HIV gp41 is a heavily glycosylated transmembrane protein. The difficulty in producing full length recombinant gp41 requires an incremental approach for structural determination. The ectodomain region, located on the outer surface of the viral membrane directly mediates membrane fusion events via an N-terminal fusion domain. In previous work both the NMR and X-ray structures of the gp41 ectodomain were determined. Both methods indicated a rod-like trimer comprising three parallel N-terminal alpha-helices assembled as a coiled-coil in the center with three antiparallel C-terminal alpha-helices packed on the outside with highly flexible loops connecting the inner and outer helices (6-helical bundle: 6HB). Previously, we reported the structure of fusion domain which consists of an N-terminal helix with a C-terminal linker region. To understand in more detail the interaction between the fusion domain, and the rest of the ectodomain, constructs which included the following four domains: N-terminal fusion domain - 6 HB - linker region - transmembrane region. This complex protein which contains two membrane associating domains (fusion and transmembrane) was expressed in bacteria and purified as a protein-detergent complex. Using analytical ultracentrifugation and other biophysical methods, the protein complex was shown to be physical homogenous and readily formed crystals. Currently, the diffraction resolution of the crystals is not high enough for detailed structure determination -this is a common occurrence with protein-detergent systems. To improve the quality of the crystals we are making various mutational changes to the protein sequence in order to improve the solubility and stability of the protein. Further, we are screening an extensive range of detergent - phospholipid combinations to determine which best maintain solubility of the membrane protein. Apart from the structural work, we have used the protein for immunizations and have initiated functional studies of protein reconstituted into lipid bilayers. It is hoped that the structural, biochemical and immunological studies will provide some insight to how the fusion and the transmembrane domain interact. This information is directly related to development of a better understanding of the mechanism of viral membrane fusion and may provide useful therapeutic targets based on structural information.
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| Desmezieres, Emmanuel; Gupta, Nidhi; Vassell, Russell et al. (2005) Human immunodeficiency virus (HIV) gp41 escape mutants: cross-resistance to peptide inhibitors of HIV fusion and altered receptor activation of gp120. J Virol 79:4774-81 |
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