Escherichia coli thioredoxin-like protein YbbN contains an atypical tetratricopeptide repeat motif and is a negative regulator of GroEL

Abstract

Many proteins contain a thioredoxin (Trx)-like domain fused with one or more partner domains that diversify protein function by the modular construction of new molecules. The Escherichia coli protein YbbN is a Trx-like protein that contains a C-terminal domain with low homology to tetratricopeptide repeat motifs. YbbN has been proposed to act as a chaperone or co-chaperone that aids in heat stress response and DNA synthesis. We report the crystal structure of YbbN, which is an elongated molecule with a mobile Trx domain and four atypical tetratricopeptide repeat motifs. The Trx domain lacks a canonical CXXC active site architecture and is not a functional oxidoreductase. A variety of proteins in E. coli interact with YbbN, including multiple ribosomal protein subunits and a strong interaction with GroEL. YbbN acts as a mild inhibitor of GroESL chaperonin function and ATPase activity, suggesting that it is a negative regulator of the GroESL system. Combined with previous observations that YbbN enhances the DnaK-DnaJ-GrpE chaperone system, we propose that YbbN coordinately regulates the activities of these two prokaryotic chaperones, thereby helping to direct client protein traffic initially to DnaK. Therefore, YbbN may play a role in integrating the activities of different chaperone pathways in E. coli and related bacteria.

FIGURE 1.

Structure, electrostatics, and disorder in YbbN. In panel A, YbbN is shown as a ribbon diagram, with the N and C termini labeled. The Trx domain is in gold, and the two TPR-containing subdomains are in blue (subdomain A) and purple (subdomain B). Helices are lettered and strands are numbered in both panels A and B. The protein is highly prolate, and the Trx domain makes no direct contacts with the TPR domain. In panel B the ribbon diagram of YbbN is shown rotated by 90 degrees about the horizontal. The electrostatic surface for YbbN is shown in panels C and D in the same orientations as in panels A and B, with red representing negative electrostatic potential and blue representing positive. YbbN presents a predominantly negatively charged surface to solution, particularly in the cleft between the two TPR subdomains. However, there is a basic (positive) patch of residues near the C-terminal region of YbbN. Units of electrostatic potential are kT/e, and the temperature was 300 K. In panel E thermal ellipsoids at 75% probability level for all Cα atoms are shown for the refined TLS model of domain mobility in YbbN, emphasizing the greatly elevated disorder of the Trx domain compared with the better-ordered TPR domains. Ellipsoid color indicates the displacement magnitude, ranging from B values of 10 Ų (blue) to 80 Ų (red).

FIGURE 2.

YbbN is a monomer in solution. Sedimentation equilibrium ultracentrifugation of YbbN was performed at three rotor speeds and protein concentrations and globally fit to determine molecular mass. A representative run is shown. The best-fit model (shown in the solid line in the lower panel) corresponds to the YbbN monomer and agrees well with the measured absorbance of the protein at 280 nm as a function of radius (open circles). The residuals (top panel) between data and model are randomly distributed and lack a systematic trend, indicating adequate fit.

FIGURE 3.

The Trx domain of YbbN lacks a conventional CXXC active site. A superposition of the Trx domain of YbbN (blue) and oxidized E. coli Trx (PDB 2TRX (49); yellow) is shown with the key cysteine residues of both proteins shown in stick representation. Residue number corresponds to YbbN, and the N and C termini of the domains are indicated. YbbN lacks the classical CXXC motif because residue 35, which is the more reactive cysteine in Trx, is a serine in YbbN. Although another cysteine residue (Cys-63) is present in YbbN, it is too distant from Cys-38 to participate in Trx-like thiol-disulfide exchange chemistry. This position is occupied by an isoleucine in Trx.

FIGURE 4.

The C-terminal domain of YbbN contains atypical TPR motifs. The YbbN TPR motifs (blue) are superimposed with the crystal structure of an idealized TPR motif (PDB code 1NA3; yellow). In panels A and B, the helices are represented as cylinders and are labeled A or B to indicate the corresponding helix of the idealized TPR motif. N and C termini of the domains are labeled. Panel A shows the close agreement between TPR subdomain A of YbbN (blue) and the idealized TPR motif (yellow). The capping helices are located on the C terminus of the domains. In panel B, the best superposition of YbbN TPR subdomain B (blue) and the idealized TPR motif (yellow) involves an unusual staggered alignment where the YbbN TPR subdomain B lacks the N-terminal A helix and has two C-terminal capping helices. Panels C and D provide a more detailed view of the superimposed TPR domains in the same orientations as panels A and B. Highly conserved TPR consensus residues that differ between YbbN and the idealized TPR motif are shown in stick representation, with standard TPR numbering as described in Main et al. (33).

FIGURE 5.

A potential binding cleft in the TPR domain. The two TPR subdomains form a solvent-rich cleft in YbbN that may form an interaction surface with other proteins. The YbbN peptide backbone is shown as a ribbon diagram and is colored as in Fig. 1. A variety of charged amino acids are located in this cleft, creating an electrostatically varied surface that may bind to other proteins. Ordered water molecules are depicted as red spheres.

FIGURE 6.

YbbN interacts with a variety of proteins in E. coli lysate. Proteins retained from sonicated E. coli lysate on a YbbN affinity resin were eluted with either a 0.6 m or a 1.0 m NaCl wash. Negative control lanes (C) were those fractions that eluted from unmodified resin that had been incubated with lysate. The gel was stained with Coomassie Blue, and numbers indicate protein bands that were excised and identified using mass spectrometry (see Table 2 for identities). Band 4 is GroEL, which is the dominant protein present in the more stringent 1 m NaCl wash and interacts robustly with YbbN.

FIGURE 7.

GroESL activity is mildly inhibited by YbbN. Panel A shows the amount of CS activity recovered from chemically denatured enzyme that was refolded by dilution into buffer containing the proteins indicated by a + sign in the table at the bottom of the graph. The reaction was followed by monitoring the production of colored 2-nitro-5-thiobenzoate by liberated CoA. Native CS, which had not been denatured before the assay, is the positive control. The GroESL chaperonin leads to a substantial recovery of CS activity that is diminished by the addition of YbbN. The asterisk indicates that the difference between values with and without YbbN is significant to a p value <0.05 by Student's t test for data generated from three independent experiments. YbbN alone is no more effective than the negative control protein lysozyme at refolding CS. Panel B shows the release of inorganic phosphate from ATP by the GroEL ATPase activity as determined using the Malachite Green assay. Samples containing the protein(s) are indicated by a + sign in the table at the bottom of the graph. The addition of YbbN results in a statistically significant (asterisk; p value < 0.05 by Student's t test) decrease in the GroEL ATPase activity, in agreement with the diminution of chaperone activity shown in panel A. Bovine serum albumin (BSA) was used as a negative control. Data are from three independent experiments.