Abstract
The fusion-active conformation of the envelope protein gp41 of HIV-1 consists of an N-terminal trimeric α-helical coiled-coil domain and three anti-parallel C-terminal helices that fold down the grooves of the coiled-coil to form a six-helix bundle. Disruption of the six-helix bundle is considered to be a key component of an effective non-peptide fusion inhibitor. In the current study, a fluorescence resonance energy transfer (FRET) experiment for the detection of inhibitor binding to the gp41 N-peptide coiled-coil of HIV-1 was performed, utilizing peptide inhibitors derived from the gp41 C-terminal helical region. The FRET acceptor is a 31-residue N-peptide containing a known deep hydrophobic pocket, stabilized into a trimer by ferrous ion ligation. The FRET donor is a 16–18-residue fluorophore-labeled C-peptide, designed to test the specificity of the N-C interaction. Low μM dissociation constants were observed, correlated to the correct sequence and helical propensity of the C-peptides. Competitive inhibition was demonstrated using the assay, allowing for rank ordering of peptide inhibitors according to their affinity in the 1–20 μM range. The assay was conducted by measuring fluorescence intensity in 384-well plates. The rapid detection of inhibitor binding may permit identification of novel drug classes from a library.
References
Caffrey, M., Cai, M., Kaufman, J., Stahl, S.J., Wingfield, P.T., Covell, D.G., Gronenborn, A.M., and Clore, G.M. (1998). Three-dimensional solution structure of the 44-kDa ectodomain of SIV gp41. EMBO J.17, 4572–4584.10.1093/emboj/17.16.4572Search in Google Scholar
Case, M.A. and McLendon, G.L. (2000). A virtual library approach to investigate protein folding and internal packing. J. Am. Chem. Soc.122, 8089–8090.10.1021/ja000722qSearch in Google Scholar
Chan, D.C., Chutkowski, C.T., and Kim, P.S. (1998). Evidence that a prominent cavity in the coiled coil of HIV type 1 gp41 is an attractive drug target. Proc. Natl. Acad. Sci. USA95, 15613–15617.10.1073/pnas.95.26.15613Search in Google Scholar
Chen, Y.-H., Xiao, Y., and Dierich, M.P. (2000). HIV-1 gp41, role in HIV entry and prevention. Immunobiology201, 308–316.10.1016/S0171-2985(00)80085-6Search in Google Scholar
Eckert, D.M., Malashkevich, V.N., Hong, L.H., Carr, P.A., and Kim, P.S. (1999). Inhibiting HIV-1 entry, discovery of D-peptide inhibitors that target the gp41 coiled-coil pocket. Cell99, 103–115.10.1016/S0092-8674(00)80066-5Search in Google Scholar
Gochin, M., Khorosheva, V., and Case, M.A. (2002). Structural characterization of a paramagnetic metal-ion-assembled three-stranded α-helical coiled coil. J. Am. Chem. Soc.124, 11018–11028.10.1021/ja020431cSearch in Google Scholar
Gochin, M., Guy, R.K., and Case, M.A. (2003). A metallopeptide assembly of the HIV-1 gp41 coiled coil is an ideal receptor in fluorescence detection of ligand binding. Angew. Chem. Int. Ed.42, 5325–5328.10.1002/anie.200352006Search in Google Scholar
Jiang, S., Lin, K., Zhang, L., and Debnath, A.K. (1999). A screening assay for antiviral compounds targeted to the HIV-1 gp41 core structure using a conformation-specific monoclonal antibody. J. Virol. Methods80, 85–96.10.1016/S0166-0934(99)00041-5Search in Google Scholar
Kilgore, N.R., Salzwedel, K., Reddick, M., Allaway, G.P., and Wild, C.T. (2003). Direct evidence that C-peptide inhibitors of human immunodeficiency virus type 1 entry bind to the gp41 N-helical domain in receptor-activated viral envelope. J. Virol.77, 7669–7672.10.1128/JVI.77.13.7669-7672.2003Search in Google Scholar PubMed PubMed Central
Lakowitz, J.R. (1999). Principles of Fluorescence Spectroscopy, 2nd Edition (New York/London: Plenum Press), pp. 237–265.Search in Google Scholar
Liu, S., Zhao, Q., and Jiang, S. (2003). Determination of the HIV-1 gp41 fusogenic core conformation modeled by synthetic peptides, applicable for identification of HIV-1 fusion inhibitors. Peptides24, 1303–1313.10.1016/j.peptides.2003.07.013Search in Google Scholar PubMed
Marme, N., Knemeyer, J.-P., Sauer, M., and Wolfrum, J. (2003). Inter- and intramolecular fluorescence quenching of organic dyes by tryptophan. Bioconjug. Chem.14, 1133–1139.10.1021/bc0341324Search in Google Scholar PubMed
Mo, H., Konstantinidis, A.K., Stewart, K.D., Dekhtyar, T., Ng, T., Swift, K., Matayoshi, E.D., Kati, W., Kohlbrenner, W., and Molla, A. (2004). Conserved residues in the coiled-coil pocket of human immunodeficiency virus type 1 gp41 are essential for viral replication and interhelical interaction. Virology329, 319–327.10.1016/j.virol.2004.08.025Search in Google Scholar PubMed
Ogihara, N.L., Weiss, M.S., Degrado, W.F., and Eisenberg, D. (1997). The crystal structure of the designed trimeric coiled coil coil-VaLd, implications for engineering crystals and supramolecular assemblies. Protein Sci.6, 80–88.10.1002/pro.5560060109Search in Google Scholar PubMed PubMed Central
Otaka, A., Nakamura, M., Daisuke, N., Kodama, E., Uchiyama, S., Nakamura, H., Kobayashi, Y., Matsuoka, M., and Fujii, N. (2002). Remodelling of gp41-C34 peptide leads to highly effective inhibitors of the fusion of HIV-1 with target cells. Angew. Chem. Int. Ed.41, 2938–2939.Search in Google Scholar
Root, M. and Steger, H. (2004). HIV-1 gp41 as a target for viral entry inhibition. Curr. Pharm. Des.10, 1805–1825.10.2174/1381612043384448Search in Google Scholar PubMed
Sia, S.K., Carr, P.A., Cochran, A.G., Malashkevich, V.N., and Kim, P.S. (2002). Short constrained peptides that inhibit HIV-1 entry. Proc. Natl. Acad. Sci. USA99, 14664–14669.10.1073/pnas.232566599Search in Google Scholar PubMed PubMed Central
Wild, C.T., Shugars, D.C., Greenwell, T.K., McDanal, C.B., and Matthews, T.J. (1994). Peptides corresponding to a predictive α-helical domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection. Proc. Natl. Acad. Sci. USA91, 9770–9774.10.1073/pnas.91.21.9770Search in Google Scholar PubMed PubMed Central
©2006 by Walter de Gruyter Berlin New York
