Crystal structure of dipeptidyl peptidase III from the human gut symbiont Bacteroides thetaiotaomicron

Abstract

Bacteroides thetaiotaomicron is a dominant member of the human intestinal microbiome. The genome of this anaerobe encodes more than 100 proteolytic enzymes, the majority of which have not been characterized. In the present study, we have produced and purified recombinant dipeptidyl peptidase III (DPP III) from B. thetaiotaomicron for the purposes of biochemical and structural investigations. DPP III is a cytosolic zinc-metallopeptidase of the M49 family, involved in protein metabolism. The biochemical results for B. thetaiotaomicron DPP III from our research showed both some similarities to, as well as certain differences from, previously characterised yeast and human DPP III. The 3D-structure of B. thetaiotaomicron DPP III was determined by X-ray crystallography and revealed a two-domain protein. The ligand-free structure (refined to 2.4 Å) was in the open conformation, while in the presence of the hydroxamate inhibitor Tyr-Phe-NHOH, the closed form (refined to 3.3 Å) was observed. Compared to the closed form, the two domains of the open form are rotated away from each other by about 28 degrees. A comparison of the crystal structure of B. thetaiotaomicron DPP III with that of the human and yeast enzymes revealed a similar overall fold. However, a significant difference with functional implications was discovered in the upper domain, farther away from the catalytic centre. In addition, our data indicate that large protein flexibility might be conserved in the M49 family.

Fig 1. SDS-PAGE and isoelectric focusing analysis of the purified BtDPP III protein variants.

(A) SDS-PAGE of purified recombinant wild-type DPP III; (B) Isoelectric focusing analysis of purified wild-type (lane 1) and cysteine variants: C11S (lanes 2–3), C158S (lanes 4–5), C189S (lanes 6–7), C425S (lanes 8–9), C450S (lane 10) and Cys-null (lane 11). Proteins were visualized by Coomassie Blue staining.

Fig 2. Structures of ligand-free Bt, yeast and human DPP III with their respective zinc binding sites.

Zinc binding sites are shown in grey squares. Amino acids coordinating the zinc ions (shown as grey spheres) and the glutamic acid residues essential for enzyme activity are shown in stick representations. The figure was prepared using the PyMol program (http://www.pymol.org/), and the PDB-deposited crystal structures of the yeast (PDB ID 3csk) and human DPP III (PDB ID 3fvy).

Fig 3. Superposition of ligand-free BtDPP III on human and yeast proteins.

The upper and lower domains were separately superimposed to the corresponding domains of human (A) and yeast (B) DPP III. Their structures are shown in cartoon representation: bacterial in magenta, human in green, and yeast in cyan. The black ellipses indicate the areas of the two main differences between the superimposed structures. The figure was prepared using the PyMol program (http://www.pymol.org/).

Fig 4. Phylogenetic tree of the M49 family.

The tree is based on a multiple sequence alignment and maximum likelihood analysis of 87 peptidases of the M49 family. The branch support values are indicated at the major branch points. Species abbreviations are given in S2 Table. Arrows represent the appearance of the loop between the two conserved active-site motifs in the upper domain and the emergence of the ETGE motif.

Fig 5. A section of a multiple sequence alignment of M49 peptidases.

Thirty M49 peptidases were selected from different eukaryotic and bacterial species. The active-site motifs I and II as well as ETGE motif are framed. The loop between the two conserved active-site motifs of the human M49 peptidase is highlighted in grey. The full names of the species are given in S2 Table.

Fig 6. Surface representation of the ligand-free structure and the structure of the closed form of BtDPP III.

The upper structural domain is shown in blue and the lower one in magenta. Amino acids coloured in red are: zinc binding residues (His448, His453, and Glu476), glutamic acid essential for enzyme activity (Glu449), and structurally equivalent amino acids residues (Glu307, Tyr309, Thr380, Ile382, Gly383, Asn385, Asn388, Asp465, His533, and Tyr627) that were shown to interact with peptide substrates in human DPP III. The figure was prepared using the PyMol program (http://www.pymol.org/). The illustration showing domain movement in human DPP III, given in the square, was taken from Bezerra et al. [12].

Fig 7. Overall cartoon representation of the active site in BtDPP III structure in the closed form.

2mF_o-DF_c electron density at 1 σ (blue) and mF_o-DF_c electron density at 3 σ (green) correspond to the substrate-binding position. The upper structural domain is shown in blue, the lower one in magenta, and the zinc atom as a grey sphere. The amino acids binding the zinc ion, Tris, and Tyr-Phe-NHOH are shown in stick representation. (A) Electron density map in the active site; (B) electron density with Tyr-Phe-NHOH included in the refinement; (C) electron density with Tris included in the refinement. The figure was prepared using the PyMol program (http://www.pymol.org/).

Fig 8. Superposition of the active sites of BtDPP III and hDPP III with the bound tynorphin.

The human DPP III ligand (tynorphin, VVYPW) is shown in yellow. The amino acids that make polar interactions with the peptide substrate (dashed lines) and the conserved Asp496 that is important for the substrate specificity are shown as stick models (hDPPIII and BtDPPIII in green and magenta, respectively). The figure was prepared using the PyMol program (http://www.pymol.org/).