Noncanonical interactions and conformational dynamics in cargo-Pex5-Pex14 ternary complex for peroxisomal import

Abstract

ABSTRACTPeroxisomes are membrane bound organelles which compartmentalize 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. In the first step, the Peroxisome Targeting Sequence 1 (PTS1)-marked cargo is recognized by soluble receptor, Pex5. The cargo-Pex5 complex then docks at the peroxisomal membrane translocon containing Pex14 and Pex13 and translocates into peroxisomal lumen, but the structural understanding is elusive. Here, we report a cryo-EM structure of cargo-Pex5-Pex14 ternary complex fromTrypanosoma cruzi, the causative agent of human Chagas disease. Malate dehydrogenase (cargo) – Pex5 interface is characterized by a swinging motion of Pex5 restricted by secondary, PTS1 independent interactions. The Pex5-Pex14 interface reveals a noncanonical contact surface adjacent to the previously identified Wxxx(F/Y) motif. Mutational analysis demonstrates that noncanonical interactions did not evolve to increase the affinity, but rather to restrict the dynamics at the cost of affinity. Consequences for the peroxisomal transport are discussed.HIGHLIGHTS1.The structure of a ternary complex (MDH-Pex5-Pex14) enabling cargo translocationviaperoxisomal membrane was determined using cryo-EM.2.Pex5 interface with the cargo (MDH) is characterized by significant conformational dynamics restricted by PTS1 independent interactions3.Wxxx(F/Y) motif mediated docking of Pex14 at Pex5 is complemented by interactions at the noncanonical site.