IceBear: an intuitive and versatile web application for research-data tracking from crystallization experiment to PDB deposition.

The web-based IceBear software is a versatile tool to monitor the results of crystallization experiments and is designed to facilitate supervisor and student communications. It also records and tracks all relevant information from crystallization setup to PDB deposition in protein crystallography projects. Fully automated data collection is now possible at several synchrotrons, which means that the number of samples tested at the synchrotron is currently increasing rapidly.

High-resolution Crystal Structure of Human pERp1, A Saposin-like Protein Involved in IgA, IgM and Integrin Maturation in the Endoplasmic Reticulum.

The folding of disulfide bond containing proteins in the endoplasmic reticulum (ER) is a complex process that requires protein folding factors, some of which are protein-specific. The ER resident saposin-like protein pERp1 (MZB1, CNPY5) is crucial for the correct folding of IgA, IgM and integrins. pERp1 also plays a role in ER calcium homeostasis and plasma cell mobility.

The crystal structure of human microsomal triglyceride transfer protein.

Microsomal triglyceride transfer protein (MTP) plays an essential role in lipid metabolism, especially in the biogenesis of very low-density lipoproteins and chylomicrons via the transfer of neutral lipids and the assembly of apoB-containing lipoproteins. Our understanding of the molecular mechanisms of MTP has been hindered by a lack of structural information of this heterodimeric complex comprising an MTPα subunit and a protein disulfide isomerase (PDI) β-subunit. The structure of MTP presented here gives important insights into the potential mechanisms of action of this essential lipid transfer molecule, structure-based rationale for previously reported disease-causing mutations, and a means for rational drug design against cardiovascular disease and obesity.

The Pro-Oncogenic Adaptor CIN85 Acts as an Inhibitory Binding Partner of Hypoxia-Inducible Factor Prolyl Hydroxylase 2.

The EGFR adaptor protein, CIN85, has been shown to promote breast cancer malignancy and hypoxia-inducible factor (HIF) stability. However, the mechanisms underlying cancer promotion remain ill defined. Here we show that CIN85 is a novel binding partner of the main HIF-prolyl hydroxylase, PHD2, but not of PHD1 or PHD3.

Structural analysis of human NHLRC2, mutations of which are associated with FINCA disease.

NHLRC2 (NHL repeat-containing protein 2) is an essential protein. Mutations of NHLRC2, including Asp148Tyr, have been recently associated with a novel FINCA disease (fibrosis, neurodegeneration, cerebral angiomatosis), which is fatal in early childhood. To gain insight into the mechanisms of action of this essential protein, we determined the crystal structure of the Trx-like and NHL repeat β-propeller domains of human NHLRC2 to a resolution of 2.

Crystal structure of human anterior gradient protein 3.

Oxidative protein folding in the endoplasmic reticulum is catalyzed by the protein disulfide isomerase family of proteins. Of the 20 recognized human family members, the structures of eight have been deposited in the PDB along with domains from six more. Three members of this family, ERp18, anterior gradient protein 2 (AGR2) and anterior gradient protein 3 (AGR3), are single-domain proteins which share sequence similarity.

Structures of Angptl3 and Angptl4, modulators of triglyceride levels and coronary artery disease.

Coronary artery disease is the most common cause of death globally and is linked to a number of risk factors including serum low density lipoprotein, high density lipoprotein, triglycerides and lipoprotein(a). Recently two proteins, angiopoietin-like protein 3 and 4, have emerged from genetic studies as being factors that significantly modulate plasma triglyceride levels and coronary artery disease. The exact function and mechanism of action of both proteins remains to be elucidated, however, mutations in these proteins results in up to 34% reduction in coronary artery disease and inhibition of function results in reduced plasma triglyceride levels.

NHLRC2 variants identified in patients with fibrosis, neurodegeneration, and cerebral angiomatosis (FINCA): characterisation of a novel cerebropulmonary disease.

A novel multi-organ disease that is fatal in early childhood was identified in three patients from two non-consanguineous families. These children were born asymptomatic but at the age of 2 months they manifested progressive multi-organ symptoms resembling no previously known disease. The main clinical features included progressive cerebropulmonary symptoms, malabsorption, progressive growth failure, recurrent infections, chronic haemolytic anaemia and transient liver dysfunction.

Purification, crystallization and preliminary X-ray crystallographic analysis of the lumenal domain of the ER-vesicle protein Erv41p from Saccharomyces cerevisiae.

