Structural analysis of YcdY, a member of the redox-enzyme maturation protein family

Proteins of the NarJ subfamily from facultatively or obligately anaerobic bacteria play key roles as chaperones in folding and cofactor insertion for complex iron–sulfur molybdoenzymes (CISMs), which mediate energy production under anaerobic conditions. YcdY was identified as a NarJ subfamily member but was proposed to increase the catalytic activity of the non-CISM enzyme YcdX phosphatase, presumably by inserting a zinc cofactor into YcdX. To elucidate the structural features of YcdY required for its chaperone function, we determined the crystal structure of Enterobacter cloacae YcdY (enYcdY). enYcdY adopts a single-domain, curved helix-bundle structure decorated with α-helices. enYcdY contains an extensive dent on its concave side. The dent in enYcdY generally forms using hydrophobic or conserved residues. Based on comparative structural and sequence analyses, we propose that enYcdY uses the dent to recognize and fold the client protein. Interestingly, enYcdY did not increase the enzymatic activity of E. cloacae YcdX (enYcdX) in the presence or absence of Zn²⁺ ions, even for partially denatured enYCdX protein. The same results were obtained for the Escherichia coli counterparts, in contrast to a previous report. These observations suggest that YcdY functions as a chaperone for proteins other than YcdX.

1. Introduction

Facultatively or obligately anaerobic bacteria use complex iron–sulfur molybdoenzymes (CISMs) to produce energy under anaerobic conditions by transferring electrons through iron–sulfur clusters and a molybdenum cofactor to non-oxygen electron acceptors such as nitrate, dimethyl sulfoxide or trimethylamine-N-oxide (Rothery et al., 2008). Owing to the complexity of CISM folding or cofactor insertion into CISM, CISM biogenesis proceeds with the assistance of a redox-enzyme maturation protein (REMP) specific for each CISM (Turner et al., 2004; Chan et al., 2014).

Within the REMP family, the NarJ subfamily has been identified as a distinct group of REMPs whose members share significant sequence and structural homology (Song, Kim et al., 2024; Stevens et al., 2009; Tranier et al., 2003). The NarJ subfamily includes NarJ, NarW, DmsD and TorD, which function as chaperones for the CISMs nitrate reductase A, nitrate reductase Z, dimethyl sulfoxide reductase and trimethyl­amine-N-oxide reductase, respectively (Bay et al., 2015; Blasco et al., 1992; Pommier et al., 1998; Oresnik et al., 2001). NarJ subfamily proteins have been shown to interact with the signal peptide of the molybdenum cofactor-containing catalytic subunit of CISM (Song, Kim et al., 2024; Zakian et al., 2010; Li & Turner, 2009; Winstone et al., 2006; Oresnik et al., 2001; Pommier et al., 1998). For example, NarJ and DmsD recognize the N-terminal signal peptides of the molybdoenzymes NarG and DmsA, respectively, for molybdenum cofactor incorporation into NarG and DmsA (Song, Kim et al., 2024; Zakian et al., 2010; Li & Turner, 2009; Winstone et al., 2006; Oresnik et al., 2001). The signal peptide-binding mechanism of NarJ subfamily proteins has recently been revealed by a structural study of NarJ and NarG (Song, Kim et al., 2024). NarJ harbors a hydrophobic groove on the concave side of its structure and uses it to accommodate the hydrophobic residues of the NarG signal peptide via dual hydrophobic interactions (Song, Kim et al., 2024).

YcdY was identified as a NarJ subfamily protein with significant sequence homology to DmsD (Redelberger et al., 2011). However, YcdY is considered to be an atypical member of the NarJ subfamily because it appears to act as a chaperone for the Zn²⁺-dependent YcdX phosphatase, which does not contain a molybdenum cofactor (Redelberger et al., 2011). YcdY was shown to improve the phosphatase activity of YcdX as a chaperone, presumably by enhancing Zn²⁺-cofactor incorporation into YcdX. However, subsequent studies of the chaperone function of YcdY have not been reported. Owing to the uniqueness of the function of YcdY in the NarJ subfamily, the chaperone–client relationship between YcdY and YcdX remains to be confirmed. Moreover, the structure of YcdY is unknown, and it is therefore unclear whether YcdY is structurally similar to other NarJ subfamily proteins and how it mediates the chaperone function for its client protein.

