Biochemical indication for myristoylation-dependent conformational changes in HIV-1 Nef.
The accessory HIV-1 Nef protein is essential for viral replication, high virus load, and progression to AIDS. These functions are mediated by the alteration of signaling and trafficking pathways and require the membrane association of Nef by its N-terminal myristoylation. However, a large portion of Nef is also found in the cytosol, in line with the observation that myristoylation is only a weak lipidation anchor for membrane attachment. We performed biochemical studies to analyze the implications of myristoylation on the conformation of Nef in aqueous solution. To establish an in vivo myristoylation assay, we first optimized the codon usage of Nef for Escherichia coli expression, which resulted in a 15-fold higher protein yield. Myristoylation was achieved by coexpression with the N-myristoyltransferase and confirmed by mass spectrometry. The myristoylated protein was soluble, and proton NMR spectra confirmed proper folding. Size exclusion chromatography revealed that myristoylated Nef appeared of smaller size than the unmodified form but not as small as an N-terminally truncated from of Nef that omits the anchor domain. Western blot stainings and limited proteolysis of both forms showed different recognition profiles and degradation pattern. Analytical ultracentrifugation revealed that myristoylated Nef prevails in a monomeric state while the unmodified form exists in an oligomeric equilibrium of monomer, dimer, and trimer associations. Finally, fluorescence correlation spectroscopy using multiphoton excitation revealed a shorter diffusion time for the lipidated protein compared to the unmodified form. Taken together, our data indicated myristoylation-dependent conformational changes in Nef, suggesting a rather compact and monomeric form for the lipidated protein in solution.
Abteilungen Physikalische Biochemie und Strukturelle Biologie, Max-Planck-Institut für Molekulare Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Sebastian
S
Breuer
Holger
H
Gerlach
Branko
B
Kolaric
Claus
C
Urbanke
Norbert
N
Opitz
Matthias
M
Geyer
D001483:Base Sequence; D015153:Blotting, Western; D002850:Chromatography, Gel; D003062:Codon; D015693:Gene Products, nef / Q000737:chemistry* / Q000302:isolation & purification; D015497:HIV-1 / Q000737:chemistry; D008969:Molecular Sequence Data; D009227:Myristic Acids / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D014461:Ultracentrifugation; D054311:nef Gene Products, Human Immunodeficiency Virus
D003062:Codon; D015693:Gene Products, nef; D009227:Myristic Acids; D054311:nef Gene Products, Human Immunodeficiency Virus