The cyclic nucleotide–binding homology domain of the integral membrane protein CNNM mediates dimerization and is required for Mg2+ efflux activity

Abstract

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-␤synthase (CBS) domain and a putative cyclic nucleotidebinding 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.

A novel pathogenic missense variant in CNNM4 underlying Jalili syndrome: Insights from molecular dynamics simulations

“…With a molecular modeling approach, it is plausible to describe that CBS domains of CNNM4 bind directly to ATP, depending on the presence of Mg 2+ . The interactions with ATP may account for immediate Mg 2+ transport (Chen, Kozlov, et al, ; Hirata et al, ). Given the small size of the metal ions, the strict positioning by Arg407 and Thr495, of the pyrophosphate chain, is hypothesized to be crucial.…”

Structure of a transporter domain emerges

“…Magnesium trafficking across the cell membrane requires dedicated transporters due to the ion's unusual radius compared with other divalent cations. One important class of Mg 2ϩ transporters is the family of cystathione ␤-synthase domain divalent metal cation transport mediators (CNNMs), 2 including CNNM1-CNNM4. Topologically, CNNM transporters contain an N-terminal extracellular domain, a transmembrane domain, and a large cytosolic sequence composed of two discrete domains.…”

“…Indeed, mutations in CNNM genes are associated with a range of inherited diseases, including familial dominant hypomagnesemia or Jalili syndrome, characterized by recessive enamel abnormalities and cone-rod dystrophy (1). A new study by Chen et al (2) provides tentative but intriguing answers to one of these questions in their structural, biochemical, and cellular analysis of one of the cytosolic sequences, the cyclic nucleotide-binding homology domain (CNBHD).…”

“…Because determining the structural details of integral membrane proteins remains a substantial challenge, Chen et al (2) focused their attention on the isolated CNBHDs of two CNNM Mg 2ϩ transporters in a divide-and-conquer approach. Specifically, the authors obtained the first structures of the CNNM3 and CNNM2 CNBHDs, solved at relatively high resolution (1.9 and 2.6 Å, respectively) and revealing important structural detail.…”

“…E-mail: chris.ulens@ kuleuven.be. 2 The abbreviations used are: CNNM, cystathione ␤-synthase domain divalent metal cation transport mediators; CNBHD, cyclic nucleotide-binding homology domain; CNBD, cyclic nucleotide-binding domain; HCN, hyperpolarization-activated/cyclic nucleotide-sensitive.…”