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Biochemistry. 1999 Mar 30;38(13):3926-35.

Structural independence of ligand-binding modules five and six of the LDL receptor.

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Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.


The low-density lipoprotein receptor (LDLR) is the primary mechanism for the uptake of plasma cholesterol into cells and serves as a prototype for a growing family of cell surface receptors. These receptors all utilize tandemly repeated LDL-A modules to bind their ligands. Each LDL-A module is about 40 residues long, has six conserved cysteine residues, and contains a conserved acidic region near the C-terminus which serves as a calcium-binding site. The structure of the interface presented for ligand binding by these modules, and the basis for their specificity and affinity in ligand binding, is not yet known. We have purified recombinant molecules corresponding to LDL-A modules five (LR5), six (LR6), and the module five-six pair (LR5-6) of the LDL receptor. Calcium is required to establish native disulfide bonds and to maintain the structural integrity of LR5, LR6, and the LR5-6 module pair. Folding studies of the I189D and D206Y mutations within LR5 indicate that each change leads to misfolding of the module, explaining the previous observation that each of these changes mimics the functional effect of deletion of the entire module [Russell, D. W., Brown, M. S., and Goldstein, J. L. (1989) J. Biol. Chem. 264, 21682-21688]. By fluorescence, the affinity of LR5 for calcium, which is crucial for folding and function of these modules, remains approximately 40 nM whether LR6 is attached. Comparison of proton and multidimensional heteronuclear NMR spectra of individual modules to those of the module pair indicates that most of the significant spectroscopic changes lie within the linker region between modules and that little structural interaction occurs between the cores of modules five and six in the 5-6 pair. These findings strongly support a model in which each module is essentially structurally independent of the other.

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