A ligand-induced conformational change in apolipoprotein(a) enhances covalent Lp(a) assembly

Abstract number: P1351

Becker L., Webb B. A., Chitayat S., Nesheim M. E., Koschinsky M. L.

Queen's University, Canada

Lipoprotein(a) (Lp(a)) assembly proceeds via a two-step mechanism in which initial noncovalent interactions between apolipoprotein(a) (apo(a)) and low density lipoprotein (LDL) precede disulfide bond formation. Apo(a) contains multiple repeats of sequences that resemble plasminogen kringle IV, followed by sequences that are homologous to the kringle V and protease domains of plasminogen. The kringle IV domains of apo(a) are further classified into 10 distinct subclasses; the kringle IV type 2 domain (KIV2) is present in variable copy number accounting for the observed isoform size heterogeneity of Lp(a). Elevated levels of Lp(a) have been correlated with an increased risk for the development of a variety of atherogenic disorders, an observation that may in part be explained by the ability of apo(a)/Lp(a) to attenuate fibrinolysis. In this study, we used analytical ultracentrifugation, differential scanning calorimetry, and intrinsic fluorescence to demonstrate that in the presence of the lysine analog e-aminocaproic acid (e-ACA), apo(a) undergoes a substantial conformational change from a ‘closed’ to ‘open’ structure that is characterized by an increase in the hydrodynamic radius (~10%), an alteration in domain stability, as well as a decrease (~10%) in tryptophan fluorescence. We further demonstrate that the ‘closed’ conformation of apo(a) is maintained by an intramolecular interaction between the strong lysine-binding site within apo(a) kringle IV type 10 and sequences within the N-terminal 30% of the protein. Although e-ACA is a well characterized inhibitor of the noncovalent interaction between apo(a) and LDL, we made the novel observation that this ligand at low concentrations (500 µM to 5 mM) actually enhances covalent Lp(a) assembly by altering the conformation of apo(a). Using apo(a) isoforms containing 1, 3, or 8 KIV2 repeats, we found that the isoform size of apo(a) is an important determinant of the conformational status of apo(a), and hence the efficiency of covalent Lp(a) assembly. Interestingly, an analogous conformational change has been previously described for plasminogen resulting in an increase in the hydrodynamic radius, an increase in tryptophan fluorescence, and an acceleration of the rate of plasminogen activation. Although the functions of apo(a) and plasminogen have diverged considerably, elements of structural and conformational homology have been retained leading to a similar regulation of two unrelated biological processes.