Mg²⁺-ATP Sensing in CNNM, a Putative Magnesium Transporter

Highlights

• Structural and functional studies of CNNM, a putative Mg²⁺ transporter in human

• Crystal structures of cytosolic fragments in open and closed conformations

• Tight correlation between Mg²⁺-ATP binding and CBS-pair domain dimerization

• Mg²⁺ efflux is regulated by conformational changes associated with Mg²⁺-ATP binding

Summary

The family of cystathionine-β-synthase (CBS)-pair domain divalent metal cation transport mediators (CNNMs) is composed of four integral membrane proteins associated with Mg²⁺ transport. Structurally, CNNMs contain large cytosolic regions composed of a CBS-pair and a cyclic nucleotide-binding homology (CNBH) domain. How these regulate Mg²⁺ transport activity is unknown. Here, we determined the crystal structures of cytosolic fragments in two conformations: Mg²⁺-ATP-analog bound and ligand free. The structures reveal open and closed conformations with functionally important contacts not observed in structures of the individual domains. We also identified a second Mg²⁺-binding region in the CBS-pair domain and a different dimerization interface for the CNBH domain. Analytical ultracentrifugation and isothermal titration calorimetry experiments revealed a tight correlation between Mg²⁺-ATP binding and protein dimerization. Mutations that blocked either function prevented cellular Mg²⁺ efflux activity. The results suggest Mg²⁺ efflux is regulated by conformational changes associated with Mg²⁺-ATP binding to CNNM CBS-pair domains.