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Published on: July 23, 2010
Design and characterization of an enhanced repressor of human papillomavirus E2 protein
Kakoli Bose1, Gretchen Meinke, Andrew Bohm
1Department of Biochemistry, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111, USA.
Abstract:
Papillomaviruses are causative agents of cervical and anogenital cancers. The viral E2 protein mediates viral DNA replication and transactivation of viral oncogenes and thus represents a specific target for therapeutic intervention. Short forms of E2, E2R, contain only the C-terminal dimerization domain, and repress the normal function of E2 due to formation of an inactive heterodimer. Using structure-guided design, we replaced conserved residues at the dimer interface to design a heterodimer with increased stability. One E2R mutant in which histidine was replaced by a glutamate residue showed preferential heterodimer formation in vitro, as well as an increase in plasticity at the interface, as a result of histidine-glutamate pair formation, as observed spectroscopically and in the crystal structure, determined to 2.2-Å resolution. In addition, the enhanced E2R showed greater repression of transcription from E2-responsive reporter plasmids in mammalian cell culture. Recent advances in protein delivery into the cell raise the possibility of using exogenously added proteins as therapeutic agents. More generally, this approach may be used to target the subunit interfaces of any multisubunit protein having a similar mechanism of action.
Insights
Researchers engineered a more stable short E2 protein (E2R) from human papillomaviruses. This enhanced E2R effectively represses viral gene activity, offering a potential therapeutic strategy against HPV-associated cancers.
Area of Science:
- Virology
- Molecular Biology
- Structural Biology
Background:
- Human papillomaviruses (HPVs) cause cervical and anogenital cancers.
- The viral E2 protein is crucial for HPV DNA replication and oncogene transactivation.
- Short E2 proteins (E2R) inhibit E2 function by forming inactive heterodimers.
Purpose of the Study:
- To design a more stable E2R mutant with enhanced inhibitory activity.
- To investigate the structural and functional consequences of mutations at the E2R dimer interface.
Main Methods:
- Structure-guided protein design.
- Site-directed mutagenesis (Histidine to Glutamate substitution).
- In vitro heterodimerization assays.
- Spectroscopic analysis and X-ray crystallography (2.2 Å resolution).
- Reporter gene assays in mammalian cell culture.
Main Results:
- A novel E2R mutant (H-E) demonstrated preferential in vitro heterodimer formation.
- The H-E mutation increased interface plasticity via histidine-glutamate pair formation.
- Structural analysis confirmed increased plasticity and stable heterodimer formation.
- The enhanced E2R exhibited significantly greater repression of E2-responsive transcription.
Conclusions:
- Engineered E2R mutants can form more stable heterodimers and enhance transcriptional repression.
- This structure-guided approach offers a promising strategy for developing therapeutic agents targeting viral protein-protein interactions.
- Exogenous protein delivery of enhanced E2R could be a viable therapeutic intervention for HPV-related cancers.
