Highly sensitive analysis of the interaction between HIV-1 Gag and phosphoinositide derivatives based on surface plasmon resonance.
Human immunodeficiency virus type 1 (HIV-1) Gag protein is the principal structural component of the HIV particle. Localization of the Pr55(Gag) protein to the plasma membrane initiates virus assembly. Recent studies indicated that d-myo-phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P2) regulates Pr55(Gag) localization and assembly. We determined the binding affinity between Pr55(Gag) or its N-terminal MA domain and various phosphoinositide derivatives using a highly sensitive surface plasmon resonance (SPR) sensor and biotinylated inositol phosphate. The equilibrium dissociation constants obtained using this approach reflected the distinct magnitude of acyl group-based and phosphate group-based interactions. The dissociation constant (K(D)) for Pr55(Gag) complexed with 1,4,5-IP3 (an inositol with divalent phosphate groups and devoid of lipid groups) was 2170 microM, while the K(D) for di-C(8)-PI (a lipid-containing inositol devoid of divalent phosphate groups) was 186 microM, and the K(D) for di-C(8)-PI(4,5)P2 (an inositol with both lipid and divalent phosphate groups) was 47.4 microM. The same trend in affinity was observed when these phosphoinositides were complexed with MA. Our results suggest that the contribution of hydrophobic acyl chains is greater than negatively charged inositol phosphates in Pr55(Gag)/MA binding. Furthermore, each inositol phosphate (devoid of lipid groups) tested showed a distinct Pr55(Gag)-binding affinity depending on the position and number of phosphate groups. However, the position and number of phosphate groups had no effect on MA-binding affinity.
Anraku
Kensaku
K
Institute of Health Sciences, Kumamoto Health Science University, 325 Izumi-machi, Kumamoto 861-5598, Japan.
Fukuda
Ryota
R
Takamune
Nobutoki
N
Misumi
Shogo
S
Okamoto
Yoshinari
Y
Otsuka
Masami
M
Fujita
Mikako
M
D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D015683:Gene Products, gag / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D010716:Phosphatidylinositols / Q000378:metabolism*; D011485:Protein Binding; D020349:Surface Plasmon Resonance
D015683:Gene Products, gag; D010716:Phosphatidylinositols