Lipid binding-induced conformational change in human apolipoprotein E. Evidence for two lipid-bound states on spherical particles.

Hiroyuki Saito,Karl H Weisgraber,Sissel Lund-Katz,Padmaja Dhanasekaran,Michael C Phillips,Faye Baldwin

doi:10.1074/jbc.m106337200

Abstract

Apolipoprotein (apo) E contains two structural domains, a 22-kDa (amino acids 1-191) N-terminal domain and a 10-kDa (amino acids 223-299) C-terminal domain. To better understand apoE-lipid interactions on lipoprotein surfaces, we determined the thermodynamic parameters for binding of apoE4 and its 22- and 10-kDa fragments to triolein-egg phosphatidylcholine emulsions using a centrifugation assay and titration calorimetry. In both large (120 nm) and small (35 nm) emulsion particles, the binding affinities decreased in the order 10-kDa fragment approximately 34-kDa intact apoE4 > 22-kDa fragment, whereas the maximal binding capacity of intact apoE4 was much larger than those of the 22- and 10-kDa fragments. These results suggest that at maximal binding, the binding behavior of intact apoE4 is different from that of each fragment and that the N-terminal domain of intact apoE4 does not contact lipid. Isothermal titration calorimetry measurements showed that apoE binding to emulsions was an exothermic process. Binding to large particles is enthalpically driven, and binding to small particles is entropically driven. At a low surface concentration of protein, the binding enthalpy of intact apoE4 (-69 kcal/mol) was approximately equal to the sum of the enthalpies for the 22- and 10-kDa fragments, indicating that both the 22- and 10-kDa fragments interact with lipids. In a saturated condition, however, the binding enthalpy of intact apoE4 (-39 kcal/mol) was less exothermic and rather similar to that of each fragment, supporting the hypothesis that only the C-terminal domain of intact apoE4 binds to lipid. We conclude that the N-terminal four-helix bundle can adopt either open or closed conformations, depending upon the surface concentration of emulsion-bound apoE.

Highlights

Apolipoprotein E contains two structural domains, a 22-kDa N-terminal domain and a 10-kDa C-terminal domain
It should be noted that a previous study of the distribution of the 22-kDa fragments of Apolipoprotein E (apoE) among plasma lipoproteins demonstrated that the 22-kDa fragment does not bind to any triglyceride-rich lipoprotein particles (33), our results clearly showed that the apoE4 22-kDa fragment can bind to some extent to the surface of emulsion particles
For high affinity binding to the low density lipoprotein (LDL) receptor, apoE must be associated with lipid

Summary

EXPERIMENTAL PROCEDURES

Materials—Egg yolk phosphatidylcholine (PC) and triolein were purchased from Sigma, and stock solutions were stored in chloroform/ methanol (2:1) under nitrogen at Ϫ20 °C. [14C]Formaldehyde (40 – 60 Ci/mol) in distilled water was purchased from PerkinElmer Life Sciences. After the bacterial pellet was sonicated and the lysate was centrifuged to remove debris, the fusion protein was cleaved with thrombin to remove thioredoxin from full-length apoE4 or the 22- or 10-kDa fragment. The dry lipids were suspended in Tris buffer (10 mM Tris-HCl, 150 mM NaCl, 0.02% NaN3, 1 mM EDTA, pH 7.4) and sonicated at 80 watts with a Branson sonifier model 350 and flat tip for 30 min at 50 – 60 °C under a stream of nitrogen. 14C-Labeled apoE4 or the 22- or 10-kDa fragment (freshly dialyzed from 1% ␤-mercaptoethanol and 6 M guanidine HCl solution into Tris buffer, pH 7.4) and emulsions (0.3 mg phospholipid/ml) were incubated for 1 h at room temperature with gentle shaking in 1.4 ml of Tris buffer (pH 7.4) containing 0.25 M sucrose.

Bmax Surfacea

RESULTS

DISCUSSION