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

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 ß-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 Šresolution, respectively, using synchrotron radiation.

Influence of the IL6 gene in susceptibility to systemic sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) is a genetically complex autoimmune disease; the genetic component has not been fully defined. Interleukin 6 (IL-6) plays a crucial role in immunity and fibrosis, both key aspects of SSc. We investigated the influence of IL6 gene in the susceptibility and phenotype expression of SSc. METHODS: We performed a large metaanalysis including a total of 2749 cases and 3189 controls from 6 white populations (Germany, The Netherlands, Norway, Spain, Sweden, and United Kingdom). Three IL6 single-nucleotide polymorphisms (SNP; rs2069827, rs1800795, and rs2069840) were selected by SNP tagging and genotyped using TaqMan(®) allele discrimination technology. RESULTS: Individual SNP metaanalysis showed no evidence of association of the 3 IL6 genetic variants with the global disease. Phenotype analyses revealed a significant association between the minor allele of rs2069840 and the limited cutaneous SSc clinical form (Bonferroni p = 0.036, OR 1.14, 95% CI 1.04-1.25). A trend of association between the minor allele of the rs1800795 and the diffuse cutaneous SSc clinical form was also evident (Bonferroni p = 0.072, OR 0.86, 95% CI 0.77-0.96). In the IL6 allelic combination analyses, the GGC allelic combination rs2069827-rs1800795-rs2069840 showed an association with overall SSc (Bonferroni p = 0.016, OR 1.13, 95% CI 1.04-1.23). CONCLUSION: Our results suggest that the IL6 gene may influence the development of SSc and its progression.

Purification, crystallization and preliminary crystallographic analysis of the CBS-domain protein MJ1004 from Methanocaldococcus jannaschii.

The purification and preliminary crystallographic analysis of the archaeal CBS-domain protein MJ1004 from Methanocaldococcus jannaschii are described. The native protein was overexpressed, purified and crystallized in the monoclinic space group P2(1), with unit-cell parameters a=54.4, b=53.8, c=82.6 Å, ß=106.1°. The crystals diffracted X-rays to 2.7 Šresolution using synchrotron radiation. Matthews-volume calculations suggested the presence of two molecules in the asymmetric unit that are likely to correspond to a dimeric species, which is also observed in solution.

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

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 Šresolution 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.

Nucleotide-induced conformational transitions in the CBS domain protein MJ0729 of Methanocaldococcus jannaschii.

Nucleotide-binding cystathionine ß-synthase (CBS) domains function as regulatory motifs in several proteins distributed through all kingdoms of life. This function has been proposed based on their affinity for adenosyl-derivatives, although the exact binding mechanisms remain largely unknown. The question of how CBS domains exactly work is relevant because in humans, several genetic diseases have been associated with mutations in those motifs. In this work, we describe the adenosyl-ligand (AMP, ATP, NADP and SAM) properties of the wild-type CBS domain protein MJ0729 from Methanocaldococcus jannaschii by using a combination of spectroscopic techniques (fluorescence, FTIR and FRET). The fluorescence results show that binding to AMP and ATP occurs with an apparent dissociation constant of ~10 µM, and interestingly enough, binding induces protein conformational changes, as shown by FTIR. On the other hand, fluorescence spectra (FRET and steady-state) did not change upon addition of NADP and SAM to MJ0729, suggesting that tryptophan and/or tyrosine residues were not involved in the recognition of those ligands; however, there were changes in the secondary structure of the protein upon addition of NADP and SAM, as shown by FTIR (thus, indicating binding to the nucleotide). Taken together, these results suggest that: (i) the adenosyl ligands bind to MJ0729 in different ways, and (ii) there are changes in the protein secondary structure upon binding of the nucleotides.

The crystal structure of protein MJ1225 from Methanocaldococcus jannaschii shows strong conservation of key structural features seen in the eukaryal gamma-AMPK.

