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Conformational changes induced by nucleotide binding in Cdc6/ORC from Aeropyrum pernix
Martin R Singleton1, Renaud Morales, Ian Grainge
1Cancer Research UK Clare Hall Laboratories, The London Research Institute, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3LD, UK.
Journal of Molecular Biology
|October 7, 2004
Summary
Archaea possess ORC/Cdc6-homologous proteins, including ORC2. Crystal structures reveal ATP binding stabilizes ORC2 conformation, and its C-terminal domain binds DNA, with a novel winged helix domain interaction model proposed.
Area of Science:
- Biochemistry
- Structural Biology
- Archaea Biology
Background:
- Archaea share proteins homologous to eukaryotic ORC/Cdc6, suggesting conserved DNA replication machinery.
- Two subfamilies, ORC1 and ORC2, are proposed based on sequence analysis.
Purpose of the Study:
- To elucidate the structural and functional characteristics of archaeal ORC2 protein.
- To investigate the role of ATP binding in ORC2 conformation and DNA binding.
Main Methods:
- X-ray crystallography was used to determine the structures of Aeropyrum pernix ORC2 in complex with ADP and ADPNP.
- DNA binding assays were performed to assess the interaction of ORC2 with DNA.
- Bioinformatic analysis and structural modeling were employed to propose a DNA interaction model.
Main Results:
- Crystal structures revealed significant conformational flexibility in ORC2 with ADP, while ATP binding (ADPNP) stabilized a single conformation.
- The ORC2 protein demonstrated DNA binding activity, localized to its C-terminal domain.
- A novel model for the interaction of the winged helix (WH) domain of ORC2 with DNA was proposed, differing from previous models.
Conclusions:
- ATP binding plays a crucial role in stabilizing the conformation of archaeal ORC2.
- The C-terminal domain, particularly the WH domain, is critical for DNA binding in ORC2.
- This study provides new insights into the structure-function relationship of archaeal replication initiation proteins.