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Proteins of the cyclin M family (CNNMs; also called ancient conserved domain proteins, or ACDPs) are represented by four integral membrane proteins that have been proposed to function as Mg2β transporters. CNNMs are associated with a number of genetic diseases affecting ion movement and cancer via their association with highly oncogenic phosphatases of regenerating liver (PRLs). Structurally, CNNMs contain an N-terminal extracellular domain, a transmembrane domain (DUF21), and a large cytosolic region containing a cystathionine-β- synthase (CBS) domain and a putative cyclic nucleotide- binding homology (CNBH) domain. Although the CBS domain has been extensively characterized, little is known about the CNBH domain. Here, we determined the first crystal structures of the CNBH domains of CNNM2 and CNNM3 at 2.6 and 1.9 Å resolutions. Contrary to expectation, these domains did not bind cyclic nucleotides, but mediated dimerization both in crystals and in solution. Analytical ultracentrifugation experiments revealed an inverse correlation between the propensity of the CNBH domains to dimerize and the ability of CNNMs to mediate Mg2β efflux. CNBH domains from active family members were observed as both dimers and monomers, whereas the inactive member,CNNM3,was observed only as a dimer. Mutational analysis revealed that the CNBH domain was required for Mg2β efflux activity of CNNM4. This work provides a structural basis for understanding the function of CNNM proteins in Mg2β transport and associated diseases. [ABSTRACT FROM AUTHOR]

The family of cystathionine-β-synthase (CBS)-pair domain divalent metal cation transport mediators (CNNMs) is composed of four integral membrane proteins associated with Mg 2+ transport. Structurally, CNNMs contain large cytosolic regions composed of a CBS-pair and a cyclic nucleotide-binding homology (CNBH) domain. How these regulate Mg 2+ transport activity is unknown. Here, we determined the crystal structures of cytosolic fragments in two conformations: Mg 2+ -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 Mg 2+ -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 Mg 2+ -ATP binding and protein dimerization. Mutations that blocked either function prevented cellular Mg 2+ efflux activity. The results suggest Mg 2+ efflux is regulated by conformational changes associated with Mg 2+ -ATP binding to CNNM CBS-pair domains., Competing Interests: Declaration of Interests The authors declare no conflict of interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Proteins of the cyclin M family (CNNMs; also called ancient conserved domain proteins, or ACDPs) are represented by four integral membrane proteins that have been proposed to function as Mg 2+ transporters. CNNMs are associated with a number of genetic diseases affecting ion movement and cancer via their association with highly oncogenic phosphatases of regenerating liver (PRLs). Structurally, CNNMs contain an N-terminal extracellular domain, a transmembrane domain (DUF21), and a large cytosolic region containing a cystathionine-β-synthase (CBS) domain and a putative cyclic nucleotide-binding homology (CNBH) domain. Although the CBS domain has been extensively characterized, little is known about the CNBH domain. Here, we determined the first crystal structures of the CNBH domains of CNNM2 and CNNM3 at 2.6 and 1.9 Å resolutions. Contrary to expectation, these domains did not bind cyclic nucleotides, but mediated dimerization both in crystals and in solution. Analytical ultracentrifugation experiments revealed an inverse correlation between the propensity of the CNBH domains to dimerize and the ability of CNNMs to mediate Mg 2+ efflux. CNBH domains from active family members were observed as both dimers and monomers, whereas the inactive member, CNNM3, was observed only as a dimer. Mutational analysis revealed that the CNBH domain was required for Mg 2+ efflux activity of CNNM4. This work provides a structural basis for understanding the function of CNNM proteins in Mg 2+ transport and associated diseases., (© 2018 Chen et al.)