Structure of Reduced DsbA from Escherichia coli in Solution

Biochemistry 1998, 37, 6263-6276

6263

Structure of Reduced DsbA from Escherichia coli in Solution†,‡ Horst Joachim Schirra,§,| Christian Renner,§ Michael Czisch,§,⊥ Martina Huber-Wunderlich,| Tad A. Holak,*,§ and Rudi Glockshuber*,| Max-Planck-Institut fu¨ r Biochemie, Am Klopferspitz 18a, D-82152 Martinsried, Germany, and Institut fu¨ r Molekularbiologie und Biophysik, Eidgeno¨ ssische Technische Hochschule Ho¨ nggerberg, CH-8093 Zu¨ rich, Switzerland ReceiVed January 20, 1998 ABSTRACT: The three-dimensional structure of reduced DsbA from Escherichia coli in aqueous solution has been determined by nuclear magnetic resonance (NMR) spectroscopy and is compared with the crystal structure of oxidized DsbA [Guddat, L. W., Bardwell, J. C. A., Zander, T., and Martin, J. L. (1997) Protein Sci. 6, 1148-1156]. DsbA is a monomeric 21 kDa protein which consists of 189 residues and is required for disulfide bond formation in the periplasm of E. coli. On the basis of sequence-specific 1H NMR assignments, 1664 nuclear Overhauser enhancement distance constraints, 118 hydrogen bond distance constraints, and 293 dihedral angle constraints were obtained as the input for the structure calculations by simulated annealing with the program X-PLOR. The enzyme is made up of two domains. The catalytic domain has a thioredoxin-like fold with a five-stranded β-sheet and three R-helices, and the second domain consists of four R-helices and is inserted into the thioredoxin motif. The active site between Cys30 and Cys33 is located at the N terminus of the first R-helix in the thioredoxin-like domain. The solution structure of reduced DsbA is rather similar to the crystal structure of the oxidized enzyme but exhibits a different relative orientation of both domains. In addition, the conformations of the active site and a loop between strand β5 and helix R7 are slightly different. These structural differences may reflect important functional requirements in the reaction cycle of DsbA as they appear to facilitate the release of oxidized polypeptides from reduced DsbA. The extremely low pKa value of the nucleophilic active site thiol of Cys30 in reduced DsbA is most likely caused by its interactions with the dipole of the active site helix and the side chain of His32, as no other charged residues are located next to the sulfur atom of Cys30 in the solution structure.

The formation of disulfide bonds in secretory proteins is often a slow and rate-limiting step during protein folding and is catalyzed by enzymes in vivo (1). In the periplasmic space of Escherichia coli, the thiol-disulfide oxidoreductase DsbA is required for the introduction of disulfide bridges into folding polypeptides (2, 3). DsbA possesses a catalytic disulfide bond and randomly oxidizes reduced, unfolded proteins in an extremely rapid disulfide exchange reaction (4-6). Reduced DsbA is then reoxidized by disulfide interchange with DsbB, a protein from the inner membrane of E. coli (7-11). DsbA is a soluble monomer of 189 amino acids (2). The crystal structure of oxidized DsbA has been determined to 1.7 Å resolution and has revealed that DsbA consists of two domains (12, 13). The catalytic domain (residues 1-62 and 139-189) has the thioredoxin fold, the common motif of † This work was supported by grants from the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 533 der Ludwig-MaximiliansUniversita¨t Mu¨nchen), the ETH Zu¨rich, and the Fonds der Chemischen Industrie. ‡ The coordinates of the 20 best conformers and the energyminimized mean structure of reduced DsbA have been deposited in the Brookhaven Protein Data Bank (accession codes 1a24 and 1a23). * Corresponding authors. For a proof query contact Rudi Glockshuber. Phone: +41 (1) 633-6819. Fax: +41 (1) 633-1036. § Max-Planck-Institut fu ¨ r Biochemie. | Eidgeno ¨ ssische Technische Hochschule Ho¨nggerberg. ⊥ Present address: Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, NL-3584 CH Utrecht, The Netherlands.

