Estrone sulfatase (ES; 562 amino acids), one of the key enzymes responsible for maintaining high levels of estrogens in breast tumor cells, is associated with the membrane of the endoplasmic reticulum (ER). The structure of ES, purified from the microsomal fraction of human placentas, has been determined at 2.60-Å resolution by x-ray crystallography. This structure shows a domain consisting of two antiparallel ␣-helices that protrude from the roughly spherical molecule, thereby giving the molecule a "mushroom-like" shape. These highly hydrophobic helices, each about 40 Å long, are capable of traversing the membrane, thus presumably anchoring the functional domain on the membrane surface facing the ER lumen. The location of the transmembrane domain is such that the opening to the active site, buried deep in a cavity of the "gill" of the "mushroom," rests near the membrane surface, thereby suggesting a role of the lipid bilayer in catalysis. This simple architecture could be a prototype utilized by the ER membrane in dictating the form and the function of ER-resident enzymes.

To assess the state of the art in antibody 3D modeling, 11 unpublished high-resolution x-ray Fab crystal structures from diverse species and covering a wide range of antigen-binding site conformations were used as a benchmark to compare Fv models generated by seven structure prediction methodologies. The participants included: Accerlys Inc, Chemical Computer Group (CCG), Schrodinger, Jeff Gray's lab at John Hopkins University, Macromoltek, Astellas Pharma/Osaka University and Prediction of ImmunoGlobulin Structure (PIGS). The sequences of benchmark structures were submitted to the modelers and PIGS, and a set of models were generated for each structure. We provide here an overview of the organization, participants and main results of this second antibody modeling assessment (AMA-II). Also, we compare the results with the first antibody assessment published in this journal (Almagro et al., 2011;79:3050).

Summary Small molecules inhibiting hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs) are the focus of drug development efforts directed toward the treatment of ischemia and metabolic imbalance. A cell-based reporter produced by fusing HIF-1α oxygen degradable domain (ODD) to luciferase was shown to work as a capture assay monitoring stability of the overexpressed luciferase-labeled HIF PHD substrate under conditions more physiological than in vitro test tubes. High throughput screening identified novel catechol and oxyquinoline pharmacophores with a “branching motif” immediately adjacent to a Fe-binding motif that fits selectively into the HIF PHD active site in in silico models. In accord with their structure-activity relationship in the primary screen, the best “hits” stabilize HIF1α, upregulate known HIF target genes in a human neuronal line, and exert neuroprotective effects in established model of oxidative stress in cortical neurons.

A blinded study to assess the state of the art in three-dimensional structure modeling of the variable region (Fv) of antibodies was conducted. Nine unpublished high-resolution x-ray Fab crystal structures covering a wide range of antigen-binding site conformations were used as benchmark to compare Fv models generated by four structure prediction methodologies. The methodologies included two homology modeling strategies independently developed by CCG (Chemical Computer Group) and Accerlys Inc, and two fully automated antibody modeling servers: PIGS (Prediction of ImmunoGlobulin Structure), based on the canonical structure model, and Rosetta Antibody Modeling, based on homology modeling and Rosetta structure prediction methodology. The benchmark structure sequences were submitted to Accelrys and CCG and a set of models for each of the nine antibody structures were generated. PIGS and Rosetta models were obtained using the default parameters of the servers. In most cases, we found good agreement between the models and x-ray structures. The average rmsd (root mean square deviation) values calculated over the backbone atoms between the models and structures were fairly consistent, around 1.2 Å. Average rmsd values of the framework and hypervariable loops with canonical structures (L1, L2, L3, H1, and H2) were close to 1.0 Å. H3 prediction yielded rmsd values around 3.0 Å for most of the models. Quality assessment of the models and the relative strengths and weaknesses of the methods are discussed. We hope this initiative will serve as a model of scientific partnership and look forward to future antibody modeling assessments.