Here, we report the crystal structure of Enterobacter cloacae YcdY (enYcdY) in the apo form. enYcdY folds into a helix-bundle structure with an extensive dent in the concave side. Based on comparative structural analysis, we propose that the dent in enYcdY is used to accommodate its client protein. Surprisingly, YcdY-mediated enhancement of YcdX phosphatase activity was not observed for either enYcdY or Escherichia coli YcdY (esYcdY) in our assay, suggesting that YcdY may function as a chaperone for proteins other than YcdX.

2. Materials and methods

2.1. Macromolecule production

To construct a plasmid that expresses enYcdY (UniProt accession No. A0A0M7F7K2), an enYcdY-encoding DNA fragment was generated by polymerase chain reaction (PCR) from the genomic DNA of E. cloacae (ATCC 13047; Table 1). The PCR product was treated with the restriction enzymes BamHI (New England Biolabs) and SalI (Enzynomics) and inserted using T4 DNA ligase (Enzynomics) into a modified pET-49b plasmid, which was engineered to express a recombinant protein with a hexahistidine tag and a subsequent Tobacco etch virus (TEV) protease cleavage site at the N-terminus. The resulting ligation product was transformed into E. coli DH5α cells. The nucleotide sequence of the enYcdY-encoding region in the enYcdY expression plasmid was confirmed via DNA sequencing.

Table 1
Macromolecule-production information for enYcdY

Source organism	Enterobacter cloacae
DNA source	Genomic DNA of E. cloacae
Expression vector	Modified pET-49b
Expression host	Escherichia coli BL21 (DE3)
Complete amino-acid sequence of the construct produced	SAKDPMNEFSILCRVLGTLYYRQPQDPLLVPLFTLIREGKLAQNWPLEQDDLLERLQKSCDMQQISTDYNALFVGEECRVSPYRSAWQEGATEAEVRAFLSERGMPLTDTPADHIGTLLLAASWIEDHAGDDENEAIETLFEMYLLPWVGTFLGKVEAHATSPFWRTLAPLTRDAIAAMWDELEEENEE

The plasmids expressing E. cloacae YcdX (enYcdX; NCBI accession No. WP_013097177 with a sequence difference at residue 238 from proline to threonine), esYcdY (UniProt accession No. P75915) or E. coli YcdX (esYcdX; UniProt accession No. B1X9E8) were constructed in an identical manner to that for enYcdY, except that they were designed from the pET-49b plasmid to express a recombinant protein with a hexahistidine tag and a thrombin cleavage site at the N-terminus, and those for enYcdY and enYcdX were generated via PCR amplification from the genomic DNA of E. coli (ATCC 23736; Song, Ki et al., 2024).

