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To assess the state of the art in antibody 3D modeling, 11 unpublished high-resolution x-ray Fab crystal structures from diverse species and covering a wide range of antigen-binding site conformations were used as a benchmark to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.
Polysialic acid is a developmentally regulated, anti-adhesive glycan that is added to the neural cell adhesion molecule, NCAM. Polysialylated NCAM is critical for brain development and plays roles in synaptic plasticity, axon guidance, and cell migration. The first fibronectin type III repeat of NCAM, FN1, is necessary for the polysialylation of N-glycans on the adjacent immunoglobulin domain. This repeat cannot be replaced by other fibronectin type III repeats. We solved the crystal structure of human NCAM FN1 and found that, in addition to a unique acidic surface patch, it possesses a novel ␣-helix that links strands 4 and 5 of its -sandwich structure. Replacement of the ␣-helix did not eliminate polysialyltransferase recognition, but shifted the addition of polysialic acid from the N-glycans modifying the adjacent immunoglobulin domain to O-glycans modifying FN1. Other experiments demonstrated that replacement of residues in the acidic surface patch alter the polysialylation of both N-and O-glycans in the same way, while the ␣-helix is only required for the polysialylation of N-glycans. Our data are consistent with a model in which the FN1 ␣-helix is involved in an Ig5-FN1 interaction that is critical for the correct positioning of Ig5 N-glycans for polysialylation.Polysialic acid is a developmentally regulated, anti-adhesive glycan that is found predominantly on the neural cell adhesion molecule, NCAM.2 Long, negatively charged polysialic acid chains decrease NCAM-dependent and -independent adhesion processes (1), thereby facilitating axon guidance and pathfinding, neurite outgrowth, synaptic plasticity, and general cell migration in the central nervous system (2-5). Polysialic acid levels are high in the embryo and neonate and decrease in the adult, except in areas of the brain such as the hippocampus and olfactory bulb that require on-going cell migration and functional plasticity (2, 3). In addition, highly polysialylated NCAM is re-expressed on the surface of some cancer cells where it is believed to promote cancer cell growth and invasion (6 -11).The polysialyltransferases ST8Sia II (STX) and ST8Sia IV (PST) are responsible for NCAM polysialylation, and their expression levels mirror the abundance of polysialic acid during different developmental stages (12, 13). Recent studies using polysialyltransferase and NCAM knock-out animals highlight the importance of polysialic acid during brain development (14 -16). While deletion of NCAM results in relatively mild morphological and behavioral effects in mice (17), the simultaneous deletion of the two polysialyltransferases results in severe alterations in brain development and premature death (14, 15). Strikingly, animals lacking NCAM and the polysialyltransferases appear normal, suggesting that the presence of polysialic acid is critical for down-regulating NCAM-dependent adhesion at specific times during brain development (15).Polysialic acid is unique among glycan modifications in that it is added to a small subset of proteins including the ␣-subunit of t...
We describe the methodology and results from our participation in the second Antibody Modeling Assessment experiment. During the experiment we predicted the structure of eleven unpublished antibody Fv fragments. Our prediction methods centered on template-based modeling; potential templates were selected from an antibody database based on their sequence similarity to the target in the framework regions. Depending on the quality of the templates, we constructed models of the antibody framework regions either using a single, chimeric or multiple template approach. The hypervariable loop regions in the initial models were rebuilt by grafting the corresponding regions from suitable templates onto the model. For the H3 loop region, we further refined models using ab initio methods. The final models were subjected to constrained energy minimization to resolve severe local structural problems. The analysis of the models submitted show that Accelrys tools allow for the construction of quite accurate models for the framework and the canonical CDR regions, with RMSDs to the X-ray structure on average below 1 Å for most of these regions. The results show that accurate prediction of the H3 hypervariable loops remains a challenge. Furthermore, model quality assessment of the submitted models show that the models are of quite high quality, with local geometry assessment scores similar to that of the target X-ray structures. Proteins 2014; 82:1583–1598. © 2014 The Authors. Proteins published by Wiley Periodicals, Inc.
Although mimics of human tumor antigens are effective immunogens to overcome host unresponsiveness to the nominal antigen, the structural basis of this mimicry remains poorly defined. Therefore, in this study we have characterized the structural basis of the human high molecular weight-melanoma-associated antigen (HMW-MAA) mimicry by the mouse anti-idiotypic (anti-id) monoclonal antibody (mAb) MK2-23. Using x-ray crystallography, we have characterized the three-dimensional structure of the anti-id mAb MK2-23 Fab and shown that its heavy chain complementarity-determining region (CDR3) (H3) and its light chain CDR1 (L1) are closely associated. These moieties are the source of HMW-MAA mimicry, since they display partial amino acid sequence homology along with a similar structural fold with the HMW-MAA core protein. Furthermore, a 15-residue peptide comprising the H3 loop of anti-id mAb MK2-23 demonstrates HMW-MAA-like in vitro and in vivo reactivity. This peptide in conjunction with the structural data will facilitate the characterization of the effect of the degree of antigen mimicry on the induction of a self-antigen-specific immune response by a mimic.Antigen mimicry has been implicated in the pathogenesis of several pathophysiological conditions such as viral immune evasion (1-3) and autoimmunity (4 -7). In these situations, the structurally similar foreign moieties either interfere with the normal biological functions mediated by their nominal counterparts or elicit unwanted immune responses against the host antigens. The consequences sometimes can be quite detrimental. Nevertheless, in the case of autoimmune responses mediated by molecular mimicry, an intriguing finding is the restriction of the damage to an organ or a tissue. This pattern is distinct from the general immune hyper-responsiveness caused by suppression of the regulatory arm of the immune system, such as cytotoxic T lymphocyte antigen-4 (CTLA-4) blockade (8, 9).The potential ability of molecular mimicry to target a specific host antigen has provided the rationale for its use to elicit and/or enhance an immune response against human tumor antigens that are mostly non-mutated self-antigens and therefore poorly or non-immunogenic in patients (for review, see Refs. 10 and 11). Various types of tumor antigen mimics have been identified (for review, see Ref. 11). Among them the most extensively utilized is represented by antiidiotypic (anti-id) 4 monoclonal antibodies (mAb), which have been developed in several human tumor antigen systems (for review, see Refs. 10 and 11). Anti-id mAb markedly differ in their immunogenicity as measured by their ability to elicit a humoral immune response to the corresponding self-tumor antigen. The cause of this variability is not known. The lack of this information reflects, at least in part, the limited knowledge about the structural basis of antigen mimicry by anti-id antibodies and about the relationship between the extent of antigen mimicry and ability of a mimic to overcome unresponsiveness to a self-tumor antig...
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