Glubodies: randomized libraries of glutathione transferase enzymes

AbstractBackground: The immunoglobulin framework has been mutagenized to engineer recombinant libraries of proteins as potential diagnostics and novel catalysts, although the often shallow binding cleft may limit the utility of this framework for binding diverse small organic molecules. By contrast, the glutathione S-transferase (GST) family of enzymes contains a deep binding cleft, which has evolved to accommodate a broad range of hydrophobic xenobiotics. We set out to determine whether GST molecules with novel ligand-binding characteristics could be produced by random mutagenesis of segments of the binding cleft.Results: We have identified two ligand-recognition segments (LRSs) in human GST P1, which are near the active site in the folded protein, but have characteristics indicating that the integrity of their sequence is not essential for the overall structure or activity of the protein. Libraries of GST P1-derived proteins were produced by substituting randomized sequences for an LRS or inserting random sequences into an LRS. The recombinant proteins in the libraries, collectively designated as ‘glubodies,’ generally retain enzymatic activity but differ markedly both from each other and from the parent enzyme in sensitivity to inhibition by diverse small organic compounds. In some instances, a glubody is inhibited by completely novel structures.Conclusions: We have shown that a non-antibody framework can be used to create large libraries of proteins with a wide range of binding specificities for small organic molecules. The glubodies provide a rich source of data for correlating the structural and functional features of proteins relevant to ligand binding. The criteria applied for identifying an LRS in GST P1 are generally applicable to other protein frameworks

A Meta-Analysis of the Existing Knowledge of Immunoreactivity against Hepatitis C Virus (HCV)

Approximately 3% of the world population is infected by HCV, which represents a major global health challenge. Almost 400 different scientific reports present immunological data related to T cell and antibody epitopes derived from HCV literature. Analysis of all HCV-related epitope hosted in the Immune Epitope Database (IEDB), a repository of freely accessible immune epitope data, revealed more than 1500 and 1900 distinct T cell and antibody epitopes, respectively. The inventory of all data revealed specific trends in terms of the host and the HCV genotypes from which sequences were derived. Upon further analysis we found that this large number of epitopes reflects overlapping structures, and homologous sequences derived from different HCV isolates. To access and visualize this information we developed a novel strategy that assembles large sets of epitope data, maps them onto reference genomes and displays the frequency of positive responses. Compilation of the HCV immune reactivity from hundreds of different studies, revealed a complex and thorough picture of HCV immune epitope data to date. The results pinpoint areas of more intense reactivity or research activities at the level of antibody, CD4 and CD8 responses for each of the individual HCV proteins. In general, the areas targeted by the different effector immune functions were distinct and antibody reactivity was positively correlated with hydrophilicity, while T cell reactivity correlated with hydrophobicity. At the sequence level, epitopes frequently recognized by both T cell and B cell correlated with low variability, and our analysis thus highlighted areas of potential interest for practical applications. The human reactivity was further analyzed to pinpoint differential patterns of reactivity associated with acute versus chronic infection, to reveal the apparent impact of glycosylation on T cell, but not antibody responses, and to highlight a paucity of studies involved antibody epitopes associated with virus neutralization

Metal-Free ALS Variants of Dimeric Human Cu,Zn-Superoxide Dismutase Have Enhanced Populations of Monomeric Species

Amino acid replacements at dozens of positions in the dimeric protein human, Cu,Zn superoxide dismutase (SOD1) can cause amyotrophic lateral sclerosis (ALS). Although it has long been hypothesized that these mutations might enhance the populations of marginally-stable aggregation-prone species responsible for cellular toxicity, there has been little quantitative evidence to support this notion. Perturbations of the folding free energy landscapes of metal-free versions of five ALS-inducing variants, A4V, L38V, G93A, L106V and S134N SOD1, were determined with a global analysis of kinetic and thermodynamic folding data for dimeric and stable monomeric versions of these variants. Utilizing this global analysis approach, the perturbations on the global stability in response to mutation can be partitioned between the monomer folding and association steps, and the effects of mutation on the populations of the folded and unfolded monomeric states can be determined. The 2- to 10-fold increase in the population of the folded monomeric state for A4V, L38V and L106V and the 80- to 480-fold increase in the population of the unfolded monomeric states for all but S134N would dramatically increase their propensity for aggregation through high-order nucleation reactions. The wild-type-like populations of these states for the metal-binding region S134N variant suggest that even wild-type SOD1 may also be prone to aggregation in the absence of metals

Cupricyclins, Novel Redox-Active Metallopeptides Based on Conotoxins Scaffold

Highly stable natural scaffolds which tolerate multiple amino acid substitutions represent the ideal starting point for the application of rational redesign strategies to develop new catalysts of potential biomedical and biotechnological interest. The knottins family of disulphide-constrained peptides display the desired characteristics, being highly stable and characterized by hypervariability of the inter-cysteine loops. The potential of knottins as scaffolds for the design of novel copper-based biocatalysts has been tested by engineering a metal binding site on two different variants of an ω-conotoxin, a neurotoxic peptide belonging to the knottins family. The binding site has been designed by computational modelling and the redesigned peptides have been synthesized and characterized by optical, fluorescence, electron spin resonance and nuclear magnetic resonance spectroscopy. The novel peptides, named Cupricyclin-1 and -2, bind one Cu2+ ion per molecule with nanomolar affinity. Cupricyclins display redox activity and catalyze the dismutation of superoxide anions with an activity comparable to that of non-peptidic superoxide dismutase mimics. We thus propose knottins as a novel scaffold for the design of catalytically-active mini metalloproteins

Clamp loader ATPases and the evolution of DNA replication machinery

Clamp loaders are pentameric ATPases of the AAA+ family that operate to ensure processive DNA replication. They do so by loading onto DNA the ring-shaped sliding clamps that tether the polymerase to the DNA. Structural and biochemical analysis of clamp loaders has shown how, despite differences in composition across different branches of life, all clamp loaders undergo the same concerted conformational transformations, which generate a binding surface for the open clamp and an internal spiral chamber into which the DNA at the replication fork can slide, triggering ATP hydrolysis, release of the clamp loader, and closure of the clamp round the DNA. We review here the current understanding of the clamp loader mechanism and discuss the implications of the differences between clamp loaders from the different branches of life

FoXS, FoXSDock and MultiFoXS: Single-state and multi-state structural modeling of proteins and their complexes based on SAXS profiles

Small Angle X-ray Scattering (SAXS) is an increasingly common and useful technique for structural characterization of molecules in solution. A SAXS experiment determines the scattering intensity of a molecule as a function of spatial frequency, termed SAXS profile. Here, we describe three web servers for modeling atomic structures based on SAXS profiles. FoXS (Fast X-Ray Scattering) rapidly computes a SAXS profile of a given atomistic model and fits it to an experimental profile. FoXSDock docks two rigid protein structures based on a SAXS profile of their complex. MultiFoXS computes a population-weighted ensemble starting from a single input structure by fitting to a SAXS profile of the protein in solution. We describe the interfaces and capabilities of the servers (salilab.org/foxs), followed by demonstrating their application on Interleukin-33 (IL-33) and its primary receptor ST2