Mutational Alterations of the Key cis Proline Residue That Cause Accumulation of Enzymatic Reaction Intermediates of DsbA, a Member of the Thioredoxin Superfamily

Abstract

The DsbA-DsbB pathway introduces disulfide bonds into newly translocated proteins. Conversion of the conserved cis proline 151 of DsbA to several hydrophilic residues results in accumulation of mixed disulfides between DsbA and its dedicated oxidant, DsbB. However, only a proline-to-threonine change causes accumulation of mixed disulfides of DsbA with its substrates.

Formation of disulfide bonds is important for the folding, stability, and activity of many proteins exported out of the cytoplasm. In both prokaryotes and eukaryotes, proteins with a thioredoxin fold are responsible for this process (17, 24, 30). In the periplasm of Escherichia coli, DsbA, a thioredoxin superfamily member, introduces disulfide bonds directly into substrate proteins by donating the disulfide bond in its active-site Cys30-Pro31-His32-Cys33 to a pair of cysteines in substrate proteins (Fig. 1, stages 1 to 3) (3, 9, 19). DsbA, in turn, is maintained in its oxidized state (20) by a membrane protein DsbB, which uses its two pairs of essential cysteines (Cys41-Cys44 and Cys104-Cys130) to transfer electrons from DsbA to quinones in the respiratory chain (Fig. 1, stages 4 to 6) (1, 2, 13, 14, 16, 22, 28). Both of these reactions are thought to occur via formation of a mixed disulfide complex between Cys30 of DsbA and one of the cysteines of a substrate (for substrate oxidation) (Fig. 1, stage 2) (6, 7, 18, 33) and between Cys30 of DsbA and Cys104 of DsbB (for reoxidation of DsbA) (Fig. 1, stage 5) (11, 13, 16, 21).

FIG. 1.

Different stages of DsbA function. See main text for description.

We have previously described two mutations which altered residue Pro151 of DsbA and resulted in defects in two different steps in the disulfide bond formation pathway (18). One mutant, P151T, appears to slow down step 2 of the reaction of DsbA with substrate (Fig. 1), thus allowing detection of DsbA in the act of substrate oxidation (Fig. 1, stage 2). The other mutant, P151S, interferes with the transfer of electrons from DsbA to DsbB, accumulating a large amount of a mixed disulfide complex of DsbA and DsbB, a presumed intermediate in the process of reoxidation of DsbA by DsbB (Fig. 1, stage 5) (8, 13, 16). This alteration appears to cause a defect in a step required for resolution of the DsbA-DsbB complex. In DsbA, Pro151 assumes the cis configuration and is positioned very close to the active-site cysteines of DsbA (5, 10, 24, 25).

These mutants are quite useful for identifying intermediates in the enzymatic reactions of DsbA, for identifying the substrates of DsbA, and for analyzing details of the electron transfer pathway. Since a similarly positioned proline is found in nearly all proteins containing thioredoxin-like domains (12, 24, 26), the characterization of similar mutants for these other proteins may be equally useful. In order to begin to determine the utility of this approach, we have examined the effects of altering Pro151 of DsbA to each of the remaining 17 amino acids. We did this in order to see which other amino acid changes, if any, in this residue would give similar phenotypes. Our results may be helpful in the study of other members of the thioredoxin superfamily.

Plasmid constructions.

To construct plasmids that express each of the DsbA mutants, substitution mutations were introduced into the dsbA gene of plasmid pHK520 by using a QuikChange site-directed mutagenesis kit (Stratagene) and appropriate mutagenic primers (Table 1). The plasmid pHK520 is a pSC101-derived low-copy-number plasmid carrying dsbA under the lac promoter. This plasmid was constructed by inserting the DsbA-encoding 0.7-kb KpnI-XbaI fragment of pCH3 (11) into pAM238 (pSC101 ori, Spec^r, lac promoter) (16). Importantly, when the cells were grown on M63 minimal glucose medium (15), pHK520 expressed DsbA at levels comparable to those of DsbA from the chromosome (data not shown). This level of expression is crucial to the success of such studies. We have found that even slight increases in expression of DsbA over wild-type levels can ameliorate or eliminate the phenotypic effects of interesting mutants (V. C. Tam, H. Kadokura, and J. Beckwith, unpublished results).

