Modulation of peroxisomal import by the PEX13 SH3 domain and a proximal FxxxF binding motif.

Import of proteins into peroxisomes depends on PEX5, PEX13 and PEX14. By combining biochemical methods and structural biology, we show that the C-terminal SH3 domain of PEX13 mediates intramolecular interactions with a proximal FxxxF motif. The SH3 domain also binds WxxxF peptide motifs in the import receptor PEX5, demonstrating evolutionary conservation of such interactions from yeast to human. Strikingly, intramolecular interaction of the PEX13 FxxxF motif regulates binding of PEX5 WxxxF/Y motifs to the PEX13 SH3 domain. Crystal structures reveal how FxxxF and WxxxF/Y motifs are recognized by a non-canonical surface on the SH3 domain. The PEX13 FxxxF motif also mediates binding to PEX14. Surprisingly, the potential PxxP binding surface of the SH3 domain does not recognize PEX14 PxxP motifs, distinct from its yeast ortholog. Our data show that the dynamic network of PEX13 interactions with PEX5 and PEX14, mediated by diaromatic peptide motifs, modulates peroxisomal matrix import.

Pharmacological perturbation of the phase-separating protein SMNDC1.

SMNDC1 is a Tudor domain protein that recognizes di-methylated arginines and controls gene expression as an essential splicing factor. Here, we study the specific contributions of the SMNDC1 Tudor domain to protein-protein interactions, subcellular localization, and molecular function. To perturb the protein function in cells, we develop small molecule inhibitors targeting the dimethylarginine binding pocket of the SMNDC1 Tudor domain. We find that SMNDC1 localizes to phase-separated membraneless organelles that partially overlap with nuclear speckles. This condensation behavior is driven by the unstructured C-terminal region of SMNDC1, depends on RNA interaction and can be recapitulated in vitro. Inhibitors of the protein's Tudor domain drastically alter protein-protein interactions and subcellular localization, causing splicing changes for SMNDC1-dependent genes. These compounds will enable further pharmacological studies on the role of SMNDC1 in the regulation of nuclear condensates, gene regulation and cell identity.

Distinct conformational and energetic features define the specific recognition of (di)aromatic peptide motifs by PEX14.

The cycling import receptor PEX5 and its membrane-located binding partner PEX14 are key constituents of the peroxisomal import machinery. Upon recognition of newly synthesized cargo proteins carrying a peroxisomal targeting signal type 1 (PTS1) in the cytosol, the PEX5/cargo complex docks at the peroxisomal membrane by binding to PEX14. The PEX14 N-terminal domain (NTD) recognizes (di)aromatic peptides, mostly corresponding to Wxxx(F/Y)-motifs, with nano-to micromolar affinity. Human PEX5 possesses eight of these conserved motifs distributed within its 320-residue disordered N-terminal region. Here, we combine biophysical (ITC, NMR, CD), biochemical and computational methods to characterize the recognition of these (di)aromatic peptides motifs and identify key features that are recognized by PEX14. Notably, the eight motifs present in human PEX5 exhibit distinct affinities and energetic contributions for the interaction with the PEX14 NTD. Computational docking and analysis of the interactions of the (di)aromatic motifs identify the specific amino acids features that stabilize a helical conformation of the peptide ligands and mediate interactions with PEX14 NTD. We propose a refined consensus motif ExWΦxE(F/Y)Φ for high affinity binding to the PEX14 NTD and discuss conservation of the (di)aromatic peptide recognition by PEX14 in other species.

A modular toolbox to generate complex polymeric ubiquitin architectures using orthogonal sortase enzymes.

The post-translational modification of proteins with ubiquitin (Ub) and Ub-like modifiers (Ubls) represents one of the most important regulators in eukaryotic biology. Polymeric Ub/Ubl chains of distinct topologies control the activity, stability, interaction and localization of almost all cellular proteins and elicit a variety of biological outputs. Our ability to characterize the roles of distinct Ub/Ubl topologies and to identify enzymes and receptors that create, recognize and remove these modifications is however hampered by the difficulty to prepare them. Here we introduce a modular toolbox (Ubl-tools) that allows the stepwise assembly of Ub/Ubl chains in a flexible and user-defined manner facilitated by orthogonal sortase enzymes. We demonstrate the universality and applicability of Ubl-tools by generating distinctly linked Ub/Ubl hybrid chains, and investigate their role in DNA damage repair. Importantly, Ubl-tools guarantees straightforward access to target proteins, site-specifically modified with distinct homo- and heterotypic (including branched) Ub chains, providing a powerful approach for studying the functional impact of these complex modifications on cellular processes.

Membrane Interactions of the Peroxisomal Proteins PEX5 and PEX14.