To overexpress the enYcdY protein, its expression plasmid was transformed into E. coli BL21 (DE3) cells (Table 1). The transformant cells were grown in LB medium (LPS Solution) in a shaking incubator at 37°C. When the optical density of the culture at 600 nm reached 0.6, the culture was supplemented with 1 mM isopropyl β-D-1-thiogalactopyranoside for recombinant protein overexpression. The cells were further cultured at 18°C overnight and then collected by centrifugation. The resulting cells were resuspended in a solution consisting of 50 mM Tris pH 8.0, 200 mM NaCl, 5 mM β-mercaptoethanol and were lysed by sonication in the presence of 1 mM phenylmethanesulfonyl fluoride (Bio Basic). The cell lysate was cleared by centrifugation, and the resulting supernatant was incubated with nickel–nitrilotriacetic acid (Ni–NTA) resin for 1 h at 4°C for affinity chromatography. The hexahistidine-tagged enYcdY protein was eluted from the resin using 150 mM imidazole (Georgiachem) and was dialyzed against 20 mM Tris pH 8.0 (or 20 mM HEPES pH 7.4), 5 mM β-mercaptoethanol. The hexahistidine tag was removed from the enYcdY protein by incubating the dialyzed protein solution with purified TEV protease protein at a molar ratio of ∼3:1 for 4 h at room temperature in a solution consisting of 20 mM Tris pH 8.0 (or 20 mM HEPES pH 7.4), 5 mM β-mercaptoethanol. The resulting protein solution was subjected to a second purification via ion-exchange chromatography, which was performed using a Mono Q 10/100 column with a 0–0.5 M NaCl gradient in 20 mM Tris pH 8.0, 5 mM β-mercaptoethanol. The tag-free enYcdY protein was concentrated using a centrifugal filter for crystallization. The protein concentration was determined by measuring the absorbance of the protein solution at 280 nm (molar extinction coefficient 40 450 M⁻¹ cm⁻¹). The enYcdY protein was alternatively purified in a tagged form for a phosphatase assay in a similar manner to that for the tag-free enYcdY protein, but without tag cleavage. The enYcdX, esYcdY and esYcdX proteins were obtained in an identical manner to the tagged enYcdY protein.

2.2. Crystallization

The enYcdY protein was crystallized via the sitting-drop vapor-diffusion method at 18°C using a 1.0 µl drop consisting of 0.5 µl enYcdY protein solution (30.9 mg ml⁻¹) and 0.5 µl reservoir solution consisting of 40% PEG 600, 0.2 M calcium acetate, 0.1 M sodium cacodylate pH 7.0 (Table 2).

Table 2
Crystallization of enYcdY

Method	Vapor diffusion, sitting drop
Plate type	Hampton Research 24-well plate
Temperature (°C)	18
Buffer composition of protein solution	20 mM Tris pH 8.0, 5 mM β-mercaptoethanol
Composition of reservoir solution	40% PEG 600, 0.2 M calcium acetate, 0.1 M sodium cacodylate pH 7.0
Volume and ratio of drop	1 µl, 1:1 ratio
Volume of reservoir	500 (µl)

2.3. Data collection and processing

The enYcdY crystal was flash-cooled under a nitrogen gas stream (−173°C) and diffraction data were collected on X-ray beamline 7A at the Pohang Accelerator Laboratory (PAL). The X-ray diffraction data were indexed, integrated, merged and scaled using HKL-2000 (Table 3; Otwinowski & Minor, 1997).

Table 3
Data collection and processing

Values in parentheses are for the outer shell.

X-ray source	Beamline 7A, PAL
Wavelength (Å)	0.9793
Temperature (°C)	−173
Detector	ADSC Quantum 270 CCD
Space group	P21
a, b, c (Å)	65.26, 88.69, 82.25
α, β, γ (°)	90, 103.69, 90
Resolution range (Å)	30.000–2.500 (2.54–2.50)
No. of unique reflections	31272 (1530)
Completeness (%)	98.0 (98.0)
Multiplicity	3.5 (3.5)
〈I/σ(I)〉	12.4 (3.0)
Rmeas	0.213 (0.921)
CC1/2	0.945 (0.548)

2.4. Structure solution and refinement

The enYcdY structure was solved by the molecular-replacement method using Phaser with a search model generated by the AlphaFold2 server (McCoy et al., 2007; Jumper et al., 2021). The molecular-replacement solution was modified into the final structure of YcdY via iterative cycles of manual model building and automatic refinement using Coot and phenix.refine, respectively (Table 4; Emsley et al., 2010; Liebschner et al., 2019).

Table 4
Structure solution and refinement

Values in parentheses are for the outer shell.

