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 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. The first obstacle is that SSCs aggregate in solution. Atomic force microscopy shows that aggregation occurs through protein-protein interactions between SSCs. This suggests that IN undergoes conformational changes upon SSC assembly. Extensive studies directed at finding solution conditions that do not permit aggregation have not proved fruitful. We are therefore pursuing an alternative strategy of screening large numbers of IN mutants with single amino acid changes to identify the protein surface responsible for this aggregation. A second round of screening will then focus on this region of the protein for identification of mutants that form complexes with a lesser propensity to aggregate. At the same time we have identified IN-solubilising buffer conditions and have charactised IN aggregation, tetramerization
| 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 |