all thiol-disulfide oxidoreductases, although there is only 10% sequence identity between the catalytic domain of DsbA and E. coli thioredoxin (12, 14). The second domain (residues 63-138) exclusively consists of R-helices and is inserted into the thioredoxin motif. The active site of DsbA (Cys30-Pro31-His32-Cys33) is located at the amino-terminal end of the first R-helix in the thioredoxin-like domain. While the thiol of Cys30 is solvent-exposed and acts as a nucleophile in reduced DsbA, the thiol of Cys33 is buried in the interior of the protein (13, 15-17). Despite the structural similarities with other thiol-disulfide oxidoreductases such as thioredoxin, glutaredoxin, thioltransferase, and protein disulfide isomerase (14, 18, 19), DsbA shows remarkable biophysical properties which make it unique in this protein family. The enzyme is the most oxidizing thiol-disulfide oxidoreductase with a redox potential (E0′) of -122 mV (20, 21), compared to a value of -270 mV for the most reducing member of the protein family, thioredoxin (22, 23). Reduced DsbA is more stable than the oxidized form (24), and the enzyme shows very high reaction rates with thiol substrates even at low pH (4, 6, 15, 25). Thus, DsbA is an ideal oxidant for reduced polypeptides in the bacterial periplasm. The oxidative force of DsbA, its destabilizing disulfide bond, and its fast disulfide exchange reactions can, at least qualitatively, be explained by the extremely low pKa of 3.5 of the nucleophilic thiol of Cys30 (26). In addition, DsbA has been shown to interact specifically with reduced, unfolded polypeptides and appears

S0006-2960(98)00136-6 CCC: $15.00 © 1998 American Chemical Society Published on Web 04/16/1998

6264 Biochemistry, Vol. 37, No. 18, 1998 to possess a peptide binding site (4-6, 25, 27) which has been attributed to a hydrophobic region in the vicinity of the active site disulfide (13). However, the molecular basis for the low pKa value of Cys30 in reduced DsbA is not yet completely understood because the structure of reduced DsbA has not been determined so far. It is generally assumed that interactions between the Cys30 thiolate and the dipole of the active site helix (26, 28-30) as well as the residues of the dipeptide between the two active site cysteines (21, 23, 31-35) are mainly responsible for the pKa of DsbA’s nucleophilic cysteine. Additional mechanisms have been discussed for DsbA and the homologous protein thioredoxin, such as a charged hydrogen bond between the thiolate and the thiol or the amide hydrogen of the buried cysteine (29, 36-39) or interactions with charged residues in the vicinity of the active site (40). Apart from this, a structural comparison of oxidized and reduced DsbA should shed light on the physicochemical properties of the enzyme. It has for example been speculated that both domains of DsbA could move relative to each other when the protein is reduced and that such a movement could be of functional significance (13, 41). On the other hand, the structural comparison of the oxidized and reduced forms of human and E. coli thioredoxin and E. coli glutaredoxin revealed only slight local differences (29, 37, 39, 42-47). Overall, knowledge of the three-dimensional structure of reduced DsbA is required for understanding the extraordinary properties of this enzyme at a molecular level. Here we report the determination of the three-dimensional structure of reduced DsbA at pH 3.7 and 300 K in solution by NMR1 spectroscopy. We compare the structure of reduced DsbA with the crystal structure of the oxidized protein (13) and discuss the structural differences between both redox forms with respect to their functional significance. MATERIALS AND METHODS Materials. 1,4-Dithio-DL-threitol (DTT), amino acids, ampicillin, and polymyxin B sulfate were purchased from Sigma (Deisenhofen, Germany). Isopropyl β-D-thiogalactoside (IPTG) was from AGS GmbH (Heidelberg, Germany). [15N]Ammonium chloride and [13C]glucose were obtained from Campro Scientific (Emmerich, Germany). D2O was from Cambridge Isotope Laboratories (Cambridge, Great Britain). DE52 cellulose was purchased from Whatman (Maidstone, United Kingdom) and phenyl Sepharose from Pharmacia (Uppsala, Sweden). All other chemicals were from Merck (Darmstadt, Germany) or Fluka (Buchs, Switzerland) and of the highest purity available. Sample Preparation. For the preparation of uniformly 15Nlabeled DsbA, the protein was overproduced in E. coli BL21 1 Abbreviations: 2D, two-dimensional; 3D, three-dimensional; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; TOCSY, total correlation spectroscopy; CBCANH, intra- and interresidue 1HN, 15N, 13Cβ, and 13CR correlation; CBCA(CO)NH, interresidue 1HN, 15N, 13Cβ, and 13CR correlation; ct, constant time; HBHA(CBCA)NH, intra- and interresidue 1HN, 15N, 1Hβ, and 1HR correlation; HBHA(CBCACO)NH, interresidue 1HN, 15N, 1Hβ, and 1HR correlation; HCACO, intraresidue 1HR, 13CR, and 13C′ correlation; HNCA, intra- and interresidue 1HN, 15N, and 13CR correlation; HN(CA)CO, intra- and interresidue 1HN, 15N, and 13C’ correlation; HNCO, interresidue 1HN, 15N, and 13C’ correlation; HNHA, intraresidue 1HN, 15N, and 1HR correlation; HSQC, heteronuclear single-quantum correlation; rmsd, root-mean-square deviation; SA, simulated annealing.