Resolution range (Å)	29.85–2.50
No. of reflections, working set	29475
No. of reflections, test set	1473
Final Rwork (%)	21.3
Final Rfree (%)	26.6
No. of non-H atoms
Protein	5734
Ion (Ca^2+)	13
Water	138
Total	5885
R.m.s. deviations
Bond lengths (Å)	0.006
Angles (°)	0.832
Average B factor (Å^2)	25.7
Ramachandran plot
Favored (%)	96.2
Outliers (%)	0.0
PDB code	9m26

2.5. Gel-filtration chromatography

To determine the oligomeric state of YcdY, gel-filtration chromatography was performed. The YcdY protein (100 µg) in 300 µl of a solution consisting of 20 mM Tris pH 8.0, 150 mM NaCl, 5 mM β-mercaptoethanol was applied onto a Superdex 200 10/300 column. Protein elution was performed using a solution consisting of 20 mM Tris pH 8.0, 150 mM NaCl, 5 mM β-mercaptoethanol and was monitored by measuring the absorbance at 280 nm. The molecular size of YcdY was estimated by comparing its elution volume with those of gel-filtration standards (Bio-Rad).

2.6. Phosphatase assay

To determine the phosphatase activity of YcdX, a p-nitrophenyl phosphate (pNPP) phosphatase assay was performed. For the assay, the purified YcdX protein (1 µM for enYcdX; 5 µM for esYcdX) was incubated with pNPP (Sigma–Aldrich) in the absence or presence of the YcdY protein (1 µM for enYcdY; 5 µM for esYcdY) in a solution consisting of zinc chloride (10 µM for enYcdX; 50 µM for esYcdX) and 50 mM Tris pH 8.0 at 37°C for 20 min (for enYcdX) or 1 h (for esYcdX) in a 96-well plate. The absorbance of the reaction mixture was measured at 410 nm using a microplate reader (Epoch). For comparative analysis, the purified YcdX protein was partially denatured by three cycles of freezing (−80°C; 10 min) and thawing (room temperature; 10 min) or by treatment with 3 M urea (70°C; 1 h) followed by dialysis against 20 mM Tris pH 8.0, 150 mM NaCl, 5 mM β-mercaptoethanol, and the phosphatase activity of the resulting protein was determined by the pNPP assay in the presence and absence of YcdY or zinc chloride.

2.7. Native polyacrylamide gel electrophoresis (PAGE)

Native PAGE was performed to determine whether enYcdY interacts with enYcdX under nondenaturing conditions. The enYcdY protein (17 µM) was incubated with the enYcdX protein at a molar ratio of 1:0, 0:1 or 1:1 at 18°C for 30 min in a solution consisting of 20 mM Tris pH 8.0, 150 mM NaCl, 5 mM β-mercaptoethanol. The resulting reaction was loaded onto a 6% native polyacrylamide gel and electrophoresed at 100 V for 100 min at pH 8.8. Protein bands on the gel were visualized with Coomassie Brilliant Blue.

3. Results and discussion

3.1. Overall structure of enYcdY

To elucidate the structural features of enYcdY that are necessary for its putative chaperone function, we expressed the full-length enYcdY protein (residues 1–184) and purified it to high purity via Ni–NTA affinity chromatography and anion-exchange chromatography (Fig. 1a and Supplementary Fig. S1). The purified enYcdY protein was crystallized in the presence of PEG 600 and calcium acetate at pH 7.0 (Tables 2 and 3). The structure of enYcdY was determined by molecular replacement in space group P2₁ and refined to R_work and R_free values of 21.3% and 26.3%, respectively, using diffraction data to 2.5 Å resolution, without outliers in the Ramachandran plot (Fig. 1b and Table 4).

Figure 1 Structure and sequence of enYcdY. (a) Amino-acid sequence of enYcdY and its comparison with that of esYcdY. The secondary structure of enYcdY is shown above the enYcdY sequence as waves (α-helices) and lines (coils). (b) Overall structure of an enYcdY monomer in rainbow ribbons (N-terminus, blue; C-terminus, red). (c) Oligomeric state analysis of enYcdY and esYcdY by gel-filtration chromatography. The elution volumes of the gel-filtration standards are indicated by gray vertical lines.

