Two-step Mechanism of Binding of Apolipoprotein E to Heparin

Abstract

The interaction of apolipoprotein E (apoE) with cellsurface heparan sulfate proteoglycans is an important step in the uptake of lipoprotein remnants by the liver. ApoE interacts predominantly with heparin through the N-terminal binding site spanning the residues around 136 -150. In this work, surface plasmon resonance analysis was employed to investigate how amphipathic ␣-helix properties and basic residue organization in this region modulate binding of apoE to heparin. The apoE/heparin interaction involves a twostep process; apoE initially binds to heparin with fast association and dissociation rates, followed by a step exhibiting much slower kinetics. Circular dichroism and surface plasmon resonance experiments using a disulfide-linked mutant, in which opening of the Nterminal helix bundle was prevented, demonstrated that there is no major secondary or tertiary structural change in apoE upon heparin binding. Mutations of Lys-146, a key residue for the heparin interaction, greatly reduced the favorable free energy of binding of the first step without affecting the second step, suggesting that electrostatic interaction is involved in the first binding step. Although lipid-free apoE2 tended to bind less than apoE3 and apoE4, there were no significant differences in rate and equilibrium constants of binding among the apoE isoforms in the lipidated state. Discoidal apoE3-phospholipid complexes using a substitution mutant (K143R/K146R) showed similar binding affinity to wild type apoE3, indicating that basic residue specificity is not required for the effective binding of apoE to heparin, unlike its binding to the low density lipoprotein receptor. In addition, disruption of the ␣-helix structure in the apoE heparin binding region led to an increased favorable free energy of binding in the second step, suggesting that hydrophobic interactions contribute to the second binding step. Based on these results, it seems that cellsurface heparan sulfate proteoglycan localizes apoEenriched remnant lipoproteins to the vicinity of receptors by fast association and dissociation.

ApoE: In Vitro Studies of a Small Molecule Effector

“…EZ-482 prevents this increase in a concentration-dependent manner indicating that EZ-482 blocks heparin binding. Figure 3 shows that the increase in fluorescence is at least biphasic and that both phases are too slow to represent the actual binding step for heparin to apoE as determined from the data of Futamura et al (25). Rather, we believe that the slow time course is the re-equilibration of different molecular weight forms of rhodamine labeled heparin.…”

“…Dong et al characterized the binding of an octasaccharide to apoE4 in this same region (24). More recently, Futamura et al, (25) using SPR, characterized binding as a two step process: apoE initially binds to heparin with fast association and dissociation rates, followed by a step exhibiting much slower kinetics.…”

Light chain somatic mutations change thermodynamics of binding and water coordination in the HyHEL-10 family of antibodies

“…H26L10 and H26L8 show increases in ΔΔG o 2 (SPR) in going to the time-dependant Complex II (Copeland et al, 2006; Futamura et al, 2005; Gooljarsingh et al, 2006; Torres et al, 2007) (Table I). Given that there were no significant differences among the ΔΔG o 1 (SPR) of the three antibodies, transition to Complex II accounts for the overall greater affinity of the chain chimeras.…”

Surface plasmon resonance analysis of the mechanism of binding of apoA-I to high density lipoprotein particles

“…The kinetic data were not fi tted well by a 1:1 Langmuir binding model, as refl ected by the large value of the goodness of fi t parameter ( 2 > 20 compared with <10 for a global fi t using the two-state model). It follows that the binding of apoA-I to HDL particles involves either a sequential two-step process or some conformational change ( 22,34,35 ). Fitting of the sensorgrams in Fig.…”