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J. Biol. Chem., Vol. 282, Issue 35, 25631-25639, August 31, 2007

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Conserved Residue Lys⁵⁷⁴ in the Cavity of HIV-1 Gp41 Coiled-coil Domain Is Critical for Six-helix Bundle Stability and Virus Entry^*

Yuxian He¹, Shuwen Liu^¶, Weiguo Jing, Hong Lu, Dongmei Cai, Darin Jeekin Chin, Asim K. Debnath, Frank Kirchhoff, and Shibo Jiang²

From the Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York 10021, the ^¶Antiviral Research Center, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong 510515, China, and the Institute of Virology, University of Ulm, 89081 Ulm, Germany

The fusion-active HIV-1 gp41 core structure is a stable six-helix bundle (6-HB) formed by its N- and C-terminal heptad-repeat sequences (NHR and CHR). A highly conserved, deep hydrophobic cavity on the surface of the N-helical trimer is important for stability of the 6-HB and serves as an ideal target for developing anti-human immunodeficiency virus (HIV) fusion inhibitors. We have recently identified several small molecule HIV-1 fusion inhibitors that bind to the gp41 cavity through hydrophobic and ionic interactions and block the gp41 6-HB formation. Molecular docking analysis reveals that these small molecules fit inside the hydrophobic cavity and interact with positively charged residue Lys⁵⁷⁴ to form a conserved salt bridge. In this study, the functionality of Lys⁵⁷⁴ has been finely characterized by mutational analysis and biophysical approaches. We found that substitutions of Lys⁵⁷⁴ with non-conserved residues (K574D, K574E, and K574V) could completely abolish virus infectivity. With a set of wild-type and mutant N36 peptides derived from the NHR sequence as a model, we demonstrated that non-conservative Lys⁵⁷⁴ substitutions severely impaired the stability and conformation of 6-HBs as detected by circular dichroism spectroscopy, native polyacrylamide gel electrophoresis, and enzyme-linked immunosorbent assay. The binding affinity of N36 mutants bearing non-conservative Lys⁵⁷⁴ substitutions to the peptide C34 derived from the CHR sequence dramatically decreased as measured by isothermal titration calorimetry. These substitutions also significantly reduced the potency of N-peptides to inhibit HIV-1 infection. Collectively, these data suggest that conserved Lys⁵⁷⁴ plays a critical role in 6-HB formation and HIV-1 infectivity, and may serve as an important target for designing anti-HIV drugs.

Received for publication, May 8, 2007 , and in revised form, June 19, 2007.

^* This work was supported by National Institutes of Health Grant RO1 AZ46221 (to S. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence may be addressed: 310 East 67th St., NY, New York 10021. Tel.: 212-570-3366; Fax: 212-570-3099; E-mail: yhe{at}nybloodcenter.org. ² To whom correspondence may be addressed: 310 East 67th St., NY, New York 10021. Tel.: 212-570-3058; E-mail: sjiang{at}nybloodcenter.org.

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