Purification, crystallization and preliminary crystallographic analysis of the CBS-domain pair of cyclin M2 (CNNM2)

Abstract

This work describes the purification and preliminary crystallographic analysis of the CBS-domain pair of the murine CNNM2 magnesium transporter (formerly known as ancient domain protein 2; ACDP2), which consists of a pair of cystathionine -synthase (CBS) motifs and has 100% sequence identity to its human homologue. CNNM proteins represent the least-studied members of the eight different types of magnesium transporters identified to date in mammals. In humans, the CNNM family is encoded by four genes: CNNM1-4. CNNM1 acts as a cytosolic copper chaperone, whereas CNNM2 and CNNM4 have been associated with magnesium handling. Interestingly, mutations in the CNNM2 gene cause familial dominant hypomagnesaemia (MIM:607803), a rare human disorder characterized by renal and intestinal magnesium (Mg 2+ ) wasting, which may lead to symptoms of Mg 2+ depletion such as tetany, seizures and cardiac arrhythmias. This manuscript describes the preliminary crystallographic analysis of two different crystal habits of a truncated form of the protein containing its regulatory CBS-domain pair, which has been reported to host the pathological mutation T568I in humans. The crystals belonged to space groups P2 1 2 1 2 and I222 (or I2 1 2 1 2 1 ) and diffracted X-rays to 2.0 and 3.6 Å resolution, respectively, using synchrotron radiation.

Structural Basis of the Oncogenic Interaction of Phosphatase PRL-1 with the Magnesium Transporter CNNM2

“…Expression and Purification of CNNM2 BAT , CNNM2 cNMP , and CNNM2 BAT-cNMP -The CNNM2 BAT protein was produced as described previously (39). Use of EDTA was avoided to preclude Mg 2ϩ and Zn 2ϩ chelation.…”

“…Pure PRL-1, -2, and -3 (peak 1; M th ϭ 22.6, 21.9, and 22.4 kDa, respectively (where M indicates mass)) eluted as monomers (V e Ϸ17 ml; M exp Ϸ13-15 kDa) when they were independently loaded into a size exclusion chromatography column. In the absence of PRLs, CNNM2 BAT (peak 2; M th Ϸ34 kDa) eluted as a dimer (V e Ϸ16 ml; M exp Ϸ25 kDa) (37,39). The molecular masses of the resulting PRL-1/-2/-3⅐CNNM2 BAT complexes (M th Ϸ40 kDa; marked with black, red and blue asterisks, respectively) were consistent with 2ϫPRLϩ2ϫCNNM2 BAT heterotetramers in solution (V e Ϸ14.7; 14.5 and 14.5 ml, respectively; M exp Ϸ43, 48, and 48 kDa, respectively).…”

“…In this study, we used the full-length human PRL proteins and an engineered protein construct, CNNM2 BAT (39), comprising residues 429 -584 and containing the Bateman module of CNNM2 and the preceding ␣-helix (H0) that connect to the DUF21 transmembrane domain (supplemental Figs. S1 and S2).…”

“…1) were pooled and concentrated to ϳ15 mg ml Ϫ1 using an Amicon Ultra-15 (5000-Da cutoff) centrifugal concentrator (Millipore) and distributed in aliquots of 25 l that were subsequently flash-frozen in liquid nitrogen and stored at Ϫ80°C. The identity of all independent proteins as well as of those forming the complex was confirmed by mass spectrometry from the corresponding SDS-polyacrylamide gel bands as described previously (39). The approximate molecular mass of PRL-1, -2, and -3 and CNNM2 BAT and the PRLs⅐CNNM2 BAT complexes was estimated from a plot representing K av versus log M w (where…”

PRL2 links magnesium flux and sex-dependent circadian metabolic rhythms

“…Thus, correlating with the enhanced BAT thermogenesis observed, the overall increase in energy dissipation can explain the reason PRL2-KO mice are underweight, even though they exhibited increased food intake and O 2 consumption, especially in female mice. Mg 2+ transporter CNNMs contain tandem pairs of cystathionine-β-synthase (CBS) domains that are conserved in all living cells from prokaryotes to eukaryotes (48,49). CBS domains act as energy-sensing modules; for instance, the γ subunit of AMPK possesses 4 CBS domains with allosteric binding sites for AMP as well as ATP, allowing AMPK activity to vary according to the intracellular ATP:AMP ratio (50).…”