One common feature of the more than 1,000 complement-type repeats (or low density lipoprotein (LDL)-A modules) found in LDL receptor and the other members of the LDL receptor superfamily is a cluster of five highly conserved acidic residues in the C-terminal region, DXXXDXXDXXDE. However, the role of the third conserved aspartate of these LDL-A modules in protein folding and ligand recognition has not been elucidated. In this report, using a model LDL-A module and several experimental approaches, we demonstrate that this acidic residue, like the other four conserved acidic residues, is involved in calcium-dependent protein folding. These results suggest an alternative calcium coordination conformation for the LDL-A modules. The proposed model provides a plausible explanation for the conservation of this acidic residue among the LDL-A modules. Furthermore, the model can explain why mutations of this residue in human LDL receptor cause familial hypercholesterolemia.The LDL 1 receptor superfamily, with human LDL receptor as its prototype, consists of a large number of proteins such as the LDL receptor-related protein (LRP), gp330, and the very low density lipoprotein receptor (1). One common feature of these proteins is that they all contain at least one modular domain (called complement-type repeat, or LDL-A module) of ϳ40 residues in length, including six invariable cysteines and the C-terminal highly conserved acidic residue motif (DXXX-DXXDXXDE). All together there are more than 1,000 known LDL-A modules found in a variety of proteins that are involved in diverse biological processes. In human LDL receptor, seven such imperfect repeats of LDL-A modules at the N terminus of the protein form the ligand binding domain, responsible for binding to its ligands, apoB and apoE (2-5). Naturally occurring point mutations in any of the conserved acidic residues of the individual LDL-A modules of human LDL receptor can cause familial hypercholesterolemia (FH), a genetic disease that ultimately leads to coronary heart disease and atherosclerosis (6). One proposed mechanism for these conserved acidic residues of LDL receptor in ligand binding is to interact with the basic residues of its ligands via ionic interactions (2, 7-9).Structural analysis of individual LDL-A modules, and recently the entire ectodomain of human LDL receptor, by x-ray crystallography revealed another mechanism by which four conserved acidic acids exert their role in protein conformation and function of LDL-A modules and thus LDL receptors (10 -12). Among the five conserved acidic residues, DXXXDXX-DXXDE, the side chains of four of them (the first, second, fourth, and fifth acidic residues, in italics), with the carbonyl oxygen groups of two non-conserved residues, are involved in calcium coordination. Thus mutations of these residues in the LDL receptor can result in folding defects of LDL-A modules and thus the overall structure of the LDL receptor, which indirectly lead to an impaired ligand-binding phenotype and eventually heart disease. However, th...

Rong et al. have demonstrated previously that with a few substitutions, the fourth repeat of human low-density lipoprotein (hLDL-A4) receptor can functionally replace the LDL-A module of Tva, the cellular receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A), in viral entry (L. Rong, K. Gendron, and P. Bates, Proc. Natl. Acad. Sci. USA 95:8467-8472, 1998). Here we have shown that swapping the amino terminus of hLDL repeat 5 (hLDL-A5) with that of Tva, in addition to the corresponding substitutions made in human LDL-A4, was required to convert hLDL-A5 into an efficient ASLV-A receptor. These results substantiated our previous findings regarding the role of the specific residues in the viral interaction domain of Tva and demonstrated the critical role of the amino terminus of the Tva LDL-A module in ASLV-A infection. Furthermore, we have shown that the residues between cysteines 2 and 3 of the Tva LDL-A module in a Tva/LDL-A5 chimeric protein can be functionally replaced by the corresponding region of another LDL-A module, human LDL receptor-related protein repeat 22 (LDL-A22), to mediate efficient ASLV-A entry. Since the only conserved feature between the C2-C3 region of LDL-A22 and the Tva LDL-A module is that both contain nine amino acids of which none are conserved, we conclude that the spacing between C2 and C3 of the LDL-A module of Tva is an important determinant for ASLV-A entry. Thus, the present study provides strong evidence to support our hypothesis that one role of the N terminus of the LDL-A module of Tva is to allow proper folding and conformation of the protein for optimal interaction with the viral glycoprotein EnvA in ASLV-A entry.