The asymmetric unit of the enYcdY crystal contains four polypeptide chains of enYcdY (chains A–D; Supplementary Fig. S2a). Oligomerization analysis using the PISA server suggested the possibility that enYcdY chains C and D form a homodimer with a buried surface area of ∼790 Ų on each chain (Krissinel & Henrick, 2007). However, our analysis via gel-filtration chromatography clearly indicated that enYcdY is a monomer (Fig. 1c). In gel-filtration chromatography, the purified enYcdY protein was eluted as a single peak between the 17 and 44 kDa standards, with an apparent molecular weight of 21.3 ± 1.0 kDa (mean ± standard deviation). Given that the calculated molecular weight of one monomer of the purified enYcdY protein is 21.6 kDa, enYcdY exists predominantly as a monomer in solution. Consistently, the esYcdY protein, which shares a sequence identity of ∼84% with enYcdY as an ortholog of enYcdY, also eluted as a single peak as a monomer in gel-filtration chromatography (apparent molecular weight 22.0 ± 0.9 kDa; calculated molecular weight of monomer 24.9 kDa; Figs. 1a and 1c). Therefore, we conclude that YcdY prefers to exist in a monomeric form.

The four enYcdY chains in the asymmetric unit of the enYcdY crystal exhibit similar structures, with root-mean-square deviation (r.m.s.d.) values of 0.15–0.52 Å (Supplementary Fig. S2b). Among the four chains, chains A and C exhibit high structural similarity to chains B and D, respectively (r.m.s.d.s of 0.15 and 0.16 Å, respectively). Because chain A has a more ordered structure than the other chains based on B-factor analysis and covers more enYcdY residues, chain A will be described primarily below unless otherwise noted.

The enYcdY structure contains enYcdY residues 1–124 and 127–184, whereas residues 125 and 126 are disordered. enYcdY folds into a single-domain structure consisting of nine α-helices (α1–α6, α7_a, α7_b and α8) without β-strands (Figs. 1a and 1b). enYcdY forms a curved α-helix-bundle structure and positions the α1 and α6 helices side by side in an antiparallel organization on the middle of the concave side. The C-terminal α7_a, α7_b and α8 helices buttress the α1 and α6 helices from the back of these helices and form the convex surface of the enYcdY structure. Given that the α7_a and α7_b helices are linked by a single residue at an angle of ∼126° and form an elongated bent helix segment (α7), the core structure of enYcdY can be described as an antiparallel four-helix bundle consisting of the α1, α6, α7 and α8 helix segments. Notably, the α7 and α8 segments are longer than the α1 and α6 helices and extend further down to interact with the α4 helix at the bottom of the enYcdY structure. The central four-helix bundle of enYcdY is decorated with the α2 and α3 helices on one side and with the α5 helix on the other side.

3.2. Structural comparison of enYcdY with other NarJ subfamily proteins

To identify the structural homologs of enYcdY, a homolog search was performed using the DALI server (Holm, 2022). enYcdY shares relatively high structural homology with other NarJ subfamily proteins, such as DmsD and NarJ (Fig. 2). Among NarJ subfamily proteins, the structures of DmsD were ranked as being the most homologous structures to enYcdY (Stevens et al., 2009; Qiu et al., 2008). The enYcdY and E. coli DmsD (esDmsD; PDB entry 3efp) structures have an r.m.s.d. value of 1.91 Å for 170 C^α atoms, with a sequence identity of ∼29% (Fig. 2a and Supplementary Fig. S3; Stevens et al., 2009). In the superimposed structures of enYcdY and esDmsD, the nine α-helices of enYcdY are observed to be the counterparts of the esDmsD structure, although the esDmsD structure has some α-helices of different lengths along with coils in different conformations and contains two additional short α-helices compared with the enYcdY structure. Other members of the NarJ subfamily share less sequence and structural homology with enYcdY. For example, E. coli NarJ (esNarJ; PDB entry 8jzd) has a higher r.m.s.d. (2.66 Å) for only 126 C^α atoms with lower sequence identity (∼15%) than esDmsD (Fig. 2b and Supplementary Fig. S3; Song, Kim et al., 2024). Moreover, many α-helices and coils are observed at different positions with different lengths between the enYcdY and esNarJ structures. These structural observations are consistent with previous phylogenetic analysis of the NarJ subfamily, which revealed that YcdY evolved from DmsD (Bay et al., 2015). Despite these structural differences, the NarJ subfamily proteins, including enYcdY, esDmsD and esNarJ, commonly form a fully α-helical structure containing a four-helix core, which is decorated by α-helices on both sides and at the bottom.

