bi9019274_si_001.pdf (39.34 kB)
Highly Sensitive Analysis of the Interaction between HIV-1 Gag and Phosphoinositide Derivatives Based on Surface Plasmon Resonance
journal contributionposted on 2010-06-29, 00:00 authored by Kensaku Anraku, Ryota Fukuda, Nobutoki Takamune, Shogo Misumi, Yoshinari Okamoto, Masami Otsuka, Mikako Fujita
Human immunodeficiency virus type 1 (HIV-1) Gag protein is the principal structural component of the HIV particle. Localization of the Pr55Gag protein to the plasma membrane initiates virus assembly. Recent studies indicated that d-myo-phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P2) regulates Pr55Gag localization and assembly. We determined the binding affinity between Pr55Gag 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 (KD) for Pr55Gag complexed with 1,4,5-IP3 (an inositol with divalent phosphate groups and devoid of lipid groups) was 2170 μM, while the KD for di-C8-PI (a lipid-containing inositol devoid of divalent phosphate groups) was 186 μM, and the KD for di-C8-PI(4,5)P2 (an inositol with both lipid and divalent phosphate groups) was 47.4 μM. 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 Pr55Gag/MA binding. Furthermore, each inositol phosphate (devoid of lipid groups) tested showed a distinct Pr55Gag-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.
SPRPr 55Gag affinitySurface Plasmon ResonanceHuman immunodeficiency virus type 1divalent phosphate groupsPr 55Gag proteinPIPr 55Gag bindingMAPr 55Gag localization186 μ MKD2170 μ MPr 55Gag complexedsurface plasmon resonance47.4 μ MHIVlipid groupsequilibrium dissociation constantsbiotinylated inositol phosphateIPphosphate groups