TABLE 1.

Strains and plasmids

Strain or plasmid	Relevant genotype or features	Reference or source
Strains
HK295	F−Δara-714 galU galK Δ(lac)X74 rpsL thi	16
HK317	HK295 ΔdsbA	18
HK320	HK295 ΔdsbB	16
Plasmids
pAM238	lac promoter, pSC101-based, Spcr	16
pDSW206	Mutations in trc promoter of pTrc99A, pBR322 based, Blar, lacIq	32
pHK520	pAM238 DsbA	This study
pHK651	pAM238 DsbA P151T	This study
pHK652	pAM238 DsbA P151S	This study
pHK653	pAM238 DsbA P151A	This study
pHK675	pDSW206 ΔlacIq	This study
pHK677	pHK675 RcsF-c-Myc	This study
pLN101	pAM238 DsbA P151F	This study
pLN102	pAM238 DsbA P151L	This study
pLN103	pAM238 DsbA P151I	This study
pLN104	pAM238 DsbA P151M	This study
pLN105	pAM238 DsbA P151V	This study
pLN106	pAM238 DsbA P151Y	This study
pLN107	pAM238 DsbA P151H	This study
pLN108	pAM238 DsbA P151Q	This study
pLN111	pAM238 DsbA P151N	This study
pLN112	pAM238 DsbA P151K	This study
pLN113	pAM238 DsbA P151D	This study
pLN114	pAM238 DsbA P151E	This study
pLN115	pAM238 DsbA P151C	This study
pLN116	pAM238 DsbA P151W	This study
pLN117	pAM238 DsbA P151R	This study
pLN118	pAM238 DsbA P151G	This study

Effects of Pro151 mutations on ability of DsbA to oxidize substrate proteins.

To examine the effect of mutations on the ability of DsbA to oxidize substrates, we assessed the oxidative state of two substrates of DsbA (β-lactamase and RcsF) (18) in the mutants. To detect RcsF, we fused it with c-Myc at its C terminus. Plasmid pHK677, which was used to express both β-lactamase and RcsF-c-Myc, was constructed by inserting the RcsF-c-Myc-encoding 460-bp KpnI-XbaI fragment of pHK646 (18) into pHK675 (pBR322 ori, bla, weakened lac promoter). The expression vector pHK675 was generated by deleting the lacI^q-containing 872-bp BssHII fragment from pDSW206 (pBR322 ori, bla, weakened trc promoter, lacI^q) (32).

We transformed E. coli strain HK317 (ΔdsbA) with both pHK677 (carrying bla and RcsF-c-Myc) and each of the dsbA mutant plasmids and examined the abilities of the mutants to promote disulfide bond formation in β-lactamase (Fig. 2A) and RcsF-c-Myc (Fig. 2B). To distinguish the oxidized (disulfide-bonded) form from the reduced form of β-lactamase and RcsF-c-Myc by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), cellular proteins were first treated with acid to inhibit thiol-disulfide reactivity and then the free cysteines were alkylated with 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS) (15). This modification retards the mobility of the reduced forms of proteins on gels. In the ΔdsbA strain expressing the wild-type dsbA from the plasmid, both β-lactamase and RcsF-c-Myc were completely oxidized (Fig. 2, lane 2). However, in the absence of the dsbA plasmid, the substrate proteins were mostly reduced (Fig. 2, lane 1), confirming that both proteins are the substrates of DsbA.

FIG. 2.