Schirra et al. cells (48) harboring the plasmid pRBI-PDI-T7 (49). The cells were grown at 26 °C in a M9 minimal medium (50) containing ampicillin (100 µg/mL) and 1 g/L 15NH4Cl as the sole nitrogen source. Selective 15N labeling of glycines and serines was achieved with M9 minimal medium that contained 1 g/L 15NH4Cl, 1 g/L [15N]glycine, and the residual unlabeled amino acids, except for serine (51). For preparation of uniformly 15N and 13C doubly labeled DsbA, cells were grown in a minimal medium (52) that contained 1 g/L 15NH4Cl and 1 g/L [13C]glucose as sole sources of nitrogen and carbon. Purification of DsbA by anion exchange chromatography on DE52 cellulose and hydrophobic chromatography on phenyl Separose was performed as previously described (20). The DsbA concentration was determined by its absorbance at 280 nm (20). The purified protein was shown to be fully oxidized. Purified DsbA was dialyzed against 20 mM H3PO4/NaOH, at pH 3.7 and concentrated to 1-3.5 mM. Finally, the protein was reduced in the NMR tube by a 5-fold molar excess of d10-DTT and kept under nitrogen. The samples contained either 5% D2O (v/v) (“samples in H2O”) or 100% D2O in a volume of 500 µL. NMR Spectroscopy. (1) General Conditions. Spectra were recorded at 300 K on Bruker AMX600, DRX600, and DMX750 spectrometers with triple-resonance self-shielded z-gradient 5 mm probes. Quadrature detection in the indirect dimensions was achieved using the TPPI (53) or the StatesTPPI method (54). Sweep widths were 14.17 ppm for 1H, 34.53 ppm for 15N, and 75.3 ppm for 13C. Carriers were positioned at 4.73 ppm (1H), 119.24 ppm (15N), and 34.11 ppm (13C) for the respective nuclei. NOESY experiments were recorded with a pulse sequence in which the last 90° pulse was either substituted by a jump-return sequence (55) or followed by a 3-9-19 WATERGATE sequence (56) to suppress the water resonance. TOCSY experiments used the MLEV16 sequence (57) for isotropic mixing and included a presaturation of the water resonance (58) for measurements in water. Essentially the same pulse sequences were used for measurements in D2O, with the exception that no water suppression was necessary. (2) Data Acquisition. The following spectra were recorded on the 15N-labeled sample: 15N-NOESY-HSQC (59) with 180 × 68 × 1K data points in f1, f2, and f3, respectively, and a mixing time of 110 ms and 15N-TOCSY-HSQC (60) with 176 × 54 × 1K data points and a spin-lock period of 61.5 ms. Additionally, a 15N-HSQC spectrum (61) was recorded with 108 × 1K* data points in f1 and f2, respectively (* denotes complex data points). For identification of glycine and serine residues, a 15N-HSQC spectrum with a size of 64 × 1K* data points was recorded on the Gly and Ser 15Nlabeled sample. The following spectra were recorded on the uniformly 15N and 13C doubly labeled sample in 5% D2O: ct-HNCA (62), HN(CA)CO (63), ct-HNCO (62, 64), CBCANH (65), CBCA(CO)NH (64), HBHA(CBCACO)NH (66), HBHA(CBCA)NH (67), HNHA (68), HNHB (69), ctHCACO (70), and HCCH-TOCSY (71) (114* × 49* × 512* data points with an 18 ms mixing time for the HCCHTOCSY (a DIPSI-3y sequence (72) was used for isotropic mixing)); HCCH-TOCSY (180* × 70* × 1K* data points with a 16 ms spin-lock time); and 13C-NOESY-HSQC (73) (172 × 124 × 512* data points with a mixing time of 100 ms). In addition, a 13C-NOESY-HSQC spectrum (216* ×

