Folding determinants of LDL receptor type A modules.
To investigate how three disulfide bonds and coordination of a calcium ion cooperate to specify the structure of an LDL-A module, we studied the interdependence of disulfide bond formation and calcium coordination in the folding of ligand-binding module 5 of the LDL receptor (LR5). In variants of LR5 containing only a single pair of cysteines normally disulfide-bonded in the native polypeptide, the addition of calcium does not alter the effective concentration of one cysteine for the other. LR5 only exhibits a calcium-dependent preference for formation of native disulfide bonds and detectable calcium-induced changes in structure when the two C-terminal disulfide bonds are present. Furthermore, when the conformation of this two-disulfide variant of LR5 is probed by NMR in the presence of calcium, only the C-terminal lobe of the module, which contains the calcium coordination site, acquires a near-native conformation; the N-terminal lobe appears to be disordered. These findings contrast with studies of other model proteins, like BPTI, in which formation of a single disulfide bond is sufficient to drive the entire domain to acquire a stable, nativelike fold.
Koduri
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Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, 75 Francis Street, Boston, Massachusetts 02115, USA.
Blacklow
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SC
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011973:Receptors, LDL / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
D004220:Disulfides; D008024:Ligands; D010455:Peptides; D011973:Receptors, LDL; D011993:Recombinant Fusion Proteins; D003545:Cysteine