In mammals, 5'-AMP-activated protein kinase (AMPK) is a heterotrimeric protein composed of a catalytic serine/threonine kinase subunit (alpha) and two regulatory subunits (beta and gamma). The gamma-subunit senses the intracellular energy status by competitively binding AMP and ATP and is thought to be responsible for allosteric regulation of the whole complex. We describe herein the crystal structure of protein MJ1225 from Methanocaldococcus jannaschii complexed to AMP, ADP, and ATP. Our data provide evidence of a strong conservation of the key functional features seen in the gamma-subunit of the eukaryotic AMPK, and more importantly, it reveals a novel AMP binding site, herein denoted as site E, which had not been previously described in cystathionine beta-synthase domains so far. Site E is located in a small cavity existing between the alpha-helices structurally equivalent to those disrupting the internal symmetry of each Bateman domain in gamma-AMPKs and shows striking similarities with a symmetry-related crevice of the mammalian enzyme that hosts the pathological mutation N488I.

The crystal structure of the novobiocin biosynthetic enzyme NovP: the first representative structure for the TylF O-methyltransferase superfamily.

NovP is an S-adenosyl-l-methionine-dependent O-methyltransferase that catalyzes the penultimate step in the biosynthesis of the aminocoumarin antibiotic novobiocin. Specifically, it methylates at 4-OH of the noviose moiety, and the resultant methoxy group is important for the potency of the mature antibiotic: previous crystallographic studies have shown that this group interacts directly with the target enzyme DNA gyrase, which is a validated drug target. We have determined the high-resolution crystal structure of NovP from Streptomyces spheroides as a binary complex with its desmethylated cosubstrate S-adenosyl-l-homocysteine. The structure displays a typical class I methyltransferase fold, in addition to motifs that are consistent with a divalent-metal-dependent mechanism. This is the first representative structure of a methyltransferase from the TylF superfamily, which includes a number of enzymes implicated in the biosynthesis of antibiotics and other therapeutics. The NovP structure reveals a number of distinctive structural features that, based on sequence conservation, are likely to be characteristic of the superfamily. These include a helical 'lid' region that gates access to the cosubstrate binding pocket and an active center that contains a 3-Asp putative metal binding site. A further conserved Asp likely acts as the general base that initiates the reaction by deprotonating the 4-OH group of the noviose unit. Using in silico docking, we have generated models of the enzyme-substrate complex that are consistent with the proposed mechanism. Furthermore, these models suggest that NovP is unlikely to tolerate significant modifications at the noviose moiety, but could show increasing substrate promiscuity as a function of the distance of the modification from the methylation site. These observations could inform future attempts to utilize NovP for methylating a range of glycosylated compounds.

Purification, crystallization and preliminary crystallographic analysis of protein MJ1225 from Methanocaldococcus jannaschii, a putative archaeal homologue of gamma-AMPK.

In mammals, AMP-activated protein kinase (AMPK) is a heterotrimeric protein composed of a catalytic serine/threonine kinase subunit (alpha) and two regulatory subunits (beta and gamma). The gamma subunit senses the intracellular energy status by competitively binding AMP and ATP and is thought to be responsible for allosteric regulation of the whole complex. This work describes the purification and preliminary crystallographic analysis of protein MJ1225 from Methanocaldococcus jannaschii, an archaeal homologue of gamma-AMPK. The purified protein was crystallized using the hanging-drop vapour-diffusion method. Diffraction data for MJ1225 were collected to 2.3 A resolution using synchrotron radiation. The crystals belonged to space group H32, with unit-cell parameters a = b = 108.95, c = 148.08 A, alpha = beta = 90.00, gamma = 120.00 degrees . Preliminary analysis of the X-ray data indicated that there was one molecule per asymmetric unit.

Crystallization and preliminary X-ray analysis of the O-carbamoyltransferase NovN from the novobiocin-biosynthetic cluster of Streptomyces spheroides.

Crystals of recombinant NovN, an O-carbamoyltransferase from Streptomyces spheroides, were grown by vapour diffusion. The protein crystallized in two different crystal forms. Crystal form I belonged to space group C2 and native data were collected to 2.9 A resolution in-house. Crystal form II had I-centred orthorhombic symmetry and native data were recorded to a resolution of 2.3 A at a synchrotron. NovN catalyses the final step in the biosynthesis of the aminocoumarin antibiotic novobiocin that targets the essential bacterial enzyme DNA gyrase.