Crystal structure of prolyl 4-hydroxylase from Bacillus anthracis

Abstract

Prolyl 4-hydroxylases (P4H) catalyze the post-translational hydroxylation of proline residues and play a role in collagen production, hypoxia response, and cell wall development. P4Hs belong to the group of Fe(II)/alphaKG oxygenases and require Fe(II), alpha-ketoglutarate (alphaKG), and O(2) for activity. We report the 1.40 A structure of a P4H from Bacillus anthracis, the causative agent of anthrax, whose immunodominant exosporium protein BclA contains collagen-like repeat sequences. The structure reveals the double-stranded beta-helix core fold characteristic of Fe(II)/alphaKG oxygenases. This fold positions Fe-binding and alphaKG-binding residues in what is expected to be catalytically competent orientations and is consistent with proline peptide substrate binding at the active site mouth. Comparisons of the anthrax P4H structure with Cr P4H-1 structures reveal similarities in a peptide surface groove. However, sequence and structural comparisons suggest differences in conformation of adjacent loops may change the interaction with peptide substrates. These differences may be the basis of a substantial disparity between the K(M) values for the Cr P4H-1 compared to the anthrax and human P4H enzymes. Additionally, while previous structures of P4H enzymes are monomers, B. anthracis P4H forms an alpha(2) homodimer and suggests residues important for interactions between the alpha(2) subunits of alpha(2)beta(2) human collagen P4H. Thus, the anthrax P4H structure provides insight into the structure and function of the alpha-subunit of human P4H, which may aid in the development of selective inhibitors of the human P4H enzyme involved in fibrotic disease.

Figure 1

Structure of the anthrax-P4H monomer. The eight β-strands of the DSBH core fold are shown as gold arrows labeled with Roman numerals, with strands I, VIII, III, and VI making up the major sheet while strands V, IV, VII, and II make up the minor strand. Additional strands are shown as blue arrows. Yellow helices are labeled as regular helices (α1, α2, α3) or 3₁₀ helices (α1′, α2′, α3′). The loop that could not be modeled due to poor electron density is shown as a dotted line.

Figure 2

Active site of anthrax-P4H. A, Anthrax-P4H active site highlighting facial triad residues (cyan sticks), residues that coordinate the facial triad Asp129 (green sticks), bound glycerol (green sticks), an active site water (cyan sphere), and other important active site residues (orange sticks). B, Comparison of anthrax-P4H active site residues (blue sticks) with the corresponding residues in the algal P4H active site (red sticks) including active site Zn (grey sphere), and with the TauD active site (yellow sticks) including Fe(II) (red sphere) and αKG (yellow sticks). Facial triad residues are labeled without the position numbers as they differ between the three enzymes.

Figure 3

Peptide binding. A, Anthrax peptide binding groove in stereo with groove residues shown in yellow over the invagination into the active site with the glycerol shown in cyan sticks. B, Anthrax-P4H groove, side view from the β5 end with groove residues shown as yellow surface. C, Corresponding side view of the algal-P4H peptide binding groove. In both B and C, dotted lines indicate missing structure. D. Structural alignment of the algal structure (red ribbons) with the bound (Ser-Pro)₅ peptide (sticks with grey carbon atoms) with the anthrax-P4H structure (blue ribbons) shows the complementarity of the surfaces, and the overlap between the channel water and the proline that is hydroxylated. E. View of anthrax-P4H and algal P4H overlap in D, but with the surface removed and individual groove residues of anthrax-P4H shown as sticks with yellow carbons. Ser81 (anthrax-P4H numbering) is not shown to simplify the figure.

Figure 4

Oligomeric state of anthrax-P4H. A, Overview of interactions between molecule A (green) and molecule B (blue) with the facial triad residues shown as magenta sticks. B, Detailed view of half of the symmetrical network of interactions between molecule A (green carbons) and molecule B (blue carbons).

Figure 5

Overall comparison of structures of anthrax-P4H (blue ribbons), the Cr-P4H-1/Zn complex (yellow ribbons), the Cr-P4H-1/Zn/inhibitor complex (orange ribbons), and the Cr-P4H-1/Zn/(Ser-Pro)₅ complex (red ribbons with peptide in sticks with cyan carbon atoms and Zn as a cyan sphere). Highly conserved regions are shown in grey.

Scheme 1

Proposed reaction mechanism of collagen-P4H. Hydroxylation of the substrate is coupled to the decarboxylation of α-ketoglutarate to yield succinate and CO₂.