Experimental and computational analysis of peroxisome protein targeting (948.10)

Abstract

The targeting of proteins from the cytoplasm to the peroxisomal matrix is determined largely by targeting signals with the sequences of the proteins to be targeted. The Peroxisomal Targeting Signal‐1 (PTS1) is particularly important and comprises sequences at the carboxyl terminus of the protein that approximate X‐X‐X‐Ser‐Lys‐Leu‐COO‐. PTS1s are recognized by the protein Pex5p which binds them and carries the targeted protein to the peroxisome membrane at which point the Pex5p‐protein complexes translocate to the peroxisomal matrix. The structures of Pex5p‐PTS1 peptide and protein complexes as well as apo‐Pex5p have been determined. An experimental human proteome‐wide survey of Pex5p‐PTS1 peptides using a fluorescence anisotropy‐based assay revealed dissociation constants for bona fide peroxisomal matrix proteins ranging from 1.6 nm to > 1 micromolar. Further analysis revealed correlation between protein tissue‐specific expression levels and these dissociation constants, suggesting co‐evolution of PTS1‐Pex5p affinities and expression levels. These observations also suggest testable differences in tissue‐specific peroxisome composition and function. The Pex5p‐PTS1 system also presents a well‐characterized system for the exploration of free energy perturbation methods for calculating binding free energies from structural data. It has been possible to calculate relative free energies with reasonable agreement with experimental results for a series of peptides with variations within the PTS1 sequence as well as some mutated forms of Pex5p.Grant Funding Source: Supported in part by NIGMS and NIDDK