Affinities between the Binding Partners of the HIV-1 Integrase Dimer-Lens Epithelium-derived Growth Factor (IN Dimer-LEDGF) Complex

• Published in: The Journal of biological chemistry 2009-11, Vol.284 (48), p.33580-33599
• Main Authors: Tsiang, Manuel, Jones, Gregg S., Hung, Magdeleine, Mukund, Susmith, Han, Bin, Liu, Xiaohong, Babaoglu, Kerim, Lansdon, Eric, Chen, Xiaowu, Todd, Jacob, Cai, Terrence, Pagratis, Nikos, Sakowicz, Roman, Geleziunas, Romas
• Format: Article
• Language: English
• Subjects: Algorithms; Amino Acid Sequence; Binding, Competitive; Enzyme Catalysis and Regulation; Fluorescence Resonance Energy Transfer; HIV Integrase - chemistry; HIV Integrase - genetics; HIV Integrase - metabolism; Human immunodeficiency virus 1; Humans; Intercellular Signaling Peptides and Proteins - chemistry; Intercellular Signaling Peptides and Proteins - genetics; Intercellular Signaling Peptides and Proteins - metabolism; Kinetics; Models, Biological; Models, Chemical; Molecular Sequence Data; Protein Binding; Protein Interaction Domains and Motifs; Protein Interaction Mapping; Protein Multimerization
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.040121

The interaction between lens epithelium-derived growth factor/transcriptional co-activator p75 (LEDGF) and human immunodeficiency virus type 1 (HIV-1) integrase (IN) is essential for HIV-1 replication. Homogeneous time-resolved fluorescence resonance energy transfer assays were developed to characterize HIV-1 integrase dimerization and the interaction between LEDGF and IN dimers. Using these assays in an equilibrium end point dose-response format with mathematical modeling, we determined the dissociation constants of IN dimers (Kdimer = 67.8 pm) and of LEDGF from IN dimers (Kd = 10.9 nm). When used in a kinetic format, the assays allowed the determination of the on- and off-rate constants for these same interactions. Integrase dimerization had a kon of 0.1247 nm−1·min−1 and a koff of 0.0080 min−1 resulting in a Kdimer of 64.5 pm. LEDGF binding to IN dimers had a kon of 0.0285 nm−1·min−1 and a koff of 0.2340 min−1 resulting in a Kd of 8.2 nm. These binding assays can also be used in an equilibrium end point competition format. In this format, the IN catalytic core domain produced a Ki of 15.2 nm while competing for integrase dimerization, confirming the very tight interaction of IN with itself. In the same format, LEDGF produced a Ki value of 35 nm when competing for LEDGF binding to IN dimers. In summary, this study describes a methodology combining homogeneous time-resolved fluorescence resonance energy transfer and mathematical modeling to derive the affinities between IN monomers and between LEDGF and IN dimers. This study revealed the significantly tighter nature of the IN-IN dimer compared with the IN-LEDGF interaction.