Solution Structure of Reduced DsbA 64* × 1K* data points) with a 120 ms mixing time was recorded on the doubly labeled sample dissolved in D2O. A homonuclear 2D NOESY spectrum (74) with a mixing time of 60 ms (960 × 2K* data points) and a 2D TOCSY spectrum with a spin-lock period of 40 ms (512 × 2K* data points) were recorded on an unlabeled DsbA sample in H2O. In addition, a 2D NOESY spectrum with a mixing time of 40 ms (1050 × 2K* data points) was recorded on an unlabeled sample in 100% D2O. An amide proton exchange experiment was performed after lyophilization of 15N-labeled, reduced DsbA in 20 mM H3PO4/NaOH at pH 3.7 and dissolving the protein in D2O. Within 24 h, 10 15N-HSQC spectra (128 × 1K* data points) were recorded. Additional 15 N-HSQC spectra were recorded after 1, 2, and 8 weeks. (3) Processing of Data. All spectra were processed and analyzed with the in-house written programs CCNMR and GLXCC (75). Zero filling or linear prediction was used to prolong the number of time domain points in f1 and f2. Before Fourier transformation, the data were multiplied with an appropriate window function (Gauss window or shifted squared sine). Chemical Shift Assignment. Unlabeled, uniformly 15Nlabeled, 15N and 13C doubly labeled, and selectively glycine and serine 15N-labeled DsbA samples were used for the assignment of proton resonances and structure determination of the reduced protein. Assignments of the NMR spectra of DsbA were made using heteronuclear triple-resonance NMR methods (76). The assignment was carried out both manually and by using the program ALFA (77) which was modified for the assignment of triple-resonance spectra. Backbone CR and Cβ resonances were assigned with HNCA, CBCA(CO)NH, and CBCANH spectra. HNCO and HN(CA)CO were used to assign the C′ resonances. HR and Hβ resonances were assigned using HBHA(CBCACO)NH and HBHA(CBCA)NH. The resonance signals of the amino acid side chains were assigned with 13C- and 15N-NOESY-HSQC spectra, a HCCH-TOCSY spectrum, and a 2D NOESY spectrum, all measured in 5% D2O, and with a 13C-NOESYHSQC spectrum in 100% D2O. The signals of the aromatic ring systems were assigned with the 2D NOESY and TOCSY spectra. Stereospecific Assignments and Coupling Constants. Stereospecific assignments of CβH protons and methyl groups of valines and leucines were obtained using procedures described by Wagner et al. (78) and Hyberts et al. (79). The assignment of CβH protons was confirmed by data from the HNHB spectrum (69). 3JHRHN coupling constants were measured from a 3D 15N-separated HN-HR correlation spectrum HNHA (68) which yielded the coupling constants from the ratio of the HN-HR cross-peak to the HN-HN diagonal peak intensity. Interproton Distance Constraints. Interproton distances were derived from the following spectra: 3D 15N-NOESYHSQC in 5% D2O, 3D 13C-NOESY-HSQC in 100% D2O, 2D NOESY in 5% D2O, and 2D NOESY in 100% D2O with mixing times of 100, 200, 60, and 40 ms, respectively. The intensities of the NOE cross-signals were converted into distance constraints as described previously (80). The distance bounds of the distance constraints were set to d ( 0.5 Å for distance constraints between 2.2 and 3.5 Å and to d ( 0.8 Å for distance constraints between 3.6 and 4.5 Å. All protons were explicitly defined in the structure calcula-

