Structural changes common to catalysis in the Tpx peroxiredoxin subfamily

Abstract

Thiol peroxidases (Tpxs) are dimeric 2-Cys peroxiredoxins from bacteria that preferentially reduce alkyl hydroperoxides. Catalysis requires two conserved residues, the peroxidatic cysteine and the resolving cysteine, which are located in helix alpha(2) and helix alpha(3), respectively. The partial unraveling of helices alpha(2) and alpha(3) during catalysis allows for the formation of an intramolecular disulfide between these two residues. Here, we present three structures of Escherichia coli Tpx representing the fully folded (peroxide binding site intact), locally unfolded (disulfide bond), and partially locally unfolded (transitional state) conformations. We also compare known Tpx crystal structures and analyze the sequence-conservation patterns among nearly 300 Tpx sequences. Twelve fully conserved Tpx-specific residues cluster at the active site and dimer interface, and an additional 37 highly conserved residues are mostly located in a cradle providing the environment for helix alpha(2). Using the structures determined here as representative fully folded, transitional, and locally unfolded Tpx conformations, we describe in detail the structural changes associated with catalysis in the Tpx subfamily. Key insights include the description of a conserved hydrophobic collar around the active site, a set of conserved packing interactions between helices alpha(2) and alpha(3) that allow the local unfolding of alpha(2) to trigger the partial unfolding of alpha(3), a conserved dimer interface that anchors the ends of helices alpha(2) and alpha(3) to stabilize the active site during structural transitions, and a conserved set of residues constituting a cradle that stabilizes the two discrete conformations of helix alpha(2) involved in catalysis. The involvement of the dimer interface in stabilizing active-site folding and in forming the hydrophobic collar implies that Tpx is an obligate homodimer and explains the high conservation of interface residues.

Figure 1

Catalytic cycle of the Tpx subfamily. The three main chemical steps universal to the peroxiredoxin catalytic cycle, (1) peroxidation, (2) resolution and (3) recycling are shown along with the local unfolding step (double headed arrow) required for the resolution reaction. Peroxidation forms a Cys-sulfenic acid (S₆₁-OH). Specific to the Tpx subfamily, the disulfide bond formed in the resolution step is intramolecular and thioredoxin (Trx) has been shown to be the most potent reductant^,,,,. S₆₁ and S₉₅ are the sulfur atoms of the peroxidatic and resolving cysteine residues, respectively, using EcTpx residue numbering. The fully folded (FF) and locally unfolded (LU) conformations are indicated.

Figure 2

Electron density quality and active site structures. A) 2F_o-F_c electron density (cyan) is shown for the disulfide bond in chain A of EcTpx. Peptide segments Gly59 - Ala63 and Arg93 - Ala97 (except, for clarity, the side chain of Phe94) are shown. The contour level is 1 ρ_rms. B) Electron density as in panel A is shown for the FF active site in EcTpxC61S. The peptide segments containing residues Val60 - Ala63 and Lys167 - Ala168 (of a symmetry mate, green) are shown; Ser61 is labeled as C_P. The contour level is 2 ρ_rms. C) Electron density as in panel A is shown for the intermolecular C_P-C_P disulfide bond of EcTpxC82,95S. Residues Gly59 - Ala63 of chains C and D are shown; the contour level is 0.8 ρ_rms. In all panels, structures are colored by atom with C=grey, N=blue, O=red and S=yellow, and the C_P and C_R residues are indicated.

Figure 3

Structure and sequence conservation within the Tpx subfamily. A) The structure of EcTpxC61S, a representative Tpx_FF, with only one chain of the dimer is shown. α-helices (cyan) and β-strands (blue) are labeled according to the common fold of all Prxs. Two features characteristic of the Tpx subfamily are the position of C_R in helix α₃ and an N-terminal extension containing a β-hairpin (β_N1 and β_N2). The C_P and C_R residues are shown as ball and stick with the Cys sulfur atom (or Ser61 oxygen atom) in yellow. B) The structure shown in panel A is rotated ~180° around the vertical axis to give the approximate views used in Figures 3D, 5 and 7. C) Tpx-specific sequence conservation is mapped onto the sequence of EcTpx. Background coloring is by residue conservation within the Tpx subfamily: red for 100% conserved, orange for > 90% conserved, pink for > 90% conserved between two amino acids and pale blue for conserved hydrophobic residues (see Table 3). Yellow text is used for the four residues conserved in all Prxs (Pro54, Thr58, C_P61 and Arg133) and white text is used for those residues 100% conserved in Tpx sequences only. Secondary structure elements for the FF conformation are shown above the sequence, rectangles for 3₁₀-helices (α₁) and α-helices, arrows for β-strands and triangles for insertions/deletions. The structure elements that locally unfold for disulfide formation (the first four residues of helix α₂ and the last 7 residues of helix α₃) are indicated by green hash marks in the secondary structure of α₂ and α₃. Solid lines underneath the sequence indicate residues that have > 5 Ų surface area buried at the dimer interface. The conservation pattern found here is based on many more sequences than previous analyses^,. D) Stereoview of the EcTpxC61S dimer, generated by crystal symmetry, shown looking down the two-fold axis. The two chains are dark grey and pale grey and the 17 residues that form the dimer interface (underlined in panel C) are shown as sticks. Coloring of the 100%, > 90%, and > 90% between two amino acids conserved residues (as in panel C) highlights their positions at the dimer interface, active site pocket, and the α₂-cradle. The asymmetric conformations for Arg93 are visible, with each chain showing one conformation.