The membrane protein Erv41p is a major component of COPII-coated vesicles and is thought to play a role in the early secretory pathway in eukaryotic cells. In this study, the full lumenal domain of Erv41p from Saccharomyces cerevisiae (ScErv41p_LD) was recombinantly expressed in Sf9 insect cells and purified by nickel-affinity, ion-exchange and size-exclusion chromatography. ScErv41p_LD crystals were obtained using the sitting-drop vapour-diffusion method and native X-ray diffraction data were collected to 2.

The crystal structure of the lumenal domain of Erv41p, a protein involved in transport between the endoplasmic reticulum and Golgi apparatus.

Erv41p is a conserved integral membrane protein that is known to play a role in transport between the endoplasmic reticulum and Golgi apparatus, part of the early secretory pathway of eukaryotes. However, the exact function of the protein is not known, and it shares very low sequence identity with proteins of known structure or function. Here we present the structure of the full lumenal domain of Erv41p from Saccharomyces cerevisiae, determined by X-ray crystallography to a resolution of 2.

The crystal structure of the human co-chaperone P58(IPK).

P58(IPK) is one of the endoplasmic reticulum- (ER-) localised DnaJ (ERdj) proteins which interact with the chaperone BiP, the mammalian ER ortholog of Hsp70, and are thought to contribute to the specificity and regulation of its diverse functions. P58(IPK), expression of which is upregulated in response to ER stress, has been suggested to act as a co-chaperone, binding un- or misfolded proteins and delivering them to BiP. In order to give further insights into the functions of P58(IPK), and the regulation of BiP by ERdj proteins, we have determined the crystal structure of human P58(IPK) to 3.

Enzymatic defects underlying hereditary glutamate cysteine ligase deficiency are mitigated by association of the catalytic and regulatory subunits.

Glutamate cysteine ligase (GCL) deficiency is a rare autosomal recessive trait that compromises production of glutathione, a critical redox buffer and enzymatic cofactor. Patients have markedly reduced levels of erythrocyte glutathione, leading to hemolytic anemia and, in some cases, impaired neurological function. Human glutamate cysteine ligase is a heterodimer comprised of a catalytic subunit (GCLC) and a regulatory subunit (GCLM), which catalyzes the initial rate-limiting step in glutathione production.

Structural basis for feedback and pharmacological inhibition of Saccharomyces cerevisiae glutamate cysteine ligase.

Structural characterization of glutamate cysteine ligase (GCL), the enzyme that catalyzes the initial, rate-limiting step in glutathione biosynthesis, has revealed many of the molecular details of substrate recognition. To further delineate the mechanistic details of this critical enzyme, we have determined the structures of two inhibited forms of Saccharomyces cerevisiae GCL (ScGCL), which shares significant sequence identity with the human enzyme. In vivo, GCL activity is feedback regulated by glutathione.

Mechanistic details of glutathione biosynthesis revealed by crystal structures of Saccharomyces cerevisiae glutamate cysteine ligase.

Glutathione is a thiol-disulfide exchange peptide critical for buffering oxidative or chemical stress, and an essential cofactor in several biosynthesis and detoxification pathways. The rate-limiting step in its de novo biosynthesis is catalyzed by glutamate cysteine ligase, a broadly expressed enzyme for which limited structural information is available in higher eukaryotic species. Structural data are critical to the understanding of clinical glutathione deficiency, as well as rational design of enzyme modulators that could impact human disease progression.

Crystal structure of acivicin-inhibited gamma-glutamyltranspeptidase reveals critical roles for its C-terminus in autoprocessing and catalysis.

Helicobacter pylori gamma-glutamyltranspeptidase (HpGT) is a general gamma-glutamyl hydrolase and a demonstrated virulence factor. The enzyme confers a growth advantage to the bacterium, providing essential amino acid precursors by initiating the degradation of extracellular glutathione and glutamine. HpGT is a member of the N-terminal nucleophile (Ntn) hydrolase superfamily and undergoes autoprocessing to generate the active form of the enzyme.

Crystal structures of oxidized and reduced mitochondrial thioredoxin reductase provide molecular details of the reaction mechanism.

Thioredoxin reductase (TrxR) is an essential enzyme required for the efficient maintenance of the cellular redox homeostasis, particularly in cancer cells that are sensitive to reactive oxygen species. In mammals, distinct isozymes function in the cytosol and mitochondria. Through an intricate mechanism, these enzymes transfer reducing equivalents from NADPH to bound FAD and subsequently to an active-site disulfide.