Structural dynamics of the TPR domain of the peroxisomal cargo receptor Pex5 in Trypanosoma.

Peroxisomal protein import has been identified as a valid target in trypanosomiases, an important health threat in Central and South America. The importomer is built of multiple peroxins (Pex) and structural characterization of these proteins facilitates rational inhibitor development. We report crystal structures of the Trypanosoma brucei and T. cruzi tetratricopeptide repeat domain (TPR) of the cytoplasmic peroxisomal targeting signal 1 (PTS1) receptor Pex5. The structure of the TPR domain of TbPex5 represents an apo-form of the receptor which, together with the previously determined structure of the complex of TbPex5 TPR and PTS1 demonstrate significant receptor dynamics associated with signal peptide recognition. The structure of the complex of TPR domain of TcPex5 with PTS1 provided in this study details the molecular interactions that guide signal peptide recognition at the atomic level in the pathogenic species currently perceived as the most relevant among Trypanosoma. Small - angle X - ray scattering (SAXS) data obtained in solution supports the crystallographic findings on the compaction of the TPR domains of TbPex5 and TcPex5 upon interaction with the cargo.

Small molecule mediated inhibition of protein cargo recognition by peroxisomal transport receptor PEX5 is toxic to Trypanosoma.

Trypanosomiases are life-threatening infections of humans and livestock, and novel effective therapeutic approaches are needed. Trypanosoma compartmentalize glycolysis into specialized organelles termed glycosomes. Most of the trypanosomal glycolytic enzymes harbor a peroxisomal targeting signal-1 (PTS1) which is recognized by the soluble receptor PEX5 to facilitate docking and translocation of the cargo into the glycosomal lumen. Given its pivotal role in the glycosomal protein import, the PEX5-PTS1 interaction represents a potential target to inhibit import of glycolytic enzymes and thus kill the parasite. We developed a fluorescence polarization (FP)-based assay for monitoring the PEX5-PTS1 interaction and performed a High Throughput Screening (HTS) campaign to identify small molecule inhibitors of the interaction. Six of the identified hits passed orthogonal selection criteria and were found to inhibit parasite growth in cell culture. Our results validate PEX5 as a target for small molecule inhibitors and provide scaffolds suitable for further pre-clinical development of novel trypanocidal compounds.

Crystal structures of apo- and FAD-bound human peroxisomal acyl-CoA oxidase provide mechanistic basis explaining clinical observations.

Peroxisomal acyl-CoA oxidase 1a (ACOX1a) catalyzes the first and rate-limiting step of fatty acid oxidation, the conversion of acyl-CoAs to 2-trans-enoyl-CoAs. The dysfunction of human ACOX1a (hACOX1a) leads to deterioration of the nervous system manifesting in myeloneuropathy, hypotonia and convulsions. Crystal structures of hACOX1a in apo- and cofactor (FAD)-bound forms were solved at 2.00 and 2.09 Å resolution, respectively. hACOX1a exists as a homo-dimer with solvation free energy gain (ΔGo) of -44.7 kcal mol-1. Two FAD molecules bind at the interface of protein monomers completing the active sites. The substrate binding cleft of hACOX1a is wider compared to human mitochondrial very-long chain specific acyl-CoA dehydrogenase. Mutations (p.G178C, p.M278V and p.N237S) reported to cause dysfunctionality of hACOX1a are analyzed on its 3D-structure to understand structure-function related perturbations and explain the associated phenotypes.