Therapeutic link between oral microbiome, periodontitis and diabetes type 2 - a structure/function study

Project Overview


Periodontitis is the most common chronic inflammatory disease in humans worldwide. Its comorbidity with systemic diseases, such as type 2 diabetes, oral cancers and cardiovascular diseases, continues to generate considerable impact on healthcare and society. Porphyromonas gingivalis, one of the most studied organisms of the human oral microbiome, is the major causative agent in periodontitis. Surprisingly, this microorganism does not ferment carbohydrates, rather it uses proteinaceous substrates as carbon and energy sources. Proteases with different specificities degrade these extracellular proteins into di- and tri-peptides, which are then incorporated by the bacterium via membrane embedded oligopeptide transporters.

Project objectives

The importance of the human microbiome in health and disease is increasingly recognized, although the underlying mechanisms remain largely understudied. This project aims to elucidate molecular aspects of the energy metabolism of P. gingivalis focussing on its therapeutic potential as novel drug targets. Innovative recombinant technologies will be employed to express, purify and characterize structurally as well as functionally: the oligopeptide transporters, which are integral membrane proteins; and proteases within the same pathway such as PgDPP4, which has been recently proposed to be the link between periodontitis and type 2 diabetes. The student will:

  1. Generate recombinant membrane and soluble proteins for structural characterization using X-ray crystallography, cryo electron microscopy, small-angle x-ray scattering and mass spectrometry;
  2. Generate mutants in order to identify important residues for the protein activity and disease mechanism;
  3. Co-crystallize the target proteins with the natural substrates to gain insights into their binding modes and catalytic mechanism;
  4. Employ state-of-art biophysical techniques such as ITC, SPR, CD and spectroscopy to identify initial chemical matters for drug discovery.

Training Opportunities

Through this project the student will:


Protein Science & Structural Biology and Endocrinology & Metabolic Medicine


Project reference number: 958

Funding and admissions information


Name Department Institution Country Email
Dr Katharina Duerr Structural Genomics Consortium Oxford University, Old Road Campus Research Building GBR
Associate Professor Wyatt W Yue Structural Genomics Consortium Oxford University, Old Road Campus Research Building GBR

Montgomery AB, Kopec J, Shrestha L, Thezenas ML, Burgess-Brown NA, Fischer R, Yue WW, Venables PJ. 2016. Crystal structure of Porphyromonas gingivalis peptidylarginine deiminase: implications for autoimmunity in rheumatoid arthritis. Ann. Rheum. Dis., 75 (6), pp. 1255-61. Read abstract | Read more

BACKGROUND: Periodontitis (PD) is a known risk factor for rheumatoid arthritis (RA) and there is increasing evidence that the link between the two diseases is due to citrullination by the unique bacterial peptidylarginine deiminase (PAD) enzyme expressed by periodontal pathogen Pophyromonas gingivalis (PPAD). However, the precise mechanism by which PPAD could generate potentially immunogenic peptides has remained controversial due to lack of information about the structural and catalytic mechanisms of the enzyme. OBJECTIVES: By solving the 3D structure of PPAD we aim to characterise activity and elucidate potential mechanisms involved in breach of tolerance to citrullinated proteins in RA. METHODS: PPAD and a catalytically inactive mutant PPAD(C351A) were crystallised and their 3D structures solved. Key residues identified from 3D structures were examined by mutations. Fibrinogen and α-enolase were incubated with PPAD and P. gingivalis arginine gingipain (RgpB) and citrullinated peptides formed were sequenced and quantified by mass spectrometry. RESULTS: Here, we solve the crystal structure of a truncated, highly active form of PPAD. We confirm catalysis is mediated by the following residues: Asp130, His236, Asp238, Asn297 and Cys351 and show Arg152 and Arg154 may determine the substrate specificity of PPAD for C-terminal arginines. We demonstrate the formation of 37 C-terminally citrullinated peptides from fibrinogen and 11 from α-enolase following incubation with tPPAD and RgpB. CONCLUSIONS: PPAD displays an unequivocal specificity for C-terminal arginine residues and readily citrullinates peptides from key RA autoantigens. The formation of these novel citrullinated peptides may be involved in breach of tolerance to citrullinated proteins in RA. Hide abstract

Bezerra GA, Dobrovetsky E, Seitova A, Fedosyuk S, Dhe-Paganon S, Gruber K. 2015. Structure of human dipeptidyl peptidase 10 (DPPY): a modulator of neuronal Kv4 channels. Sci Rep, 5 pp. 8769. Read abstract | Read more

The voltage-gated potassium channel family (Kv) constitutes the most diverse class of ion channels in the nervous system. Dipeptidyl peptidase 10 (DPP10) is an inactive peptidase that modulates the electrophysiological properties, cell-surface expression and subcellular localization of voltage-gated potassium channels. As a consequence, DPP10 malfunctioning is associated with neurodegenerative conditions like Alzheimer and fronto-temporal dementia, making this protein an attractive drug target. In this work, we report the crystal structure of DPP10 and compare it to that of DPP6 and DPP4. DPP10 belongs to the S9B serine protease subfamily and contains two domains with two distinct folds: a β-propeller and a classical α/β-hydrolase fold. The catalytic serine, however, is replaced by a glycine, rendering the protein enzymatically inactive. Difference in the entrance channels to the active sites between DPP10 and DPP4 provide an additional rationale for the lack of activity. We also characterize the DPP10 dimer interface focusing on the alternative approach for designing drugs able to target protein-protein interactions. Hide abstract

