Affinities between the binding partners of the HIV-1 integrase dimer-lens epithelium-derived growth factor (IN dimer-LEDGF) complex.
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 (K(dimer) = 67.8 pm) and of LEDGF from IN dimers (K(d) = 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 k(on) of 0.1247 nm(-1) x min(-1) and a k(off) of 0.0080 min(-1) resulting in a K(dimer) of 64.5 pm. LEDGF binding to IN dimers had a k(on) of 0.0285 nm(-1).min(-1) and a k(off) of 0.2340 min(-1) resulting in a K(d) 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 K(i) 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 K(i) 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.
Gilead Sciences, Inc., Foster City, California 94404, USA. MTsiang@gilead.com
Manuel
M
Tsiang
Gregg S
GS
Jones
Magdeleine
M
Hung
Susmith
S
Mukund
Bin
B
Han
Xiaohong
X
Liu
Kerim
K
Babaoglu
Eric
E
Lansdon
Xiaowu
X
Chen
Jacob
J
Todd
Terrence
T
Cai
Nikos
N
Pagratis
Roman
R
Sakowicz
Romas
R
Geleziunas
D000465:Algorithms; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D031541:Fluorescence Resonance Energy Transfer; D019427:HIV Integrase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D008956:Models, Chemical; D008969:Molecular Sequence Data; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D055503:Protein Multimerization
D036341:Intercellular Signaling Peptides and Proteins; C119380:lens epithelium-derived growth factor; D019427:HIV Integrase; C514801:p31 integrase protein, Human immunodeficiency virus 1