (green), 2.5 mM AMP (blue) and 5 mM AMP (red). Note: no additional spectral changes are observed in the range 0.25–2.5 mM AMP. The protein concentration is 200 μM in all cases. The exact position of each group of signals (1–5) within the corresponding spectra is marked in Figures S1–S5.

Figure 3. Crystal structure of the CNNM2^429–584 monomer

(A) CNNM₂^429–584 adopts the overall fold of a Bateman module and contains two consecutive CBS motifs (CBS1, residues 445–508; CBS2, residues 509–578). A long loop, which is disordered in most of the crystals analysed, connects strands β5 and β6. Of note, the equivalent loop in the magnesium transporter CNNM3 mediates the interaction with protein tyrosine phosphatase PRL-2 [58]. The H0 helix connects the flexible linker preceding the Bateman module with the DUF21 transmembrane region in the full-length protein. Of the two main cavities present in the protein (referred to as S1 and S2), only S2 hosts nucleotides (see Figure 4). The pseudo two-fold symmetry axis relating CBS1 and CBS2 is indicated by a broken line. (B) Sequence alignment of the Bateman modules region in CNNM2 and its closest homologue CNNM4. The secondary structure elements shown on top of the sequences are extracted from the crystal structures. Vertical red broken lines indicate the few residues that are different between the CNNM2 and CNNM4 proteins in this region. Location of Thr568 affected by the pathogenic mutation T568I is marked with an asterisk.

Figure 4. Stereo pairs of the S2 nucleotide-binding site of CNNM2^429–584 in the apoprotein (yellow), and in the AMP (blue), ADP (green) and Mg-ATP (silver, where the Mg²⁺ ion bound to ATP is represented with a grey sphere) complexes

The lowest stereo pair corresponds to the S2 site of mutant T568I.

Figure 5. Conformations of the apo and nucleotide-bound CNNM2^429–584 complexes

The Figure shows three different views of the two different types of CNNM2^429–584 dimers that are present in our crystals: (A), a ‘twisted’ dimer in which one of the Bateman modules rotates away from the original plane containing the protein disc (see also Figure 6) and (B), a ‘flat’ disc-like dimer formed upon binding of AMP/ADP or Mg-ATP (the ADP is represented). These conformers represent the initial and final steps of the conformational change suffered by the CNNM2^429–584 dimer upon nucleotide binding. α-Helices H0 (red) and H4 (marine blue) are the main structural elements affected upon nucleotide binding in each monomer. The two complementary monomers are represented in cyan and green respectively

Figure 6. Effect of ligands and mutations on CNNM2429–584

(A) Top panel: overlap of apo-CNNM2^429–584 (yellow), AMP-, ADP- and MgATP-bound CNNM2 (blue, green and silver respectively), and T568I mutant (red). The main distinct feature is the orientation of α-helices H0 and H4, and also of the short helical region HA₁. Bottom panel: magnified view of a section of two different views showing the rotation of the ribose ring of the bound nucleotides around the C1′–N9 bond. (B) Side view of the CNNM2^429–584 dimer showing the conformational change suffered upon binding of nucleotides to both monomers. The broken line represents a plane perpendicular to the image that contains the disc-like CNNM2^429–584 flat dimer. Of note, mutation T568I mimics the effect of nucleotide binding to site S2.