Purification, crystallization and preliminary crystallographic analysis of the CBS pair of the human metal transporter CNNM4

Abstract

This work describes the purification and preliminary crystallographic analysis of the CBS-pair regulatory domain of the human ancient domain protein 4 (ACDP4), also known as CNNM4. ACDP proteins represent the least-studied members of the eight different types of magnesium transporters that have been identified in mammals to date. In humans the ACDP family includes four members: CNNM1-4. CNNM1 acts as a cytosolic copper chaperone and has been associated with urofacial syndrome, whereas CNNM2 and CNNM4 have been identified as magnesium transporters. Interestingly, mutations in the CNNM4 gene have clinical consequences that are limited to retinal function and biomineralization and are considered to be the cause of Jalili syndrome, which consists of autosomal recessive cone-rod dystrophy and amelogenesis imperfecta. The truncated protein was overexpressed, purified and crystallized in the orthorhombic space group C222. The crystals diffracted X-rays to 3.6 Å resolution using synchrotron radiation. Matthews volume calculations suggested the presence of two molecules in the asymmetric unit, which were likely to correspond to a CBS module of the CBS pair of CNNM4.

Structural Insights into the Intracellular Region of the Human Magnesium Transport Mediator CNNM4

“…Crystals of CNNM4 BAT were grown as described previously [40]. All crystallographic parameters and refinement statistics are listed in Table 1.…”

“…Human CNNM4 BAT was obtained by previously published protocols [40]. For NMR studies, an alternative construct of hCNNM4 BAT , containing a N-terminal His6-tag with V5 epitope and Tobacco Etch Virus (TEV) proteinase cleavage site, was cloned in a pET151-D/TOPO vector (Invitrogen).…”

“…The crystallizability of all protein constructs was evaluated with XtalPred [65]. CNNM4 BAT crystals were obtained as described previously [40] while crystals of SeMet labeled CNNM4 cNMP were grown by sitting drop vapor diffusion at 18 • C, mixing 0.5 µL protein (8 mg/mL) with 0.5 µL precipitant solution (0.5 M sodium formate, 5% (w/v) polyethylene glycol (PEG) 3350, 0.1 M Tris pH 7.2). All crystals were cryoprotected by immersion in mother liquor containing 20% (v/v) glycerol, and flash cooled in liquid nitrogen.…”

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

“…However, their overall biochemical mechanism of action and structural elucidation of the CBS domains of CNNM4 is presently unknown (Gómez García, Oyenarte, Oyenarte, & Martínez‐Cruz, ). 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+ .…”

“…CNNM4 (Q6P4Q7), has a DUF21 domain (residues 184–358) comprising four transmembrane helices (residues 182–204, 239–261, 265–287, and 294–316) and a leucine‐zipper (residues 188–209). Further composition of this protein contains one cyclic nucleotide monophosphate (cNMP)‐binding domain (residues 575–695), one cyclin‐box motif domain similar to the one present in ion channels and cNMP‐dependent kinases from residues 548–578, and two CBS domains (residues 377–438 and 445–511), which are believed to have a regulatory role in its biological activity (Gómez García et al, ).…”