BioSight targets it rightly - Structural homology and beyond in E. Coli DHODH

August 23, 2017

Download PDF

SUMMARY

BioSight* is a BIONEXT’s web platform dedicated to the retrieval and assessment of local surface similarities. Here, BioSight was challenged on a structure of the protein E. coli dihydroorotate dehydrogenase (E. coli DHODH) complexed with orotate. Among the first hits retrieved were legitimate DHODH homologs and also the similarly folded dihydropyrimidine dehydrogenase. Surprisingly, the molybdenum storage protein was also significantly highly ranked by BioSight despite its structural differences with the DHODH query. Overall, in a couple of hours, BioSight was not only able to retrieve all known homologous DHODH proteins but also to retrieve proteins with a different fold, nonetheless relevant.

(*) https://biosight.bionext.com/

A.Dihydroorotate dehydrogenase (PDB ID 1F76 3 , chain A) complexed with its substrate orotate (ORO, spheres) - B.Superposition of three DHODH’s binding site. Binding site residues (1F76 in blue) are displayed in lines, atoms defining the query site in balls & sticks. In some structures (in yellow) a flexible loop is in a different conformation, in others (in cyan) it is missing. - C. MoSto protein complexed with two phosphate ions (spheres) in the UMP binding site.

Fig. 1

A.Dihydroorotate dehydrogenase (PDB ID 1F76 3 , chain A) complexed with its substrate orotate (ORO, spheres) - B.Superposition of three DHODH’s binding site. Binding site residues (1F76 in blue) are displayed in lines, atoms defining the query site in balls & sticks. In some structures (in yellow) a flexible loop is in a different conformation, in others (in cyan) it is missing. - C. MoSto protein complexed with two phosphate ions (spheres) in the UMP binding site.

INTRODUCTION

Dihydroorotate dehydrogenase (DHODH) is a mitochondrial enzyme involved in the de novo biosynthesis of Uridine MonoPhosphate (UMP) as it catalyzes the oxidation of dihydroorotate to orotate (ORO). Besides, DHODH has been extensively studied for therapeutic purposes and is currently known as the therapeutic target of leflunomide, a treatment for rheumatoid and psoriatic arthitis1. The present study challenges BioBind, an application for the detection and ranking of similar sites of biological macromolecules, on the active site of E. coli DHODH. Researches on DHODH active site revealed that the binding of ORO is strongly dependent on a flexible loop bearing a serine, anchor point for the binding.

METHODOLOGY

BioSight embeddes BioBind, a surface similarity based algorithm using models borrowed to the alpha-shape theory. The algorithm takes a given surface pattern as query, detects motifs at the surface of target macromolecules, seeks for the best superpositions and ranks them by similarity using a scoring function2: best results have the highest scores and lowest ranks. The query pattern can be automatically defined using atoms nearby a given ligand. Here, the E. coli DHODH (PYRD_ECOLI, PDB ID 1F763) crystallized with its endogenous ligand ORO was used. To build the query pattern, atoms were chosen within a radius of about 6.5 Å around the ligand (chain A). The analysis was performed on all structures from the PDB database.

RESULTS

As expected, the top ranked structures retrieved by BioBind (highlighted in red in Fig.2) correspond to the E. coli DHODH chains A, B, D, E. Human DHODH shares 41% sequence identity and a highly similar folding pattern with E. coli DHODH. For 44 out of the 46 human DHODH structures (PYRD_HUMAN) listed in the PDB database, BioBind attributed an excellent similarity score (see orange bars in Fig.2) and accurately spotted the similar surface corresponding to the counterpart site of ORO. For other DHODH structures (yellow bars in Fig. 2), 129 out of the 260 chains corresponding to 17 unique homologs are in the leading pack. In the other structures, either the critical loop is in a different conformation, or it has not been resolved (respectively in yellow and cyan Fig. 1B).

Biobind’s score distribution for all PDB structures. BioBind’s results are scored between 0 and 1. A score of 1 corresponds to structural identity.

Fig. 2

Biobind’s score distribution for all PDB structures. BioBind’s results are scored between 0 and 1. A score of 1 corresponds to structural identity.

The lower BioBind ’s scores is then inevitable since seven atoms of the loop are used by BioBind to define the query site (see Fig. 1B). Moreover, among the best ranked structures, BioBind further identified dihydropyrimidine dehydrogenase [NADP(+)] (DPYD_PIG, in magenta in Fig. 2), whose fold around the binding site is similar to the DHODH’s fold despite different function and amino acid sequence (21.6% identity, 35.9% similarity). BioBind also retrieved the molybdenum storage (MoSto) protein (MOSB_AZOVD, in green in Fig. 2) involved in the pyrimidine nucleotide biosynthetic process, known to display an uridylate kinase function and thus an UMP binding site. Despite its broadly different fold compared to the query (see Fig.1, A and C), BioBind spotted the UMP binding site, relevant concidering that UMP and ORO ligands share the uracil substructure (see Fig. 3, green substructure).

2D structures of ORO and UMP. The common uracil substructure is displayed in green.

Fig. 3

2D structures of ORO and UMP. The common uracil substructure is displayed in green.

CONCLUSION

Based on ORO binding site of the E. coli DHODH and within a couple of hours, BioBind appears strongly efficient to retrieve structures of all DHODH homologs provided that the critical loop of the binding site was complete enough and in a similar conformation. Besides, BioBind further retrieved poorly related as well as a non related yet relevant proteins. This analysis brings further evidence of the potential of BioBind for the analysis of proteins of therapeutic interest. Our fully secured platform can be accessed and challenged from https://biosight.bionext.com.

REFERENCES


  1. Leban, J., & Vitt, D. (2011). Human dihydroorotate dehydrogenase inhibitors, a novel approach for the treatment of autoimmune and inflammatory diseases. Arzneimittelforschung, 61(01), 66-72. [return]
  2. Bionext submitted publication to BioInformatics. Available on demand for private communication. [return]
  3. Nørager, S. et al. (2002). E. coli dihydroorotate dehydrogenase reveals structural and functional distinctions between different classes of dihydroorotate dehydrogenases. Structure, 10(9), 1211-1223. [return]