Intradomain Confinement of Disulfides in the Folding of Two Consecutive Modules of the LDL Receptor

Abstract

The LDL receptor internalizes circulating LDL and VLDL particles for degradation. Its extracellular binding domain contains ten (seven LA and three EGF) cysteine-rich modules, each bearing three disulfide bonds. Despite the enormous number of disulfide combinations possible, LDLR oxidative folding leads to a single native species with 30 unique intradomain disulfides. Previous folding studies of the LDLR have shown that non native disulfides are initially formed that lead to compact species. Accordingly, the folding of the LDLR has been described as a "coordinated nonvectorial" reaction, and it has been proposed that early compaction funnels the reaction toward the native structure. Here we analyze the oxidative folding of LA4 and LA5, the modules critical for ApoE binding, isolated and in the LA45 tandem. Compared to LA5, LA4 folding is slow and inefficient, resembling that of LA5 disease-linked mutants. Without Ca++, it leads to a mixture of many two-disulfide scrambled species and, with Ca++, to the native form plus two three-disulfide intermediates. The folding of the LA45 tandem seems to recapitulate that of the individual repeats. Importantly, although the folding of the LA45 tandem takes place through formation of scrambled isomers, no interdomain disulfides are detected, i.e. the two adjacent modules fold independently without the assistance of interdomain covalent interactions. Reduction of incredibly large disulfide combinatorial spaces, such as that in the LDLR, by intradomain confinement of disulfide bond formation might be also essential for the efficient folding of other homologous disulfide-rich receptors.

Fig 1. The LDL receptor and the LA45 pair of its lipoprotein binding domain.

(a) Schematic representation of the LDL receptor. The LA domains (circles) are numbered from 1 to 7. Domains LA4 (in red) and LA5 (in green) are highlighted. The epidermal grow factor motifs (A, B and C) are shown as blue squares and the β-propeller motif is represented as a 6-bladed blue flower. The glycosylated (Gly; brown rectangle), transmembrane (Tra; black square) and cytoplasmatic (Cyt; orange rectangle) domains are also shown. (b) Representation of LA45 (PDB:2LGP) using Pymol. The LA4 residues are marked in red and those of LA5 are shown in green. The linker between LA4 and LA5 is shown in black and arginines are highlighted in dark blue. Cysteines and calcium ions are shown in yellow and native disulfide bonds are shown as yellow sticks. ARG-C proteinase cleavage sites are present in the C-terminal of arginine residues. (c) Amino acid sequence of recombinant LA45. The first two residues (GS; in grey) were added in the cloning and are not present in wild type LA45. All other residues are presented in the same colors used in the structural representation. Disulfide bonds are shown as black lines.

Fig 2. Oxidative folding of LA4 and LA45.

(a, d) RP-HPLC traces of acid trapped intermediates in the folding of reduced LA4 (a) or LA45 (d) in Tris-HCl buffer, pH 8.4, with either 0.1 mM EDTA or 10 mM CaCl₂. In panel a, N, Xa4 and X4b correspond, respectively, to native LA4, a major and a minor LA4 scrambled isomers, while R corresponds to the fully reduced form. In panel d, N and X45 correspond to native LA45 and a major scrambled isomer, respectively, while R corresponds to reduced LA45. (b, e) Chromatograms after 48 hours folding of LA4 (b) or LA45 (e) in Tris-HCl buffer, pH 8.4 with 10 mM CaCl₂ and either 0.25 mM 2-mercaptoethanol, 0.5 mM GSSG (GSSG), 150 mM NaCl and 2 μm PDI (PDI) or no additives (-). (c, f) Chromatograms after 48 hours folding of LA4 (c) or LA45 (f) in Tris-HCl buffer, pH 8.4 with 10 mM CaCl₂ at 4, 15, 23 or 37°C.

Fig 3. Analysis of disulfide species in LA4 folding.

(a) MALDI-TOF spectra of derivatizated LA4 intermediates along LA4 folding in Tris-HCl buffer, pH 8.4 with either 0.1 mM EDTA or 10 mM CaCl₂. Expected mass of LA4 species with 3, 2, 1 or none disulfide bonds (3S, 2S, 1S or 0S) are, respectively 4744, 4952, 5160 and 5368 Daltons. Spectra where the major species are 3S are shown in blue, red is used for 2S, green for 1S and purple for 0S. (b, c) Percentages of disulfide species found during LA4 folding in Tris-HCl buffer, pH 8.4 with either 0.1 mM EDTA (b) or 10 mM CaCl₂ (c). Percentages were calculated based on relative intensities of the peaks in the MALDI-TOF spectra. 3S, 2S 1S and 0S species are shown respectively in blue, red, green and purple.

Fig 4. Oxidative folding of LA4 and LA45.