Figure 2 Structural comparison of enYcdY with its homologs (esDmsD and esNarJ). (a) Overlay of the enYcdY (green ribbons) and esDmsD (magenta ribbons; PDB entry 3efp) structures. (b) Overlay of the enYcdY (green ribbons) and esNarJ (yellow ribbons; PDB entry 8jzd) structures.

3.3. Dented region of enYcdY on the concave side

The most distinctive structural property of enYcdY is that enYcdY contains a dented region in its concave side (Figs. 1b and 3). The dent consists of residues from the α1 helix, α1–α2 loop, α2 helix, α2–α3 loop, α5–α6 loop, α6 helix and α7_a helix. The α1 and α6 helices are found at the base of the dent to the left and right of the dent, respectively. Residues from the α2 helix and its flanking loops (α1–α2 loop and α2–α3 loop) form the left edge of the dent and those from the α5–α6 loop, α6 helix and α7_a helix form the right edge. Notably, the Arg9 residue from the α1 helix is located in the center of the dent and protrudes its side chain into the solvent, dividing the dent into left and right portions (Figs. 3b and 3c).

Figure 3 Structural analysis of the enYcdY dent. (a) The relatively neutral property of the enYcdY dent in the electrostatic potential surface of the enYcdY structure (positive, blue; neutral, white; negative, red). The electrostatic potential was calculated using the APBS module implemented in PyMOL (https://pymol.org/). The enYcdY dent is indicated by a yellow dashed ellipse. (b) High sequence conservation of the enYcdY dent. Sequence conservation calculated by the ConSurf server (https://consurf.tau.ac.il/) is proportional to the magenta intensity in the enYcdY structure (surfaces). The enYcdY dent is indicated by a yellow dashed ellipse. (c) enYcdY dent residues. On the enYcdY structure (gray ribbons), the enYcdY residues in the dent are indicated by sticks of different colors on the basis of residue polarity and conservation (yellow, apolar; orange, polar and highly conserved). (d) Structure of enYcdY (surfaces) with the esNarG signal peptide (cyan coils) from the esNarJ–esNarG complex structure (PDB entry 8jzd) overlaid on the enYcdY structure. The enYcdY structure is shown as sequence conservation-coded surfaces, as depicted in Fig. 3(b). The enYcdY dent is indicated by a yellow dashed ellipse.

NarJ is the only member of the NarJ subfamily that has been structurally analyzed for substrate recognition (Song, Kim et al., 2024). NarJ was shown to interact with the NarG signal peptide through hydrophobic interactions by inserting the hydrophobic residues of the NarG signal peptide into the hydrophobic groove of NarJ. Interestingly, the dent in YcdY is in a position similar to that of the groove in NarJ. Moreover, as shown for the NarJ groove, the dent in enYcdY generally has electrically neutral surfaces according to electrostatic potential analysis, and ∼70% of its residues are hydrophobic (Figs. 3a and 3c). These hydrophobic residues are generally conserved as hydrophobic residues across YcdY orthologs (Figs. 3b and 3c and Supplementary Fig. S4). Even hydrophilic residues in the enYcdY dent are highly conserved (Figs. 3b and 3c and Supplementary Fig. S4). For example, the central residue of the dent, Arg9, is invariant across YcdY orthologs. These observations suggest that the dent plays a critical role in the biological function of YcdY, such as client protein binding as a chaperone. Consistently, in the superimposed structures of enYcdY and the esNarJ–esNarG complex, the NarG signal peptide is located right above the dent in enYcdY (Fig. 3d). Therefore, we propose that YcdY employs its dent to recognize its substrate protein.

