Total chemical synthesis of N-myristoylated HIV-1 matrix protein p17: structural and mechanistic implications of p17 myristoylation.
The HIV-1 matrix protein p17, excised proteolytically from the N terminus of the Gag polyprotein, forms a protective shell attached to the inner surface of the plasma membrane of the virus. During the late stages of the HIV-1 replication cycle, the N-terminally myristoylated p17 domain targets the Gag polyprotein to the host-cell membrane for particle assembly. In the early stages of HIV-1 replication, however, some p17 molecules dissociate from the viral membrane to direct the preintegration complex to the host-cell nucleus. These two opposing targeting functions of p17 require that the protein be capable of reversible membrane interaction. It is postulated that a significant structural change in p17 triggered by proteolytic cleavage of the Gag polyprotein sequesters the N-terminal myristoyl group, resulting in a weaker membrane binding by the matrix protein than the Gag precursor. To test this "myristoyl switch" hypothesis, we obtained highly purified synthetic HIV-1 p17 of 131 amino acid residues and its N-myristoylated form in large quantity. Both forms of p17 were characterized by circular dichroism spectroscopy, protein chemical denaturation, and analytical centrifugal sedimentation. Our results indicate that although N-myristoylation causes no spectroscopically discernible conformational change in p17, it stabilizes the protein by 1 kcal/mol and promotes protein trimerization in solution. These findings support the premise that the myristoyl switch in p17 is triggered not by a structural change associated with proteolysis, but rather by the destabilization of oligomeric structures of membrane-bound p17 in the absence of downstream Gag subdomains.
Wu
Zhibin
Z
Institute of Human Virology, University of Maryland Biotechnology Institute, and School of Medicine, University of Maryland, 725 West Lombard Street, Baltimore, MD 21201, USA.
Alexandratos
Jerry
J
Ericksen
Bryan
B
Lubkowski
Jacek
J
Gallo
Robert C
RC
Lu
Wuyuan
W
D002462:Cell Membrane / Q000378:metabolism; D019281:Dimerization; D015683:Gene Products, gag / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D015488:HIV Antigens / Q000737:chemistry / Q000378:metabolism; D009226:Myristates / Q000138:chemical synthesis*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D013057:Spectrum Analysis; D014764:Viral Proteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D054301:gag Gene Products, Human Immunodeficiency Virus
D015683:Gene Products, gag; D015488:HIV Antigens; D009226:Myristates; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C054858:p17 protein, Human Immunodeficiency Virus Type 1