Biochemistry, Vol. 37, No. 18, 1998 6265 tions. However, in some cases, additional terms were added to the upper bounds corresponding to the pseudoatom correction introduced by Wu¨thrich (81). Torsion Angle and Hydrogen Bond Constraints. The distance constraints were supplemented with 131 backbone φ torsion angle constraints derived from the 3JHRHN coupling constant data and with 5 ψ angles for the trans peptide bond of the Xaa-Pro residues (81). The φ constraints were introduced in two stages. At the initial stage of structure calculation, only those φ constraints were used which corresponded to 3JHRHN coupling constants larger than 8 Hz (φ ) -120 ( 40°) (81). The other φ constraints and the 94 additional ψ constraints were introduced at a late stage of the refinement procedure (see below). Sixty χ1 and 5 χ2 torsion angle constraints were derived from stereochemical assignment of side chain protons. The minimum range employed was (50° for ψ and (20° for the other torsion angles. Hydrogen bonds were identified on the basis of NOE patterns and deuterium exchange data during secondary structure determination and later during examination of the calculated 3D structures (80, 81). Structure Calculations and Refinement. A total of 20 structures were calculated with the program X-PLOR 3.851 (82) using a simulated annealing protocol that starts from random coordinates or an extended conformation followed by several rounds of short SA refinement. The basic protocol used with minor modifications for the calculations has been described previously (80). For the final refinement, the NOE tables were supplemented with 118 constraints for 59 hydrogen bonds. In addition, the NOE tables were supplemented at this stage with the φ constraints derived from the 3 R JH HN constants of 7 Hz) squares. The chemical shift index (90) is also indicated below the sequence with filled and open squares designating R-helices and β-sheets, respectively. The arrows and helices at the top of the figure indicate the position of regular secondary structure elements in reduced DsbA as determined from all NMR data.

nonredundant NOEs (Figure 3A,B). The average number of interresidue constraints per residue is 9.4 which results from the extensive assignment of cross-signals in the NOESY spectra. A total of 20 structures were calculated by a simulated annealing method with the program X-PLOR 3.851 (82). All structures satisfy the experimental constraints with small deviations from idealized covalent geometry (Table 1). Figure 3B shows the ensemble of 20 three-dimensional structures of reduced DsbA. The average rmsd from the mean structure for heavy atoms in the well-defined core of DsbA (residues 3-185, except for residues 47-53, 64-66, 97-99, and 163-173) was 0.63 Å for the backbone atoms

and 0.96 Å for all atoms (Figure 4). The average rmsd from the mean structure for the φ and ψ angles was 19 and 15°, respectively. Larger rmsd values (about 2 Å for the backbone atoms and about 36° for the φ backbone torsion angles) were observed for the N and C termini of the protein (residues 1, 2, and 186-189). Figure 4 shows that the backbone of reduced DsbA is well defined with the exception of residues 47-53, 64-66, 9799, and 163-173 and both termini of the protein. In general, the conformations of the side chains are also well defined. Most of the backbone torsion angles for non-glycine residues lie within allowed regions of the Ramachandran plot. The few non-glycine residues outside these regions (Tyr3, Asp5,

6268 Biochemistry, Vol. 37, No. 18, 1998

Schirra et al.

Table 1: Parameters Characterizing the Structure Determination of Reduced DsbA in Solution at pH 3.7 and 300 Ka parameter

〈SA〉

deviations from idealized geometry bond lengths (Å) (2984) 0.003 ( 0.000 angles (deg) (5397) 0.572 ( 0.006 impropers (deg) (1564) 0.383 ( 0.008 energies (kcal mol-1) ENOE 154.5 ( 9.4 Etor 3.5 ( 0.6 EvdW 27.9 ( 2.2 number of residual constraint violations for all distance constraints 0.3 Å e rmsd e 0.4 Å 6.0 ( 1.0 0.4 Å < rmsd e 0.5 Å 0.1 ( 0.2 >0.5 Å 0 rmsd from experimental distance constraints (Å) all (1782) 0.041 ( 0.001 sequential (664) 0.054 ( 0.002 medium-range (483) 0.040 ( 0.002 long-range (517) 0.022 ( 0.006 H-bond distances (118) 0.028 ( 0.002 〈SA〉 represents the ensemble of the 20 final structures. The number of bonds, angles, and impropers is given in parentheses. Force constants used to calculate energy terms are the same as reported previously (80). The rmsd of the interproton distance constraints was calculated as described in Holak et al. (80). The number of distance constraints is given in parentheses. Besides the 1782 distance constraints, 293 dihedral angle constraints were used as input data for the structure calculations. a