Bezerra GA, Dobrovetsky E, Viertlmayr R, Dong A, Binter A, Abramic M, Macheroux P, Dhe-Paganon S, Gruber K. 2012. Entropy-driven binding of opioid peptides induces a large domain motion in human dipeptidyl peptidase III. Proc. Natl. Acad. Sci. U.S.A., 109 (17), pp. 6525-30. Read abstract | Read more

Opioid peptides are involved in various essential physiological processes, most notably nociception. Dipeptidyl peptidase III (DPP III) is one of the most important enkephalin-degrading enzymes associated with the mammalian pain modulatory system. Here we describe the X-ray structures of human DPP III and its complex with the opioid peptide tynorphin, which rationalize the enzyme's substrate specificity and reveal an exceptionally large domain motion upon ligand binding. Microcalorimetric analyses point at an entropy-dominated process, with the release of water molecules from the binding cleft ("entropy reservoir") as the major thermodynamic driving force. Our results provide the basis for the design of specific inhibitors that enable the elucidation of the exact role of DPP III and the exploration of its potential as a target of pain intervention strategies. Hide abstract

Nemoto TK, Ohara-Nemoto Y, Bezerra GA, Shimoyama Y, Kimura S. 2016. A Porphyromonas gingivalis Periplasmic Novel Exopeptidase, Acylpeptidyl Oligopeptidase, Releases N-Acylated Di- and Tripeptides from Oligopeptides. J. Biol. Chem., 291 (11), pp. 5913-25. Read abstract | Read more

Exopeptidases, including dipeptidyl- and tripeptidylpeptidase, are crucial for the growth of Porphyromonas gingivalis, a periodontopathic asaccharolytic bacterium that incorporates amino acids mainly as di- and tripeptides. In this study, we identified a novel exopeptidase, designated acylpeptidyl oligopeptidase (AOP), composed of 759 amino acid residues with active Ser(615) and encoded by PGN_1349 in P. gingivalis ATCC 33277. AOP is currently listed as an unassigned S9 family peptidase or prolyl oligopeptidase. Recombinant AOP did not hydrolyze a Pro-Xaa bond. In addition, although sequence similarities to human and archaea-type acylaminoacyl peptidase sequences were observed, its enzymatic properties were apparently distinct from those, because AOP scarcely released an N-acyl-amino acid as compared with di- and tripeptides, especially with N-terminal modification. The kcat/Km value against benzyloxycarbonyl-Val-Lys-Met-4-methycoumaryl-7-amide, the most potent substrate, was 123.3 ± 17.3 μm(-1) s(-1), optimal pH was 7-8.5, and the activity was decreased with increased NaCl concentrations. AOP existed predominantly in the periplasmic fraction as a monomer, whereas equilibrium between monomers and oligomers was observed with a recombinant molecule, suggesting a tendency of oligomerization mediated by the N-terminal region (Met(16)-Glu(101)). Three-dimensional modeling revealed the three domain structures (residues Met(16)-Ala(126), which has no similar homologue with known structure; residues Leu(127)-Met(495) (β-propeller domain); and residues Ala(496)-Phe(736) (α/β-hydrolase domain)) and further indicated the hydrophobic S1 site of AOP in accord with its hydrophobic P1 preference. AOP orthologues are widely distributed in bacteria, archaea, and eukaryotes, suggesting its importance for processing of nutritional and/or bioactive oligopeptides. Hide abstract

Nemoto TK, Bezerra GA, Ono T, Nishimata H, Fujiwara T, Ohara-Nemoto Y. 2018. Identification of a new subtype of dipeptidyl peptidase 11 and a third group of the S46-family members specifically present in the genus Bacteroides. Biochimie, 147 pp. 25-35. Read abstract | Read more

Peptidase family S46 consists of two types of dipeptidyl-peptidases (DPPs), DPP7 and DPP11, which liberate dipeptides from the N-termini of polypeptides along with the penultimate hydrophobic and acidic residues, respectively. Their specificities are primarily defined by a single amino acid residue, Gly in DPP7 and Arg in DPP11 (numbering for Porphyromonas gingivalis DPP11). Bacterial species in the phyla Proteobacteria and Bacteroidetes generally possess one gene for each, while Bacteroides species exceptionally possess three genes, one gene as DPP7 and two genes as DPP11, annotated based on the full-length similarities. In the present study, we aimed to characterize the above-mentioned Bacteroides S46 DPPs. A recombinant protein of the putative DPP11 gene BF9343_2924 from Bacteroides fragilis harboring Gly exhibited DPP7 activity by hydrolyzing Leu-Leu-4-methylcoumaryl-7-amide (MCA). Another gene, BF9343_2925, as well as the Bacteroides vulgatus gene (BVU_2252) with Arg was confirmed to encode DPP11. These results demonstrated that classification of S46 peptidase is enforceable by the S1 essential residues. Bacteroides DPP11 showed a decreased level of activity towards the substrates, especially with P1-position Glu. Findings of 3D structural modeling indicated three potential amino acid substitutions responsible for the reduction, one of which, Asn650Thr substitution, actually recovered the hydrolyzing activity of Leu-Glu-MCA. On the other hand, the gene currently annotated as DPP7 carrying Gly from B. fragilis (BF9343_0130) and Bacteroides ovatus (Bovatus_03382) did not hydrolyze any of the examined substrates. The existence of a phylogenic branch of these putative Bacteroides DPP7 genes classified by the C-terminal conserved region (Ser-Leu) strongly suggests that Bacteroides species expresses a DPP with an unknown property. In conclusion, the genus Bacteroides exceptionally expresses three S46-family members; authentic DPP7, a new subtype of DPP11 with substantially reduced specificity for Glu, and a third group of S46 family members. Hide abstract