Human PEX5 and PEX14 are essential components of the peroxisomal translocon, which mediates import of cargo enzymes into peroxisomes. PEX5 is a soluble receptor for cargo enzymes comprised of an N-terminal intrinsically disordered domain (NTD) and a C-terminal tetratricopeptide (TPR) domain, which recognizes peroxisomal targeting signal 1 (PTS1) peptide motif in cargo proteins. The PEX5 NTD harbors multiple WF peptide motifs (WxxxF/Y or related motifs) that are recognized by a small globular domain in the NTD of the membrane-associated protein PEX14. How the PEX5 or PEX14 NTDs bind to the peroxisomal membrane and how the interaction between the two proteins is modulated at the membrane is unknown. Here, we characterize the membrane interactions of the PEX5 NTD and PEX14 NTD in vitro by membrane mimicking bicelles and nanodiscs using NMR spectroscopy and isothermal titration calorimetry. The PEX14 NTD weakly interacts with membrane mimicking bicelles with a surface that partially overlaps with the WxxxF/Y binding site. The PEX5 NTD harbors multiple interaction sites with the membrane that involve a number of amphipathic α-helical regions, which include some of the WxxxF/Y-motifs. The partially formed α-helical conformation of these regions is stabilized in the presence of bicelles. Notably, ITC data show that the interaction between the PEX5 and PEX14 NTDs is largely unaffected by the presence of the membrane. The PEX5/PEX14 interaction exhibits similar free binding enthalpies, where reduced binding enthalpy in the presence of bicelles is compensated by a reduced entropy loss. This demonstrates that docking of PEX5 to PEX14 at the membrane does not reduce the overall binding affinity between the two proteins, providing insights into the initial phase of PEX5-PEX14 docking in the assembly of the peroxisome translocon.

Competitive Microtubule Binding of PEX14 Coordinates Peroxisomal Protein Import and Motility.

Human PEX14 plays a dual role as docking protein in peroxisomal protein import and as peroxisomal anchor for microtubules (MT), which relates to peroxisome motility. For docking, the conserved N-terminal domain of PEX14 (PEX14-NTD) binds amphipathic alpha-helical ligands, typically comprising one or two aromatic residues, of which human PEX5 possesses eight. Here, we show that the PEX14-NTD also binds to microtubular filaments in vitro with a dissociation constant in nanomolar range. PEX14 interacts with two motifs in the C-terminal region of human ß-tubulin. At least one of the binding motifs is in spatial proximity to the binding site of microtubules (MT) for kinesin. Both PEX14 and kinesin can bind to MT simultaneously. Notably, binding of PEX14 to tubulin can be prevented by its association with PEX5. The data suggest that PEX5 competes peroxisome anchoring to MT by occupying the ß-tubulin-binding site of PEX14. The competitive correlation of matrix protein import and motility may facilitate the homogeneous dispersion of peroxisomes in mammalian cells.

Novel Trypanocidal Inhibitors that Block Glycosome Biogenesis by Targeting PEX3-PEX19 Interaction.

Human pathogenic trypanosomatid parasites harbor a unique form of peroxisomes termed glycosomes that are essential for parasite viability. We and others previously identified and characterized the essential Trypanosoma brucei ortholog TbPEX3, which is the membrane-docking factor for the cytosolic receptor PEX19 bound to the glycosomal membrane proteins. Knockdown of TbPEX3 expression leads to mislocalization of glycosomal membrane and matrix proteins, and subsequent cell death. As an early step in glycosome biogenesis, the PEX3-PEX19 interaction is an attractive drug target. We established a high-throughput assay for TbPEX3-TbPEX19 interaction and screened a compound library for small-molecule inhibitors. Hits from the screen were further validated using an in vitro ELISA assay. We identified three compounds, which exhibit significant trypanocidal activity but show no apparent toxicity to human cells. Furthermore, we show that these compounds lead to mislocalization of glycosomal proteins, which is toxic to the trypanosomes. Moreover, NMR-based experiments indicate that the inhibitors bind to PEX3. The inhibitors interfering with glycosomal biogenesis by targeting the TbPEX3-TbPEX19 interaction serve as starting points for further optimization and anti-trypanosomal drug development.

Evolutionary divergent PEX3 is essential for glycosome biogenesis and survival of trypanosomatid parasites.

Trypanosomatid parasites cause devastating African sleeping sickness, Chagas disease, and Leishmaniasis that affect about 18 million people worldwide. Recently, we showed that the biogenesis of glycosomes could be the "Achilles' heel" of trypanosomatids suitable for the development of new therapies against trypanosomiases. This was shown for inhibitors of the import machinery of matrix proteins, while the distinct machinery for the topogenesis of glycosomal membrane proteins evaded investigation due to the lack of a druggable interface. Here we report on the identification of the highly divergent trypanosomal PEX3, a central component of the transport machinery of peroxisomal membrane proteins and the master regulator of peroxisome biogenesis. The trypanosomatid PEX3 shows very low degree of conservation and its identification was made possible by a combinatory approach identifying of PEX19-interacting proteins and secondary structure homology screening. The trypanosomal PEX3 localizes to glycosomes and directly interacts with the membrane protein import receptor PEX19. RNAi-studies revealed that the PEX3 is essential and that its depletion results in mislocalization of glycosomal proteins to the cytosol and a severe growth defect. Comparison of the parasites and human PEX3-PEX19 interface disclosed differences that might be accessible for drug development. The absolute requirement for biogenesis of glycosomes and its structural distinction from its human counterpart make PEX3 a prime drug target for the development of novel therapies against trypanosomiases. The identification paves the way for future drug development targeting PEX3, and for the analysis of additional partners involved in this crucial step of glycosome biogenesis.