Mg²⁺-dependent interactions of ATP with the cystathionine- β-synthase (CBS) domains of a magnesium transporter

Ancient conserved domain protein/cyclin M (CNNM) family proteins are evolutionarily conserved Mg²⁺ transporters. However, their biochemical mechanism of action remains unknown. Here, we show the functional importance of the commonly conserved cystathionine- β-synthase (CBS) domains and reveal their unique binding ability to ATP. Deletion mutants of CNNM2 and CNNM4, lacking the CBS domains, are unable to promote Mg²⁺ efflux. Furthermore, the substitution of one amino acid residue in the CBS domains of CNNM2, which is associated with human hereditary hypomagnesemia, abrogates Mg²⁺ efflux. Binding analyses reveal that the CBS domains of CNNM2 bind directly to ATP and not AMP in a manner dependent on the presence of Mg²⁺, which is inhibited in a similar pattern by the disease-associated amino acid substitution. The requirement of Mg²⁺ for these interactions is a unique feature among CBS domains, which can be explained by the presence of highly electronegative surface potentials around the ATP binding site on CNNM2. These results demonstrate that the CBS domains play essential roles in Mg²⁺ efflux, probably through interactions with ATP. Interactions with ATP, which mostly forms complexes with Mg ²⁺ in cells, may account for the rapid Mg²⁺ transport by CNNM family proteins.