The molecular mechanism of lipoprotein binding by the low-density lipoprotein (LDL) receptor (LDLR) is poorly understood, one reason being that structures of lipoprotein-receptor complexes are not available. LDLR uses calcium-binding repeats (LRs) to interact with apolipoprotein B and apolipoprotein E (ApoB and ApoE). We have used NMR and SPR to characterize the complexes formed by LR5 and three peptides encompassing the putative binding regions of ApoB (site A and site B) and ApoE. The three peptides bind at the hydrophilic convex face of LR5, forming complexes that are weakened at low [Ca 2+ ] and low pH. Thus, endosomal conditions favour dissociation of LDLR/lipoprotein complexes regardless of whether active displacement of bound lipoproteins by the b-propeller in LDLR takes place. The multiple ApoE copies in b very low density lipoproteins (b-VLDLs), and the presence of two competent binding sites (A and B) in LDLs, suggest that LDLR chelates lipoproteins and enhances complex affinity by using more than one LR.

The ligand binding module five (LA5) of the low density lipoprotein receptor is a small, single-domain protein of 40 residues and three disulfide bonds with a calcium binding motif that is essential for its structure and function. Several mutations in LA5 have been reported to cause familial hypercholesterolemia by impairing a proper folding of the module. The current study reports the oxidative folding and reductive unfolding pathways of wild type and mutant LA5 modules through kinetic and structural analysis of the trapped intermediates. Wild type LA5 folding involves an initial phase of nonspecific packing where the sequential oxidation of its cysteines gives rise to complex equilibrated populations of intermediates. In the presence of calcium, the attainment of a coordination-competent conformation becomes the rate-limiting step of folding while binding of the ion funnels both thermodynamically and kinetically the folding reaction toward the native state. In the absence of calcium, a scrambled isomer (termed Xa) constitutes the global free energy minimum of the folding process. Xa and the native form are stable, inter-convertible species whose relative populations at equilibrium appear displaced in disease-linked mutants toward the scrambled form. Because stable scrambled isomers such as Xa avoid the exposition of reactive cysteines in misfolded modules, they might constitute a strategy to prevent wrong interactions with other domains during folding of the receptor. Comparison of the folding pathways of wild type and mutant LA5 provides the molecular basis to understand how LA modules fold into a functional conformation or upon mutation misfold and lead to disease.

The low‐density lipoprotein receptor (LDLR) captures circulating lipoproteins and delivers them in the endosome for degradation. Its function is essential for cholesterol homeostasis, and mutations in the LDLR are the major cause of familiar hypercholesterolemia. The release of LDL is usually attributed to endosome acidification. As the pH drops, the affinity of the LDLR/LDL complex is reduced, whereas the strength of a self‐complex formed between two domains of the receptor (i.e. the LDL binding domain and the β‐propeller domain) increases. However, an alternative model states that, as a consequence of a drop in both pH and Ca2+ concentration, the LDLR binding domain is destabilized in the endosome, which weakens the LDLR/LDL complex, thus liberating the LDL particles. In the present study, we test a key underlying assumption of the second model, namely that the lipoprotein binding repeats of the receptor (specifically repeats 4 and 5, LR4 and LR5) rapidly sense endosomal changes in Ca2+ concentration. Our kinetic and thermodynamic analysis of Ca2+ and Mg2+ binding to LR4 and LR5, as well as to the tandem of the two (LR4–5), shows that both repeats spontaneously release Ca2+ in a time scale much shorter than endosomal delivery of LDL, thus acting as Ca2+ sensors that become unfolded under endosomal conditions. Our analysis additionally explains the lower Ca2+ affinity of repeat LR4, compared to LR5, as arising from a very slow Ca2+ binding reaction in the former, most likely related to the lower conformational stability of apolipoprotein LR4, compared to apolipoprotein LR5, as determined from thermal unfolding experiments and molecular dynamics simulations.