Zinc-dependent dimerization of the folding catalyst, protein disulfide isomerase

Abstract

Protein disulfide isomerase (PDI), an essential folding catalyst and chaperone of the endoplasmic reticulum (ER), has four structural domains (a-b-b'-a'-) of approximately equal size. Each domain has sequence or structural homology with thioredoxin. Sedimentation equilibrium and velocity experiments show that PDI is an elongated monomer (axial ratio 5.7), suggesting that the four thioredoxin domains are extended. In the presence of physiological levels (<1 mM) of Zn(2+) and other thiophilic divalent cations such as Cd(2+) and Hg(2+), PDI forms a stable dimer that aggregates into much larger oligomeric forms with time. The dimer is also elongated (axial ratio 7.1). Oligomerization involves the interaction of Zn(2+) with the cysteines of PDI. PDI has active sites in the N-terminal (a) and C-terminal (a')thioredoxin domains, each with two cysteines (CGHC). Two other cysteines are found in one of the internal domains (b'). Cysteine to serine mutations show that Zn(2+)-dependent dimerization occurs predominantly by bridging an active site cysteine from either one of the active sites with one of the cysteines in the internal domain (b'). The dimer incorporates two atoms of Zn(2+) and exhibits 50% of the isomerase activity of PDI. At longer times and higher PDI concentrations, the dimer forms oligomers and aggregates of high molecular weight (>600 kDa). Because of a very high concentration of PDI in the ER, its interaction with divalent ions could play a role in regulating the effective concentration of these metal ions, protecting against metal toxicity, or affecting the activity of other (ER) proteins that use Zn(2+) as a cofactor.

Figure 1.

Oligomerization of PDI in the presence of Zn²⁺. PDI (4.4 μM) was incubated with 1 mM zinc acetate at room temperature for the indicated time and then separated by gel-filtration HPLC. Initially (0 h, top left), PDI was all monomeric. After 1 h, three peaks could be observed (top right), corresponding to aggregate (A), dimer (D), and monomer (M). After 3 or 24 h of incubation (bottom), more PDI is converted into dimer and eventually aggregates.

Figure 2.

Dependence of the disappearance of PDI monomer on the concentration of Zn²⁺. PDI (20 μM) was incubated in 50 mM HEPES (pH 7.0) with different concentrations of Zn²⁺ for 17 h at room temperature, and the amount of residual monomer determined by gel-filtration HPLC is shown. The curve is a rectangular hyperbola with a Zn½ of 0.2 ± 0.03 mM.

Figure 3.

Time dependence of PDI oligomerization. PDI (4.4 μM) was incubated with 1 mM Zn²⁺ for various times, and the amounts of 100-kDa species, 200-kDa species, and aggregates (>600 kDa) were determined by gel-filtration HPLC: monomer (solid circles), dimer (solid squares), and aggregate (open circles).

Figure 4.

Oligomerization of PDI mutants with cysteine to serine mutations. The amount of the PDI present as dimer after 1-h incubation with 1 mM Zn²⁺ was determined by HPLC. The reaction is not kinetically complete at this time point, and the amount of dimer formed is a complex function between aggregation and dimerization. The mutants are designated by a symbol showing the three different types of cysteine residues, including those of the CGHC sequence at the two active sites and the two located in the b′ domain (dotted lines). An empty box indicates that both cysteines have been mutated to serine, and CX indicates that the active site cysteine nearer the C terminus (CGHC) of each active site has been mutated to ser (CGHS).

Figure 5.

PDI aggregation in the presence of Zn²⁺. PDI at different concentrations was incubated with 1 mM Zn²⁺ at pH 7.0 (50 mM HEPES and 0.2 M NaCl) for 1 h. The relative amount of monomer (solid circles), dimer (solid squares), and aggregate (open circles) was determined by gel-filtration HPLC.

Figure 6.

Models for PDI dimerization. Dimer formation can occur through Zn²⁺-dependent cross-linking of domains a-b′ or domains a′ -b′ . For each type of dimer, the two PDI molecules could be oriented either in parallel or antiparallel directions. This gives rise to the possibility of four different dimers. The monomers are drawn with an axial ratio of 5.7, as suggested by sedimentation velocity experiments.