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Title
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Calcium is essential for the structural integrity of the cysteine-rich, ligand-binding repeat of the low-density lipoprotein receptor.
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Authors
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A.R.Atkins,
I.M.Brereton,
P.A.Kroon,
H.T.Lee,
R.Smith.
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Ref.
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Biochemistry, 1998,
37,
1662-1670.
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PubMed id
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Abstract
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Seven cysteine-rich repeats form the ligand-binding region of the low-density
lipoprotein (LDL) receptor. Each of these repeats is assumed to bind a calcium
ion, which is needed for association of the receptor with its ligands, LDL and
beta-VLDL. The effects of metal ions on the folding of the reduced N-terminal
cysteine-rich repeat have been examined by using reverse-phase high-performance
liquid chromatography to follow the formation of fully oxidized isomers with
different disulfide connectivities. In the absence of calcium many of the 15
possible isomers formed on oxidation, whereas in its presence the predominant
product at equilibrium had the native disulfide bond connectivities. Other
metals were far less effective at directing disulfide bond formation: Mn2+
partly mimicked the action of Ca2+, but Ba2+, Sr2+, and Mg2+ had little effect.
This metal-ion specificity was also observed in two-dimensional 1H NMR spectral
studies; only Ca2+ induced the native three-dimensional fold. The two
paramagnetic ions, Gd3+ and Mn2+, and Cd2+ did not promote adoption of a
well-defined structure, and the two paramagnetic ions did not displace calcium
ions. The location of calcium ion binding sites in the repeat was also explored
by NMR spectroscopy. The absence of chemical shift changes for the side chain
proton resonances of Asp26, Asp36, and Glu37 from pH 3.9 to 6.8 in the presence
of calcium ions and their proximal location in the NMR structures implicated
these side chains as calcium ligands. Deuterium exchange NMR experiments also
revealed a network of hydrogen bonds that stabilizes the putative
calcium-binding loop.
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