Noncanonical interactions and conformational dynamics in cargo-Pex5-Pex14 ternary complex for peroxisomal import
Peroxisomes are membrane enclosed organelles hosting diverse metabolic processes in eukaryotic cells. Having no protein synthetic abilities, peroxisomes import all required enzymes from the cytosol through a peroxin (pex) import system. Peroxisome targeting sequence 1 (PTS1)-tagged cargo is recognized by soluble receptor, Pex5. The cargo-Pex5 complex docks at the peroxisomal membrane translocon, composed of Pex14 and Pex13, facilitating translocation into the peroxisomal lumen. Despite its significance, the structural basis of this process is poorly understood. Here, we present a cryo-electron microscopy (cryo-EM) structure of cargo-Pex5-Pex14 ternary complex from Trypanosoma cruzi, the causative agent of human Chagas disease. The atomic model of the Pex5 (residues 327-653) bound to malate dehydrogenase (MDH, cargo) tetramer and Pex14NTD (residues 21-85) reveals dynamic motions and secondary interfaces. Specifically, we observe a swinging motion of Pex5 at the MDH-Pex5 interface. Additionally, the noncanonical contact surfaces are observed at the MDH-Pex5 and Pex5-Pex14 interfaces. Mutational analysis of the former interface indicates that it does not significantly enhance the affinity, underscoring the dynamic nature of cargo-Pex5 interactions. The latter interface constitutes an extended binding site of Pex14NTD over Pex5. We discuss the implications of these findings for understanding peroxisomal transport mechanisms. HIGHLIGHTSO_LIThe structure of the peroxisomal import ternary complex (MDH-Pex5TPR-Pex14NTD) is determined using cryo-EM. C_LIO_LIPex5 interface with the cargo (MDH) is characterized by significant conformational dynamics. C_LIO_LIThe extended binding site of Pex14NTD on Pex5 is revealed. C_LI