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.
Institute of Human Virology, University of Maryland Biotechnology Institute, and School of Medicine, University of Maryland, 725 West Lombard Street, Baltimore, MD 21201, USA.
Zhibin
Z
Wu
Jerry
J
Alexandratos
Bryan
B
Ericksen
Jacek
J
Lubkowski
Robert C
RC
Gallo
Wuyuan
W
Lu
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