Figure 4

Packing interactions at the dimer interface. Stereoview of half of the EcTpx dimer interface with the two chains colored dark grey (chain A) and light grey (chain B, residue numbers contain a prime). One of every residue buried at the dimer interface is shown with the exception of two copies for residues Asp57, Asp86, and the water labeled W. Nearby residues not buried at the dimer interface, such as Asp37, are not shown. Of the three waters conserved at the dimer interface (red spheres), one is on the two-fold axis. Hydrogen bonding interactions are indicated by cyan dashes. The two-fold axis is marked by a dotted green line. Since the side chain of Arg93 is on the two-fold, Arg93‘ (not shown) cannot simultaneously occupy the symmetry-related position; this creates an asymmetric hydrogen bonding network.

Figure 5

The FF, PLU and LU conformations. Shown are the overlays of the Tpx structures in the A) FF (Tpx_FF), B) PLU (C_P-C_P disulfide, Tpx_PLU) and C) LU (C_P-C_R disulfide, Tpx_LU) conformations. In panel A, the distorted C_P-loop of the EcTpx_FF structure is marked by an asterisk. In panel C, a notable difference is seen for MtTpx_LU (PDB code 1XVQ), which has an extended N-terminus resulting from a crystallization artifact. Each chain is colored by B-factor with red indicating higher values. The C_P and C_R residues are labeled and shown as ball and stick with sulfur atoms (or oxygen for Ser mutants) colored yellow. D) Stereoview overlay of the representative Tpx_FF (composite EcTpx_FF, grey), Tpx_PLU (EcTpx_PLU, magenta), and Tpx_LU (EcTpx_LU, cyan) structures. The distorted C_P-loop in EcTpx_FF (green) is included to show its similarity to the PLU structure. The side chains of C_P and C_R residues are shown as in panels A through C.

Figure 6

The hydrophobic collar of the Tpx active site. A) Stereoview of the composite EcTpx_FF active-site surface. The active-site pocket containing the C_P, Pro54, Thr58, and Arg133, is surrounded by a hydrophobic collar that tailors the substrate specificity of Tpxs to alkyl hydroperoxides. A bound acetate molecule (green sticks) modeled from pdb code 1Y25, mimics substrate binding and shows the methyl group pointing toward the collar. Collar residues are labeled with the asterisk representing Asp34‘ and Leu35‘ surfaces making up the right-hand wall of the collar. Asp34‘, Leu35‘, and Phe89‘ (white text) are from the second chain. Coloring is by atom with C=grey, N=blue, O=red and S=yellow. B) Sequence conservation for residues that form the hydrophobic collar. The relative size of the letters indicates conservation and all are plotted on the same scale. Hydrophobic residues are colored black. The nominally polar side chains of Asp34‘ and Thr58 contribute carbon atoms to the collar, and the importance of only the Cα and Cβ atoms for Asp34‘ is consistent with its lower sequence conservation.

Figure 7

Catalysis-associated structural transitions in the Tpx subfamily. A) Stereoview of the composite EcTpx_FF. Conserved residues (Table 3) in α₂ and the cradle are shown as sticks and colored by atom with C=grey, O=red, N=blue, and S=yellow. The Oγ of Ser61 (C_P) is colored yellow. Hydrogen bonds involving the side chains of conserved residues are indicated by cyan dashes. Select residues are labeled. B) Stereoview of EcTpx_LU oriented and shown as in panel A. C) Stereoview of the interpolated conformational pathway between the Tpx_FF and Tpx_LU conformations. The structure is oriented as in A) but with α₂ removed. The starting (FF, blue) and ending (LU, red) conformations of the side chains (sticks) and backbone (cartoon) are shown, and the side chains for the interpolated intermediate conformations are shown in semi-transparent rainbow colors (blue to red).