Structure of a Thyrotropin Receptor Monoclonal Antibody Variable Region Provides Insight into Potential Mechanisms for its Inverse Agonist Activity.

Background: The high constitutive, or ligand-independent, activity of the thyrotropin receptor (TSHR) is of clinical importance in some thyroid conditions, particularly well-differentiated thyroid carcinoma remnants following incomplete ablative therapy (surgery and radioiodine). Under these conditions, even total suppression of TSH by thyroid hormone administration does not fully reduce TSHR activity, a driver of thyrocyte growth.

Methods: CS-17 is a murine monoclonal antibody that has inverse agonist activity in that it suppresses TSHR constitutive activity.

Reinvestigation of the Branimycin Stereochemistry at Position 17-C.

A conformational study of branimycin was performed using single-crystal X-ray crystallography to characterize the solid-state form, while a combination of NMR spectroscopy and molecular modeling was employed to gain information about the solution structure. Comparison of the crystal structure with its solution counterpart showed no significant differences in conformation, confirming the relative rigidity of the tricyclic system. However, these experiments revealed that the formerly proposed stereochemistry of branimycin at 17-C should be revised.

Visualization of a radical B12 enzyme with its G-protein chaperone.

G-protein metallochaperones ensure fidelity during cofactor assembly for a variety of metalloproteins, including adenosylcobalamin (AdoCbl)-dependent methylmalonyl-CoA mutase and hydrogenase, and thus have both medical and biofuel development applications. Here, we present crystal structures of IcmF, a natural fusion protein of AdoCbl-dependent isobutyryl-CoA mutase and its corresponding G-protein chaperone, which reveal the molecular architecture of a G-protein metallochaperone in complex with its target protein. These structures show that conserved G-protein elements become ordered upon target protein association, creating the molecular pathways that both sense and report on the cofactor loading state.

Allosteric modulation of Ras and the PI3K/AKT/mTOR pathway: emerging therapeutic opportunities.

GTPases and kinases are two predominant signaling modules that regulate cell fate. Dysregulation of Ras, a GTPase, and the three eponymous kinases that form key nodes of the associated phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K)/AKT/mTOR pathway have been implicated in many cancers, including pancreatic cancer, a disease noted for its current lack of effective therapeutics. The K-Ras isoform of Ras is mutated in over 90% of pancreatic ductal adenocarcinomas (PDAC) and there is growing evidence linking aberrant PI3K/AKT/mTOR pathway activity to PDAC.

Crystal structure of a TSH receptor monoclonal antibody: insight into Graves' disease pathogenesis.

The TSH receptor (TSHR) A-subunit is more effective than the holoreceptor in inducing thyroid-stimulating antibodies (TSAb) that cause Graves' disease. A puzzling phenomenon is that 2 recombinant, eukaryotic forms of A-subunits (residues 22-289), termed active and inactive, are recognized mutually exclusively by pathogenic TSAb and mouse monoclonal antibody 3BD10, respectively. Understanding the structural difference between these TSHR A-subunit forms could provide insight into Graves' disease pathogenesis.

A comparative study of fragment screening methods on the p38α kinase: new methods, new insights.

The stress-activated kinase p38α was used to evaluate a fragment-based drug discovery approach using the BioFocus fragment library. Compounds were screened by surface plasmon resonance (SPR) on a Biacore(™) T100 against p38α and two selectivity targets. A sub-set of our library was the focus of detailed follow-up analyses that included hit confirmation, affinity determination on 24 confirmed, selective hits and competition assays of these hits with respect to a known ATP binding site inhibitor.

Two distinct proton donors at the active site of Escherichia coli 2,4-dienoyl-CoA reductase are responsible for the formation of different products.

NADPH-dependent 2,4-dienoyl-CoA reductase (DCR) is one of the auxiliary enzymes required for the beta-oxidation of unsaturated fatty acids. Mutants of Escherichia coli DCR were generated by site-directed mutagenesis to explore the molecular mechanism of this enzyme. The Tyr166Phe mutant, which was expected to be inactive due to the loss of its putative proton donor residue, exhibited 27% of the wild-type activity.

Crystal structure and mutagenesis of the metallochaperone MeaB: insight into the causes of methylmalonic aciduria.

MeaB is an auxiliary protein that plays a crucial role in the protection and assembly of the B(12)-dependent enzyme methylmalonyl-CoA mutase. Impairments in the human homologue of MeaB, MMAA, lead to methylmalonic aciduria, an inborn error of metabolism. To explore the role of this metallochaperone, its structure was solved in the nucleotide-free form, as well as in the presence of product, GDP.

Domain swapping in the low-similarity isomerase/hydratase superfamily: the crystal structure of rat mitochondrial Delta3, Delta2-enoyl-CoA isomerase.

Two monofunctional Delta(3), Delta(2)-enoyl-CoA isomerases, one in mitochondria (mECI) and the other in both mitochondria and peroxisomes (pECI), belong to the low-similarity isomerase/hydratase superfamily. Both enzymes catalyze the movement of a double bond from C3 to C2 of an unsaturated acyl-CoA substrate for re-entry into the beta-oxidation pathway. Mutagenesis has shown that Glu165 of rat mECI is involved in catalysis; however, the putative catalytic residue in yeast pECI, Glu158, is not conserved in mECI.

The crystal structure and reaction mechanism of Escherichia coli 2,4-dienoyl-CoA reductase.

Escherichia coli 2,4-dienoyl-CoA reductase is an iron-sulfur flavoenzyme required for the metabolism of unsaturated fatty acids with double bonds at even carbon positions. The enzyme contains FMN, FAD, and a 4Fe-4S cluster and exhibits sequence homology to another iron-sulfur flavoprotein, trimethylamine dehydrogenase. It also requires NADPH as an electron source, resulting in reduction of the C4-C5 double bond of the acyl chain of the CoA thioester substrate.