Capabilities of the DsbA P151 mutants to oxidize the substrate proteins in vivo. Strain HK317 (ΔdsbA) was transformed with both pHK677 (carrying bla and rcsF-c-Myc) and one of the following plasmids: pAM238 (empty vector; lane 1), pHK520 (wild-type DsbA; lane 2), pLN101 (P151F; lane 3), pLN102 (P151L; lane 4), pLN103 (P151I; lane 5), pLN104 (P151M; lane 6), pLN105 (P151V; lane 7), pLN106 (P151Y; lane 8), pLN107 (P151H; lane 9), pLN108 (P151Q; lane 10), pLN111 (P151N; lane 11), pLN112 (P151K; lane 12), pLN113 (P151D; lane 13), pLN114 (P151E; lane 14), pLN115 (P151C; lane 15), pLN116 (P151W; lane 16), pLN117 (P151R; lane 17), pLN118 (P151G; lane 18), pHK651 (P151T; lane 19), pHK652 (P151S; lane 20), and pHK653 (P151A; lane 21). Cells were grown at 30°C in M63 minimal glucose medium, and cellular proteins from the exponential culture were alkylated with AMS and separated with SDS-PAGE. Disulfide bond formation in β-lactamase (Bla; two cysteines) (A) and RcsF-c-Myc (RcsF; four cysteines) (B) was visualized with Western blotting by using anti-Bla (5 Prime 3 Prime, Inc., Boulder, Colo.) and anti-c-Myc (A-14; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) antibodies.

Three mutants (P151K, P151C, and P151R) showed strong defects, leaving a large portion of the substrates in their reduced forms (Fig. 2, lanes 12, 15, and 17) and exhibiting phenotypes that suggest defects in multiple steps of the enzyme reaction (data not shown). One of these, P151C, introduces a third cysteine into the active site region of the protein and may, thus, interfere with the normal redox activity of the protein. The other two, P151R and P151K, introduce basic amino acids into the region, which could alter the ionic state of the cysteines in reduced DsbA (4, 23) and/or disrupt the structure of this region.

However, the rest of the mutants showed weak or no detectable defects in substrate oxidation: they oxidized more than 50% of the two substrates. Notably, the P151F, P151L, P151I, P151M, P151V, P151Y, P151Q, P151E, and P151A mutants oxidized substrates almost as well as the wild-type enzyme (Fig. 2).

In many of the mutants, defects in disulfide bond formation were more pronounced in β-lactamase than in RcsF-c-Myc (e.g., P151S [Fig. 2, lane 20]). This observation may be explained by the fact that β-lactamase can rapidly fold into its active folded structure even in the absence of disulfide bond formation and, once folded, its cysteines are inaccessible to DsbA (3).

To characterize the 16 mutants that showed substantial in vivo activity, we examined the redox state of DsbA by probing the AMS-alkylated lysate with antibody to DsbA (Fig. 3). We made the following observations on the implications of the combined data for this collection of mutants.

FIG. 3.

In vivo redox states of the DsbA mutants. AMS-alkylated lysates of the following strains were separated by SDS-PAGE and analyzed with Western blotting using antibody to DsbA (15). Lanes: 1, HK317/pAM238; 2, HK320 (ΔdsbB)/pAM238; 3, HK317/pHK520; 4, HK317/pLN101; 5, HK317/pLN102; 6, HK317/pLN103; 7, HK317/pLN104; 8, HK317/pLN105; 9, HK317/pLN106; 10, HK317/pLN107; 11, HK317/pLN108; 12, HK317/pLN111; 13, HK317/pLN113; 14, HK317/pLN114; 15, HK317/pLN116; 16, HK317/pLN118; 17, HK317/pHK651; 18, HK317/pHK652; 19, HK317/pHK653. Open arrowheads, nonspecific bands; asterisks, a mixed-disulfide complex formed between DsbA and another protein (H. Kadokura and J. Beckwith, unpublished results); closed arrowheads in lane 17, DsbA-substrate complexes.

