E2 proteins

Examples of the application of SAR-by-NMR include the design of stromelysin and human papillomavirus E2 protein inhibitors [7, 8]. 

Hajduk PJ, Dinges J, Miknis GF et al (1997) NMR-based discovery of lead inhibitors that block DNA binding of the human papillomavirus E2 protein. J Med Chem 40 3144-3150... 

Modified E2 protein, now containing the IgG binding domain of Protein A... 

Budding is probably initiated by the viral nucleocapsid binding to a cluster of spike proteins at the cell surface. The binding must be mediated by the cytoplasmic domain of the E2 protein attaching to a capsid... 

While there is usually only one El enzyme, many species of E2 proteins and multiple families of E3 enzymes or E3 multiprotein complexes exist. Selection of substrates for ubiquitin-ligation occurs mainly by specific E3 enzymes which target substrate proteins that contain specific recognition signals (fig. 2.15B). E3 enzymes also can bind indirectly to the substrate, via an adaptor protein. 

In other families of E3 enzymes, no intermediate E3-ubiquitin linkage can be demonstrated. In this case ubiquitin is transferred directly from E2 to the substrate protein. The E3 enzymes are nevertheless required for ubiquitinylation since the E3 enzymes are responsible for substrate selection and are foimd in tight complexes with the cognate E2 proteins. 

CONTENTS Preface, C. Allen Bush. Thermodynamic Solvent Isotope Effects and Molecular Hydrophobicity, Terrence G. Oas and Eric J. Toone. Membrane Interactions of Hemolytic and Antibacterial Peptides, Karl Lohner and Richard M. Epand. Spin-Labeled Metabolite Analogs as Probes of Enzyme Structure, Chakravarthy Narasimhan and Henry M. Miziorko. Current Perspectives on the Mechanism of Catalysis by the Enzyme Enolase, John M. Brewer and Lukasz Leb-ioda. Protein-DNA Interactions The Papillomavirus E2 Proteins as a Model System, Rashmi S. Hedge. NMR-Based Structure Determination for Unlabeled RNA and DNA, Philip N. Borer, Lucia Pappalardo, Deborah J. Kenwood, and Istvan Pelczer. Evolution of Mononuclear to Binuclear CuA An EPR Study, William E. Antholine. Index. 

Perham and coworkers70 overexpressed the lipoyl domain of the E2 protein from E. coli. They reported that the ThDP-El-bound enamine had a ca 10,000 larger kcJKu for the reductive acetylation of lipoamide covalently attached to this lipoyl domain than for the reductive acetylation of lipoamide perse. Apparently, there is recognition between certain protein segments of El and the lipoyl domain of E2 that enhances the interaction and facilitates the reaction. 

Interpretation of the membrane proteins in an envelope virus can be assisted by the identification of transmembrane a helices. Membrane proteins represent a special class of proteins because of the predominant presence of transmembrane helices connected by extramembrane loops and domains. For example, even at 10.5-A resolution, a pair of transmembrane helices could be identified in the Semliki Forest virus El and E2 proteins (Eig. 10 see Color Insert). 

Although most eukaryotes have only one or a small number of distinct El enzymes, all eukaryotes have many distinct E2 and E3 enzymes. Moreover, there appears to be only a single family of evolutionarily related E2 proteins but many distinct families of E3 proteins. Although the E3 component provides most of the substrate specificity for ubiquitination, the multiple combinations of the E2-E3 complex allow for more finely tuned substrate discrimination. 

To date, the most successful antiviral targets have been directed against viral-specific enzymes. Therefore, the ATPase and helicase activities of the El protein are attractive targets. Papillomavirus DNA replication may also be inhibited by compounds that interfere with the ability of El to bind DNA or to interact with the E2 protein. This chapter will describe a method to assay for specific El DNA binding and cooperative origin binding with the E2 protein and a method to transiently assay papillomavirus DNA replication. The methods described are for bovine papillomavirus type 1 (BPV-1), which has been the molecular prototype of the papillomaviruses. However, the methods can easily be adapted to assay for human papillomavirus replication. 

In this assay, El protein (and E2 protein) is incubated with a mixture of 32P-labeled DNA fragments, one of which contains the viral replication origin... 

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