Conformational flexibility of the N-terminal domain of apolipoprotein a-I bound to spherical lipid particles.
Lipid binding of human apolipoprotein A-I (apoA-I) occurs initially through the C-terminal alpha-helices followed by conformational reorganization of the N-terminal helix bundle. This led us to hypothesize that apoA-I has multiple lipid-bound conformations, in which the N-terminal helix bundle adopts either open or closed conformations anchored by the C-terminal domain. To investigate such possible conformations of apoA-I at the surface of a spherical lipid particle, site-specific labeling of the N- and C-terminal helices in apoA-I by N-(1-pyrene)maleimide was employed after substitution of a Cys residue for Val-53 or Phe-229. Neither mutagenesis nor the pyrene labeling caused discernible changes in the lipid-free structure and lipid interaction of apoA-I. Taking advantage of a significant increase in fluorescence when a pyrene-labeled helix is in contact with the lipid surface, we monitored the behaviors of the N- and C-terminal helices upon binding of apoA-I to egg PC small unilamellar vesicles. Comparison of the binding isotherms for pyrene-labeled apoA-I as well as a C-terminal helical peptide suggests that an increase in surface concentration of apoA-I causes dissociation of the N-terminal helix from the surface leaving the C-terminal helix attached. Consistent with this, isothermal titration calorimetry measurements showed that the enthalpy of apoA-I binding to the lipid surface under near saturated conditions is much less exothermic than that for binding at a low surface concentration, indicating the N-terminal helix bundle is out of contact with lipid at high apoA-I surface concentrations. Interestingly, the presence of cholesterol significantly induces the open conformation of the helix bundle. These results provide insight into the multiple lipid-bound conformations that the N-terminal helix bundle of apoA-I can adopt on a lipid or lipoprotein particle, depending upon the availability of space on the surface and the surface composition.
Kono
Momoe
M
Department of Biophysical Chemistry, Kobe Pharmaceutical University, Kobe 658-8558, Japan.
Okumura
Yusuke
Y
Tanaka
Masafumi
M
Nguyen
David
D
Dhanasekaran
Padmaja
P
Lund-Katz
Sissel
S
Phillips
Michael C
MC
Saito
Hiroyuki
H
D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D008081:Liposomes / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence
C505693:APOA1 protein, human; D016632:Apolipoprotein A-I; D008055:Lipids; D008081:Liposomes