Co-translational myristoylation alters the quaternary structure of HIV-1 Nef in solution.
We have studied the solution properties of Nef, a 24-kDa cotranslationally myristoylated protein produced by HIV-1 and other primate lentiviruses. Nef is found in the cytosol and also in association with cytoplasmic membranes, the latter, mediated in part by the myristoyl group attached to the N-terminal glycine. Recombinant Nef was coexpressed in Escherichia coli in tandem with N-myristoyl-transferase and is fully myristoylated. Analysis by circular dichroism showed the myristoylated form to contain a greater alpha-helical content than the nonmyristoylated form. Analysis of modified and unmodified Nef in solution using small angle X-ray scattering, dynamic laser light scattering and analytical ultracentrifugation consistently showed differences in the oligomeric states of the two forms of Nef. Myristoylated Nef is predominantly monomeric and small oligomers which are also present, can be converted to the monomeric form under reducing conditions. By contrast, the nonmyristoylated form exists as a stable hexadecamer in solution which disassociates into tetramers upon addition of reducing agents. Shape reconstructions from small angle scattering curves of nonmyristoylated Nef are compatible with a large disc-like structure in the hexadecameric oligomer consisting of four Nef tetramers. From these findings, we hypothesize that Nef undergoes a substantial conformational change from an "open" into a "closed" form whereby the myristate group is sequestered in a hydrophobic pocket. The myristoylated protein can switch to the open conformation by association of the N-terminal region of molecule with membranes. These changes would allow Nef to carry out various functions depending on the conformational and oligomeric states.
Dennis
Caitríona A
CA
Astbury Centre for Structural Molecular Biology, University of Leeds, United Kingdom.
Baron
Andrew
A
Grossmann
J Günter
JG
Mazaleyrat
Sabine
S
Harris
Mark
M
Jaeger
Joachim
J
D001483:Base Sequence; D001665:Binding Sites; D015693:Gene Products, nef / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000737:chemistry; D008969:Molecular Sequence Data; D009227:Myristic Acids; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012996:Solutions; D054311:nef Gene Products, Human Immunodeficiency Virus
D015693:Gene Products, nef; D009227:Myristic Acids; D011994:Recombinant Proteins; D012996:Solutions; D054311:nef Gene Products, Human Immunodeficiency Virus