Thr99, Leu147, and Gln146) are located at the structural interfaces of helices and sheets and connecting loops. Figure 3B shows an ensemble of the three-dimensional structures of reduced DsbA, calculated from 1782 approximate interproton distance constraints. All structures are basically similar to the crystal structure of the oxidized protein (Figure 5). Local differences are observed in loop regions at the surface of the protein. Especially the loop between residues 162 and 171, which is located between the end of strand β5 and the beginning of helix R7, has different conformations in the ensemble of NMR models compared to the X-ray models. The exact conformation in the solution structure could not be determined as only a few mediumand long-range NOE signals could be identified in the loop, but the energy-minimized mean solution structure which approximates the average structure over time is clearly different (Figure 6). In general, the backbone of the loop runs in the plane which is defined by strand β5 and helix R7. However, in the crystal structure of oxidized DsbA, a part of the loop comprising residues 163-169 is bent and lies below this plane with an angle of roughly 90° between this part of the loop and the β5-R7 reference plane. In the solution structure of reduced DsbA, the part of the loop which is folded “down” in the crystal structure of oxidized DsbA is folded upward into the β5-R7 plane with residues Pro163 and Ser169 being the “hinge points” of this conformational difference so that the backbone runs more or less within the plane from the end of strand β5 to the beginning of the helix R7. Domain Association in Reduced DsbA. A small but clear difference in the relative positions of the two domains was detected in the refined crystal structure of oxidized DsbA between the two DsbA monomers in the asymmetric unit (13). We performed a detailed analysis of the relative domain orientations in the solution structures of reduced DsbA and compared them with those in the two monomers

in the crystal structure of oxidized DsbA. First, the energyminimized mean solution structure of reduced DsbA was compared with the refined crystal structure of oxidized DsbA. Superposition of the structures of reduced and oxidized DsbA (using the CR atoms of residues 9-11, 22-25, 31-37, 4248, 56-59, 68-82, 85-97, 105-114, 119-126, 152-155, 159-161, and 171-185) yields an rmsd of 1.86 Å for the CR atoms. When the thioredoxin-like domain of reduced DsbA (CR atoms of residues 9-11, 22-25, 31-37, 42-48, 56-59, 152-155, 159-161, and 171-185) is superimposed onto that of oxidized DsbA, the rmsd in CR position drops to 1.20 Å. Similarly, the helical domains (CR atoms of residues 68-82, 85-97, 105-114, and 119-126) can be superimposed with an rmsd of 1.22 Å for the CR positions. However, when the helical domain of reduced DsbA is first aligned with that of the oxidized protein, significant movements are necessary for a subsequent superposition of their thioredoxin-like domains, corresponding to different orientations of the two domains (Figure 7). For reduced DsbA and monomer A of oxidized DsbA, these movements involve a translation of 1.76, -12.5, and 11.8 Å along the x, y, and z axes, respectively, of the coordinate frame and a rotation κ of 20.2° about a rotation axis defined by spherical polar coordinates of ψ ) 84.2° (inclination against the y axis) and φ ) 6.0° (azimuth, angle between the x axis and projection of the rotation axis onto the x-z plane). If the same fitting procedure for reduced DsbA is applied using monomer B from the crystal structure of oxidized DsbA, the corresponding values are translation of x ) 3.5 Å, y ) -12.2 Å, and z ) 10.5 Å and a rotation of κ ) 18.9° about an axis with ψ ) 78.2° and φ ) 9.3°. Comparison of monomer A and B of the crystal structure of oxidized DsbA yields the following values: translation of x ) -1.5 Å, y ) -1.2 Å, and z ) 1.25 Å and a significantly smaller rotation κ of -2.9° about an axis with ψ ) 68.2° and φ ) 148.8°. The large κ value for reduced DsbA corresponds to an orientation in which the two domains of DsbA are more apart, thereby enlarging the wedge-shaped cleft between them. To examine the different domain orientations further, we checked the angles between secondary structure elements of the two DsbA domains in the two structures. There is a cleft between the two domains which is formed on one hand by helix R1 and sheets β2 and β3 of the thioredoxin-like domain and on the other hand by helices R2 and R3 of the helical domain. We fitted cylinders to all secondary structure elements and subsequently determined the angles between these cylinders with the program MOLMOL (83). The results are summarized in Table 2. In general, the intradomain angle differences are small for monomers A and B of the crystal structure of oxidized DsbA (