title: Conserved residue Lys574 in the cavity of HIV-1 Gp41 coiled-coil domain is critical for six-helix bundle stability and virus entry.
abstract: 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.
authors.affiliation: Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York 10021, USA. yhe@nybloodcenter.org
authors.forename: Yuxian
authors.initials: Y
authors.lastname: He
authors.forename: Shuwen
authors.initials: S
authors.lastname: Liu
authors.forename: Weiguo
authors.initials: W
authors.lastname: Jing
authors.forename: Hong
authors.initials: H
authors.lastname: Lu
authors.forename: Dongmei
authors.initials: D
authors.lastname: Cai
authors.forename: Darin Jeekin
authors.initials: DJ
authors.lastname: Chin
authors.forename: Asim K
authors.initials: AK
authors.lastname: Debnath
authors.forename: Frank
authors.initials: F
authors.lastname: Kirchhoff
authors.forename: Shibo
authors.initials: S
authors.lastname: Jiang
mesh_terms: D019943:Amino Acid Substitution; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D015195:Drug Design; D015700:HIV Envelope Protein gp41 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D023581:HIV Fusion Inhibitors / Q000737:chemistry; D015497:HIV-1 / Q000737:chemistry* / Q000502:physiology; D057927:Hydrophobic and Hydrophilic Interactions; D008239:Lysine / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D020125:Mutation, Missense; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000235:genetics; D053586:Virus Internalization / Q000187:drug effects
chemical_list: D015700:HIV Envelope Protein gp41; D023581:HIV Fusion Inhibitors; D008239:Lysine