Oligomerization of the HIV type 2 Nef protein: mutational analysis of the heptad leucine repeat motif and cysteine residues.
The human immunodeficiency virus type 2 (HIV-2) Nef protein expressed in Escherichia coli forms highly stable homooligomeric complexes in vitro. Similarly, the native protein synthesized in the persistently infected H9 T cell line also forms stable homooligomers in vivo. To determine whether homooligomer formation is mediated by the leucine zipper-type sequence located in the middle region of the protein, site-directed mutagenesis was used to introduce double and triple point mutations at heptad leucine positions L1, L2, and L4 within the HIV-2NIHZ Nef protein sequence. Here, we show that substitution of a serine residue for the L1 (residue 108) and L2 (residue 115) heptad leucines, and a glutamine residue for the L4 (residue 129) heptad leucine, did not prevent Nef homooligomer formation in vitro. However, a more drastic substitution of alpha-helix-breaking proline residue for the L2 and L4 heptad leucines significantly abrogated ability of the protein to form stable homooligomers. In addition, because significantly higher levels of the Nef oligomers were consistently observed under the nonreducing SDS-PAGE condition, site-specific mutagenesis was also used to examine the role of cysteine residues in generating disulfide-linked Nef dimers in vitro. Here, we also show that single cysteine-to-glycine substitutions at positions 28, 32, or 55 drastically reduced covalent Nef dimer formation and thermal stability of the Nef protein in vitro. Therefore, these results demonstrate that the leucine zipper-type motif in the HIV-2 Nef protein mediates stable homooligomer formation in vitro, and also establish a role for covalent disulfide bonds in the formation of linked Nef dimers and thermal stability of the monomer Nef in vitro.
Hodge
D R
DR
Laboratory of Molecular Oncology, National Cancer Institute, Frederick Research and Development Center, Maryland 21702, USA.
Chen
Y M
YM
Samuel
K P
KP
D000595:Amino Acid Sequence; D001483:Base Sequence; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015693:Gene Products, nef / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D015498:HIV-2 / Q000378:metabolism*; D006801:Humans; D016350:Leucine Zippers / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D054311:nef Gene Products, Human Immunodeficiency Virus
D015693:Gene Products, nef; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; D003545:Cysteine