IStructures of each of the individual domains of HIV-1 integrase have been obtained by X-ray crystallography and/or NMR. Structures of pairs of domains have also been determined. However, the organization of these domains in the functional synaptic complex with DNA is unknown. Having established methodologies to assemble synaptic complexes in vitro with purified integrase and HIV-1 DNA substrate, we are attempting to solve this problem. High-resolution structural information on the synaptic complex would be invaluable because the most promising class of HIV-1 integrase inhibitors recognizes the complex rather than free integrase protein. The first of these dikto-acid-derived compounds is in advanced clinical trials and structures of integrase in complex with DNA are required to understand the molecular mechanism of inhibition. At the same time as establishing the groundwork high-resolution studies we are concurrently pursuing lower resolution approaches. In collaboration with Svetlana Kotova and Emelios Dimidriadis we have visualized HIV-1 synaptic complexes by atomic force microscopy. At physiological ionic strength the complexes are highly aggregated. However, when bound to mica at high ionic strength the predominant species are pairs of viral DNAs bridged at their ends by a tetramer of integrase. We plan to collaborate with Alasdair Steven to obtain more detail of the organization of integrase within the synaptic complex by cry-electron microscopy and image reconstruction. ? We are preparing the groundwork higher resolution structural studies in collaboration with Wei Yang. At this stage a number of major obstacles must be overcome before such studies become feasible. Unlike related synaptic complexes of transposases with DNA, several hundred base pairs of DNA are required to maintain stability of the complex after assembly. We suspect that additional protomers of loosely bound integrase contribute to the stability of the complex. We are attempting to find conditions abrogate this requirement. A further obstacle that needs to be overcome is the tendency of integrase/DNA synaptic complex to aggregate. A second approach we are following is attempts to exploit structurally modified DNAs to enhance the stability of synaptic complexes.
| Craigie, Robert (2010) Structural biology: When four become one. Nature 464:167-8 |
| Li, Min; Craigie, Robert (2009) Nucleoprotein complex intermediates in HIV-1 integration. Methods 47:237-42 |
