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 Mg2+ 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 Mg2+ 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 Mg2+ efflux activity of CNNM4. This work provides a structural basis for understanding the function of CNNM proteins in Mg2+ transport and associated diseases.
Highlights
Proteins of the cyclin M family (CNNMs; called ancient conserved domain proteins, or ACDPs) are represented by four integral membrane proteins that have been proposed to function as Mg2؉ transporters
To gain insight into CNNM proteins, we endeavored to determine the three-dimensional structures of their cyclic nucleotide– binding homology (CNBH) domains
Attempts to solve the structure by molecular replacement were unsuccessful; we chose to label the protein with selenomethionine for experimental phasing. This required the introduction of additional methionine residues by site-directed mutagenesis, as there is only one methionine in the CNNM3 CNBH domain and its location was predicted to be in a mobile region
Summary
Proteins of the cyclin M family (CNNMs; called ancient conserved domain proteins, or ACDPs) are represented by four integral membrane proteins that have been proposed to function as Mg2؉ transporters. 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. Mutational analysis revealed that the CNBH domain was required for Mg2؉ efflux activity of CNNM4. The CBS-pair domain is the site of PRL binding (16 –18) and required for Mg2ϩ efflux [19]. The CBS-pair domains dimerize and are likely a site of regulation through ATP-Mg2ϩ binding. Whereas CBS-pair domains have been extensively characterized, little is known about the CNNM C termini other than the sequence similarity to cyclic nucleotide– binding domains [5]. Our results suggest that CNBH dimerization may function to inhibit and regulate CNNM activity across the different isoforms
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