In Chapter 3, we learned that both intramolecular and inter molecular disulfide bonds (–S–S–) help stabilize the tertiary and quaternary structure of many proteins. These covalent bonds form by the oxidative linkage of sulfhydryl groups (–SH), also known as thiol groups, on two cysteine residues in the same or different polypeptide chains. This reaction can proceed only when a suitable oxidant is present. In eukaryotic cells, disulfide bonds are formed only in the lumen of the rough ER. Thus disulfide bonds are found only in soluble secretory proteins and in the exoplasmic domains of membrane proteins. Cytosolic proteins and organelle proteins synthesized on free ribosomes (i.e., those destined for mitochondria, chloroplasts, peroxisomes, etc.) usually lack disulfide bonds.
The efficient formation of disulfide bonds in the lumen of the ER depends on the enzyme protein disulfide isomerase (PDI), which is present in all eukaryotic cells. This enzyme is especially abundant in the ER of secretory cells in organs such as the liver and pancreas, where large quantities of proteins that contain disulfide bonds are produced. As shown in Figure 13-19a, the disulfide bond in the active site of PDI can be readily transferred to a protein by two sequential thiol-disulfide transfer reactions. The reduced PDI generated by this reaction is returned to an oxidized form by the action of an ER-resident protein, called Ero1, which carries a disulfide bond that can be transferred to PDI. Ero1 itself becomes oxidized by reaction with molecular oxygen that has diffused into the ER.
In proteins that contain more than one disulfide bond, the proper pairing of cysteine residues is essential for nor mal structure and activity. Disulfide bonds are commonly formed between cysteines that occur sequentially in the amino acid sequence while a polypeptide is still growing on the ribosome. Such sequential formation, however, sometimes yields disulfide bonds between the wrong cysteines. For example, proinsulin, a precursor to the pep tide hormone insulin, has three disulfide bonds that link cysteines 1 and 4, 2 and 6, and 3 and 5. In this case, a disulfide bond that initially formed sequentially (e.g., between cysteines 1 and 2) would have to be rearranged for the protein to achieve its proper folded conformation. In cells, the rearrangement of disulfide bonds is also accelerated by PDI, which acts on a broad range of protein substrates, al lowing them to reach their thermodynamically most stable conformations (Figure 1b). Disulfide bonds generally form in a specific order, first stabilizing small domains of a polypeptide, then stabilizing the interactions of more dis tant segments; this phenomenon is illustrated by the folding of the influenza hemagglutinin (HA) protein, discussed in the next section.
Fig1. Action of protein disulfide isomerase (PDI). PDI forms and rearranges disulfide bonds via an active site with two closely spaced cysteine residues that are easily interconverted between the reduced dithiol form and the oxidized disulfide form. Numbered red arrows indicate the sequence of electron transfers. Yellow bars represent disulfide bonds. (a) In the formation of disulfide bonds, the ionized (–S−) form of a cysteine thiol in the substrate protein reacts with the disulfide (S–S) bond in oxidized PDI to form a disulfide- bonded PDI–substrate protein intermediate. A second ionized thiol in the substrate protein then reacts with this intermediate, forming a disulfide bond within the substrate protein and releasing reduced PDI. PDI, in turn, transfers electrons to a disulfide bond in the luminal protein Ero1, thereby regenerating the oxidized form of PDI. (b) Reduced PDI can catalyze rearrangement of improperly formed disulfide bonds by similar thiol-disulfide transfer reactions. In this case, reduced PDI both initiates and is regenerated in the reaction pathway. These reactions are repeated until the most stable conformation of the protein is achieved. See M. M. Lyles and H. F. Gilbert, 1991, Biochemistry 30:619.
