Proposed mechanisms for binding of apo[a] kringle type 9 to apo B-100 in human lipoprotein[a].
The protein component of human lipoprotein[a] consists primarily of two apolipoproteins, apo[a] and apo B-100, linked through a cystine disulfide(s). In the amino acid sequence of apo bd, Cys4057 located within a plasminogen kringle 4-like repeat sequence (3991-4068) is believed to form a disulfide bond with a specific cysteine residue in apo B-100. Our fluorescence-labeling experiments and molecular modeling studies have provided evidence for possible interactions between this apo[a] kringle type and apo B-100. The fluorescent probe, fluorescein-5-maleimide, was used in parallel experiments to label free sulfhydryl moieties in lipoprotein[a] and low-density lipoprotein (LDL). In apo B-100 of LDL, Cys3734 was labeled with the probe, but this site was not labeled in autologous lipoprotein[a]. The result strongly implicates Cys3734 of apo B-100 as the residue forming the disulfide linkage with Cys4057 of apo[a]. To explore possible noncovalent interactions between apo B-100 and apo[a], the crystallographic coordinates for plasminogen kringle 4 were used to generate molecular models of the apo[a] kringle-repeat sequence (3991-4068, LPaK9), the only plasminogen kringle 4 type repeat in apo[a] having an extra cysteine residue not involved in an intramolecular disulfide bond. The Cys4057 residue (henceforth designated as Cys67 in the LPaK9 sequence) is believed to form an intermolecular disulfide bond with a cysteine of apo B-100. In computer graphics molecular models of LPaK9, Cys67 is located on the surface of the kringle near the lysine ligand binding site. Selected segments of the LDL apo B-100 sequence that contain free sulfhydryl cysteines were subjected to energy minimization and docking with the ligand binding site and adjacent regions of the LPaK9 model. In the docking experiments, apo B-100 segment 3732-3745 (PSCKLDFREIQIYK) displayed the best fit and the largest number of van der Waals contacts with models of LPaK9. Other apo B-100 peptides with sulfhydryl cysteine were found to be less compatible when minimized with this kringle. These results support and extend previously suggested mechanisms for a complex interaction between apo[a] and apo B-100 that involve more than a simple covalent disulfide bond.
Department of Medicine, Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030.
J
J
Guevara
J
J
Spurlino
A Y
AY
Jan
C Y
CY
Yang
A
A
Tulinsky
B V
BV
Prasad
J W
JW
Gaubatz
J D
JD
Morrisett
D000595:Amino Acid Sequence; D053299:Apolipoprotein B-100; D001054:Apolipoproteins A / Q000737:chemistry / Q000378:metabolism*; D001055:Apolipoproteins B / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D005456:Fluorescent Dyes; D006801:Humans; D066298:In Vitro Techniques; D017270:Lipoprotein(a) / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D013816:Thermodynamics
D053299:Apolipoprotein B-100; D001054:Apolipoproteins A; D001055:Apolipoproteins B; D005456:Fluorescent Dyes; D017270:Lipoprotein(a); D010446:Peptide Fragments