Protein-protein interactions play a central role in most biological processes. One such biological process is the maintenance of a reducing environment inside the cell. To maintain an internal reducing environment, living cells have evolved two enzymatic systems (glutathione and thioredoxin (Trx) systems). The Trx system is composed of the enzyme TrxR and its substrate Trx. The two proteins constitute an important thiol-dependent redox system that catalyzes the reduction of many proteins that are responsible for a variety of cellular functions. The system relies on transient protein-protein interactions between Trx and TrxR for its function.
Cross-reactivity of components of the Trx system between species has been shown to be medically relevant. For example, Helicobacter pylori Trx (HP Trx) is thought to mediate catalytic reduction of human immunoglobulins and thus facilitate immune evasion. It has also been proposed that Helicobacter pylori gains access to the impenetrable gastric mucous layer by using secreted HP Trx to reduce the disulfide bonds present in the cysteine-rich mucin regions that are responsible for cross-linking mucin monomers. Therefore, disruption of secreted HP Trx-host protein interaction may result in restoration of the viscoelastic and hydrophobic protective properties of mucus. Previous studies aimed at understanding the nature of cross-reactivity of Trx system components among various species have shown that Trxs have higher affinity for cognate TrxRs (same species), than for TrxRs from different species. However, the basis for this specificity is not known. A growing body of evidence suggests that most protein-protein interactions are mediated by a small number of protein-protein interface residues, referred to as “hot spot” residues or binding epitopes. Therefore, understanding the biochemical basis of the affinity of proteins for their partners usually begins by identifying the hot spot residues responsible for the protein complex interactions.
In this study, the crystal structures of Deinococcus radiodurans thioredoxin reductase (DR TrxR) and Helicobacter pylori TrxR (HP TrxR) were determined at 1.9 Å and 2.4 Å respectively. Analysis of the Trx-binding sites of both structures suggests that the basis of affinity and specificity of Trx for TrxR is primarily due to the shape rather than the charge of the surface. In addition, the complex between Escherichia coli thioredoxin reductase (EC TrxR) and its substrate thioredoxin (EC Trx) was used to identify residues that are responsible for TrxR-Trx interface stability. Using computational alanine scanning mutagenesis and visual inspection of the EC TrxR-Trx interface, 22 EC TrxR side chains were shown to make contact across the TrxR-Trx interface. Although more than 20 EC TrxR side chains make contact across the TrxR-Trx interface, our results suggest that only 4 residues (F81, R130, F141, and F142) account for the majority of the EC TrxR-Trx interface stability. Individual replacement of equivalent DR TrxR residues (M84, K137, F148, F149) with alanine resulted in drastic changes in binding affinity, confirming that the four residues account for most of TrxR-Trx interface stability. These hot spot residues are surrounded by less important residues (hydrophobic and hydrophilic) that are also predicted to contribute to interface stability. F148 and F149 are invariant across bacterial TrxRs, however other residues that contact Trx are less conserved including M84 and K137. When M84 and K137 were changed to match equivalent E. coli TrxR residues (K137R, M84F); D. radiodurans TrxR substrate specificity was altered from its own Trx to that of E. coli Trx. The results suggest that a small subset of the TrxR-Trx interface residues are responsible for the majority of Trx binding affinity and specificity, a property that has been shown to general to protein-protein interfaces.||en_US