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The ligand-binding domain of the low-density lipoprotein receptor comprises
seven cysteine-rich repeats, which have been highly conserved through evolution.
This domain mediates interactions of the receptor with two lipoprotein
apoproteins, apo E and apo B-100, putatively through a calcium-dependent
association of the ligands with a cluster of acidic residues on the receptor.
The second repeat (rLB2) of the receptor binding domain has been expressed as a
thrombin-cleavable GST fusion protein, cleaved, and purified. On oxidation the
protein refolded to give a single peak on reverse-phase HPLC. The aqueous
solution structure of rLB2 has been determined using two-dimensional 1H NMR
spectroscopy. In contrast to the amino-terminal repeat, rLB1, rLB2 has a very
flexible structure in water. However, the conformation of rLB2 is markedly more
ordered in the presence of a 4-fold molar excess of calcium chloride; the proton
resonance dispersion and the number of NOESY cross-peaks are greatly enhanced.
The three-dimensional structure of rLB2, obtained from the NMR data by molecular
geometry and restrained molecular dynamics methods, parallels that of rLB1, with
an amino-terminal hairpin structure followed by a succession of turns. However,
there are clear differences in the backbone topology and structural flexibility.
As for rLB1, the acidic residues are clustered on one face of the module. The
side chain of Asp 37, which is part of a completely conserved SDE sequence
thought to be involved in ligand binding, is buried, as is its counterpart (Asp
36) in rLB1. These results provide the first experimental support for the
hypothesis that each of the repeats in the ligand-binding domain has a similar
global fold but also highlight significant differences in structure and internal
dynamics.
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