P151T.

This amino acid alteration, characterized previously (18), is the only one of all those tested here to cause a significant defect in resolution of DsbA-substrate complexes (Fig. 1, step 2). The mutant strain, thus, accumulates numerous DsbA-substrate complexes (Fig. 3, lane 17) (18). The P151T and P151S proteins, which exhibit very different phenotypes, showed the same standard redox potential of −152 and −151 mV, respectively, indicating that the redox potentials alone cannot explain the phenotypes of these two mutations (18). Thus, some structural feature resulting from this specific amino acid change appears to prevent the second cysteine of the substrate from resolving this complex (12, 18).

P151H, P151S, P151N, P151W, and P151G.

These five mutants (P151S was characterized previously [18]) accumulate a major band with an apparent molecular mass of 36 kDa, in addition to the reduced and oxidized forms of DsbA (Fig. 3, lanes 10, 12, 15, 16, and 18). Since this band was recognized by anti-DsbB antibody and disappeared when samples were treated with reductant (data not shown), it represents a mixed disulfide complex between DsbB and DsbA. Further coexpression of wild-type DsbA from the chromosome to compete for oxidation of substrate proteins resulted in disappearance of most of the complex (data not shown), indicating that the DsbA-DsbB complex accumulates when mutant DsbA is actively oxidizing substrate proteins. Thus, the DsbA-DsbB complex forms in the process of oxidation of DsbA by DsbB.

The P151H and P151S changes result in the greatest accumulation of DsbA-DsbB complex. The side chains of serine, threonine, or histidine residues each have the potential to form a hydrogen bond with the sulfhydryl group of a cysteine residue. Such bonding, in the context of a DsbA-DsbB or DsbA-substrate complex, may alter resolvability of mixed disulfides.

P151F, P151L, P151I, P151V, P151Y, and P151M.

Five of these six hydrophobic substitutions exhibited the least effect on DsbA activity, showing efficient oxidation of substrates (Fig. 2) and no alteration of the redox state of DsbA (Fig. 3). Nevertheless, they, as well as the other substitution mutants, did show varying degrees of hypersensitivity to Cd²⁺, a phenotype typical of dsbA and dsbB mutants (data not included) (29, 31). In the structures that have been reported for mixed disulfide complexes between human thioredoxin and two substrate peptides, the ring of the analogous proline forms van der Waals contacts with the sulfur of the cysteine of the substrate involved in the mixed disulfide bond (27). Thus, the hydrophobic amino acids substituted for DsbA's Pro151 may suffice for formation of similar contacts. These contacts may be important for the proper DsbA-DsbB interactions (see below; P151M) and resolution of mixed disulfides between either DsbA and substrate or DsbA and DsbB.

The exception to the lack of effects of the hydrophobic substitutions is the methionine substitution, which accumulates a larger amount of the reduced form of DsbA without accumulation of DsbA-DsbB complex (P151M; Fig. 3, lane 7), suggesting a defect in a step required for DsbA-DsbB complex formation. This difference may be related to the large size of the methionine residue.

Concluding remarks.

Our previous work suggested that mutating the conserved cis proline residue of thioredoxin superfamily members might be useful for studies on the mechanisms involved in electron transfer pathways, particularly in the detection of mixed disulfide intermediates in these pathways. Here we have shown that alteration of proline 151 to five other amino acid residues causes the accumulation of mixed disulfide complexes between DsbA and its dedicated oxidant DsbB. Thus, the specificity of the defect is not limited to a single specific mutational alteration, and such mutants may have similarly useful properties in other systems. The same is not true for alterations that cause accumulation of DsbA-substrate complexes, where only the P151T change shows such a phenotype. Further studies with other thioredoxin family members will be necessary to determine whether the change from proline to threonine has similar effects, whether other changes might work, or whether the phenomenon is only seen in a subset of these proteins.