3.4. No improvement in YcdX phosphatase activity by YcdY

esYcdY was shown to interact with esYcdX as a chaperone and improve the phosphatase activity of esYcdX, presumably by facilitating Zn²⁺ incorporation into esYcdX (Redelberger et al., 2011). To confirm the chaperone function of enYcdY for enYcdX, we analyzed the interaction of enYcdY with enYcdX by native PAGE and cross-linking (Supplementary Fig. S1). However, none of these analyses allowed us to detect the enYcdY–enYcdX complex. In native PAGE, only bands corresponding to enYcdY alone and to enYcdX alone were observed, without additional bands for the mixture of the enYcdY and enYcdX proteins (Supplementary Fig. S1a). Furthermore, when the enYcdY and enYcdX proteins were incubated with chemical cross-linkers, no band corresponding to the cross-linked protein of enYcdY and enYcdX was observed via sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

To further examine whether enYcdY functions as a chaperone for enYcdX, the phosphatase activity of YcdX was analyzed in the presence and absence of enYcdY. We incubated enYcdX with enYcdY and measured the phosphatase activity of enYcdX using pNPP as reported for the E. coli counterparts (Redelberger et al., 2011). enYcdY did not improve the phosphatase activity of the native enYcdX protein in the absence of Zn²⁺ ions, but rather slightly decreased the activity (Fig. 4a). Zn²⁺ ions increased the phosphatase activity of enYcdX, presumably because YcdX is a Zn²⁺-dependent enzyme (Figs. 4a and 4b). However, as shown in the absence of Zn²⁺ ions, YcdY slightly reduced the activity of enYcdX in the presence of Zn²⁺ ions (Fig. 4b). To examine the effect of enYcdY on the partially denatured enYcdX protein, denaturation of the enYcdX protein was induced by freeze–thaw cycles or urea. As observed for the native enYcdX protein, the activity of the partially denatured enYcdX protein was not enhanced by enYcdY in the absence of Zn²⁺ ions (Fig. 4a). Furthermore, the inclusion of Zn²⁺ ions in the assay did not change the pattern, indicating that enYcdY does not promote the incorporation of Zn²⁺ into enYcdX (Fig. 4b). These observations allow us to propose that enYcdY does not function as a chaperone for enYcdX.

Figure 4 YcdY (`Y' in the figure) did not significantly improve the phosphatase activity of YcdX (`X' in the figure). To assess the effect of YcdY on the renaturation of YcdX, YcdX denaturation was induced by three cycles of freezing and thawing or by urea before the phosphatase reaction. (a) Analysis of the effects of enYcdY on the phosphatase activity of enYcdX in the absence of Zn^2+ ions. (b) Analysis of the effects of enYcdY on the phosphatase activity of enYcdX in the presence of Zn^2+ ions. (c) Analysis of the effects of esYcdY on the phosphatase activity of esYcdX in the absence of Zn^2+ ions. (d) Analysis of the effects of esYcdY on the phosphatase activity of esYcdX in the presence of Zn^2+ ions.

To investigate whether the discrepancy between our results for enYcdY and enYcdX and the previous report for esYcdY and esYcdX is due to species differences (E. cloacae versus E. coli), we repeated the assays using esYcdY and esYcdX (Figs. 4c and 4d; Redelberger et al., 2011). As shown for enYcdY, esYcdY did not improve the phosphatase activity of the native and partially denatured esYcdX proteins in the absence or presence of Zn²⁺ ions, suggesting that esYcdX does not improve the folding of esYcdX or Zn²⁺ incorporation into esYcdX. These similar results between the E. cloacae and E. coli proteins are consistent with the high sequence identity (∼84%) between enYcdY and esYcdY and between esYcdX and enYcdX (Fig. 1a). Therefore, we propose that YcdY is likely to function as a chaperone for proteins other than YcdX.

In conclusion, YcdY forms an entirely α-helical structure with an extensive dent on the concave side of the YcdY structure. Our comparative structural analysis suggested that the dent in YcdY displays high hydrophobicity and conservation and is expected to contribute to client-protein recognition. Furthermore, based on the YcdX phosphatase assay in the presence and absence of YcdY, we propose that YcdY may not be a chaperone for YcdX.

Footnotes

‡Co-first authors.