(a,b) RP-HPLC traces of acid trapped intermediates in the folding of reduced LA4 (a) or LA45 (b) inTris-HCl buffer, pH 8.4 with 10 mM CaCl₂ and either 0.25 mM BME, 0.5 mM GSSG (GSSG) or 150 mM NaCl and 2 μm PDI (PDI). In panel a, N, Xa4 and X4b correspond respectively to native LA4, and its major and minor scrambled isomers. R corresponds to the fully reduced form of LA4. In panel b, N and X45 correspond to native LA45 and the major scrambled isomer of LA45, and R corresponds to reduced LA45.

Fig 5. Analysis of disulfide species after LA45 folding.

(a) Red and blue plots correspond to replicate MALDI-TOF spectra of derivatizated intermediates after 48 hours folding of reduced LA45 in Tris-HCl buffer, pH 8.4 with 10 mM CaCl₂. Expected mass is 9782 daltons for species with 6 disulfide bonds and 208 daltons more per disulfide bond unformed. There is only one peak corresponding to fully oxidized LA45 forms, without free cysteines. (b) RP-HPLC traces of the same LA45 intermediates analyzed in a. N and X45 correspond to native LA45 and the major scrambled isomer of LA45, respectively.

Fig 6. Analysis of interdomain disulfide bonds formation in LA45.

(a) MALDI-TOF spectra of LA45 folding intermediate fragments obtained after derivatization and cleavage with ARG-C proteinase. Red, fragment 74–90 0S; Dark Blue, fragment 44–73 1S; Green, fragment 1–41 2S; Orange 1–41 1S; Black 1–41 + 42–43 1S or, less probable, 44–73 + 74–90 3S, Light Blue, 42–43 + 44–73 + 74–90 3S; Purple, 1–41 + 42–43 + 44–73 + 74–90 4S or 5S. All fragments correspond to fragments without interdomain formation except the small signals detected after 15 min at 5456 (Light Blue) and 10173 daltons (purple). ±0,5–1% SD is expected in the mass data. The fragments that appear in this figure are highlighted in bold type in Tables 1 and 2. (b) RP-HPLC traces of the same LA45 intermediates analyzed in a.

Fig 7. Reversibility of LA4 and LA45 intermediates formed in the absence of calcium.

(a,b) RP-HPLC traces of LA4 (a) or LA45 (b) species formed after 48 hours folding of fully reduced LA4 (a) or LA45 (b) in Tris-HCl buffer, pH 8.4 with 1 to 1000 mM CaCl₂ and either 0.25 mM 2-mercaptoethanol, 0.5 mM GSSG (GSSG) or no additives (-). The chromatograms of native LA4 (a) and native LA45 (b) are also shown in the lower row for comparison. (c,d) RP-HPLC traces of LA4 (c) or LA45 (d) species formed after 24 hours folding of fully reduced LA4 (c) or LA45 (d) in Tris-HCl buffer, pH 8.4 with 0.1 mM EDTA and then 48 hours in Tris-HCl buffer, pH 8.4 with 1 to 1000 mM CaCl₂ and either 0.25 mM 2-BME, 0.5 mM GSSG (GSSG) or no additives (-). The chromatograms of native LA4 (c) and native LA45 (d) are also shown in the lower row for comparison. In the upper row, the chromatograms of LA4 intermediates after 24 hours folding of fully reduced LA4 (c) or LA45 (d) inTris-HCl buffer, pH 8.4 with 0.1 mM EDTA are shown for comparison.

Fig 8. Stop and go of the LA4 scrambled isomer Xa4 and reshuffling of native LA4.

(a,b) RP-HPLC of time course intermediates along the native LA4 reshuffling in Tris-HCl buffer, pH 8.4, 0.1 mM EDTA, without (a) or with (b) 0.25 mM 2-mercaptoethanol reducing agent. (a1, a2) LA4 isomers after 24 hours reshuffling of native LA4 in Tris-HCl buffer with 1 (a1) or 10 (a2) mM CaCl₂. (b1, b2) LA4 isomers after 24 hours reshuffling of native LA4 in Tris-HCl buffer with 0.25 mM 2-mercaptoethanol and 1 (b1) or 10 (b2) mM CaCl₂. (c,d) RP-HPLC of time course intermediates along the Xa4 scrambled isomer of LA4 stop and go in Tris-HCl buffer, pH 8.4, 10 mM CaCl₂, without (c) or with (d) 0.25 mM 2-mercaptoethanol reducing agent. (c1, c2) LA4 isomers after 24 hours reshuffling of Xa4 in Tris-HCl buffer with 100 (c1) or 1000 (c2) mM CaCl₂. (d1, d2) LA4 isomers after 24 hours reshuffling of Xa4 in Tris-HCl buffer with 0.25 mM 2-mercaptoethanol and 100 (d1) or 1000 (d2) mM CaCl₂.