Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5.

Cellular functions rely on proper actions of organelles such as peroxisomes. These organelles rely on the import of proteins from the cytosol. The peroxisomal import receptor PEX5 takes up target proteins in the cytosol and transports them to the peroxisomal matrix. However, its cytosolic molecular interactions have so far not directly been disclosed. Here, we combined advanced optical microscopy and spectroscopy techniques such as fluorescence correlation spectroscopy and stimulated emission depletion microscopy with biochemical tools to present a detailed characterization of the cytosolic diffusion and interaction dynamics of PEX5. Among other features, we highlight a slow diffusion of PEX5, independent of aggregation or target binding, but associated with cytosolic interaction partners via its N-terminal domain. This sheds new light on the functionality of the receptor in the cytosol as well as highlighting the potential of using complementary microscopy tools to decipher molecular interactions in the cytosol by studying their diffusion dynamics.

Inhibitors of PEX14 disrupt protein import into glycosomes and kill Trypanosoma parasites.

The parasitic protists of the Trypanosoma genus infect humans and domestic mammals, causing severe mortality and huge economic losses. The most threatening trypanosomiasis is Chagas disease, affecting up to 12 million people in the Americas. We report a way to selectively kill Trypanosoma by blocking glycosomal/peroxisomal import that depends on the PEX14-PEX5 protein-protein interaction. We developed small molecules that efficiently disrupt the PEX14-PEX5 interaction. This results in mislocalization of glycosomal enzymes, causing metabolic catastrophe, and it kills the parasite. High-resolution x-ray structures and nuclear magnetic resonance data enabled the efficient design of inhibitors with trypanocidal activities comparable to approved medications. These results identify PEX14 as an "Achilles' heel" of the Trypanosoma suitable for the development of new therapies against trypanosomiases and provide the structural basis for their development.

Yarrowia lipolytica Pex20p, Saccharomyces cerevisiae Pex18p/Pex21p and mammalian Pex5pL fulfil a common function in the early steps of the peroxisomal PTS2 import pathway.

Import of peroxisomal matrix proteins is essential for peroxisome biogenesis. Genetic and biochemical studies using a variety of different model systems have led to the discovery of 23 PEX genes required for this process. Although it is generally believed that, in contrast to mitochondria and chloroplasts, translocation of proteins into peroxisomes involves a receptor cycle, there are reported differences of an evolutionary conservation of this cycle either with respect to the components or the steps involved in different organisms. We show here that the early steps of protein import into peroxisomes exhibit a greater similarity than was thought previously to be the case. Pex20p of Yarrowia lipolytica, Pex18p and Pex21p of Saccharomyces cerevisiae and mammalian Pex5pL fulfil a common function in the PTS2 pathway of their respective organisms. These non-orthologous proteins possess a conserved sequence region that most likely represents a common PTS2-receptor binding site and di-aromatic pentapeptide motifs that could be involved in binding of the putative docking proteins. We propose that not necessarily the same proteins but functional modules of them are conserved in the early steps of peroxisomal protein import.

The di-aromatic pentapeptide repeats of the human peroxisome import receptor PEX5 are separate high affinity binding sites for the peroxisomal membrane protein PEX14.

PEX5 functions as a mobile import receptor for peroxisomal matrix proteins with a peroxisomal targeting signal 1 (PTS1). A critical step within the PTS1-import pathway is the interaction between PEX5 and the peroxisome membrane-associated protein PEX14. Based on two-hybrid analyses in mammalian cells and complementary in vitro binding assays, we demonstrate that the evolutionarily conserved pentapeptide repeat motifs, WX(E/D/Q/A/S)(E/D/Q)(F/Y), in PEX5 bind to PEX14 with high affinity. The results obtained indicate that each of the seven di-aromatic pentapeptides of human PEX5 interacts separately at the same binding site in the N terminus of PEX14 with equilibrium dissociation constants in the low nanomolar range. Mutational analysis of the PEX14-binding motifs reveals that the conserved aromatic amino acids at position 1 or 5 are essential for high affinity binding. We propose that the side chains of the aromatic amino acids are in close proximity as part of an amphipathic alpha-helix and together form hydrophobic anchors for binding PEX5 to individual PEX14 molecules.

Recombinant human peroxisomal targeting signal receptor PEX5. Structural basis for interaction of PEX5 with PEX14.

Import of matrix proteins into peroxisomes requires two targeting signal-specific import receptors, Pex5p and Pex7p, and their binding partners at the peroxisomal membrane, Pex13p and Pex14p. Several constructs of human PEX5 have been overexpressed and purified by affinity chromatography in order to determine functionally important interactions and provide initial structural information. Sizing chromatography and electron microscopy suggest that the two isoforms of the human PTS1 receptor, PEX5L and PEX5S, form homotetramers. Surface plasmon resonance analysis indicates that PEX5 binds to the N-terminal fragment of PEX14-(1-78) with a very high affinity in the low nanomolar range. Stable complexes between recombinant PEX14-(1-78) and both the full-length and truncated versions of PEX5 were formed in vitro. Analysis of these complexes revealed that PEX5 possesses multiple binding sites for PEX14, which appear to be distributed throughout its N-terminal half. Coincidentally, this part of the molecule is also responsible for oligomerization, whereas the C-terminal half with its seven tetratricopeptide repeats has been reported to bind PTS1-proteins. A pentapeptide motif that is reiterated seven times in PEX5 is proposed as a determinant for the interaction with PEX14.

A double mutation at the tip of the dimer interface loop of triosephosphate isomerase generates active monomers with reduced stability.

Triosephosphate isomerase (TIM) is a very stable dimer. In order to understand better the importance of dimerization for stability and catalytic activity, we have constructed a monomeric double-mutation variant. The dimer interface residues Thr75 and Gly76, which are at the tip of loop 3, have been substituted by an arginine and a glutamate, respectively. In wild type TIM, these two residues are at a distance of 27 A from the active site (as measured within the same subunit). This new variant, RE-TIM, was expressed in Escherichia coli, purified to homogeneity, and biochemically characterized. Sedimentation equilibrium ultracentrifugation runs showed that RE-TIM is a monomer in solution. Far-UV CD spectra indicate that this new variant is folded properly and that the secondary structure contents of RE-TIM are similar to those of wild type TIM. The monomeric RE-TIM has residual TIM activity. The thermal stability of RE-TIM is lower than that for wild type TIM. CD melting curves for RE-TIM and wild type TIM show Tm values of 52 and 57 degrees C, respectively, in the presence of the active site ligand 2-phosphoglycolate at 1 mM. Previously, we have characterized two other monomeric forms of TIM: monoTIM and H47N-TIM. The properties of RE-TIM, H47N-TIM, and monoTIM are compared, and it is argued that the properties of RE-TIM will be very similar to those of wild type monomeric subunits. This implies that wild type monomeric subunits have some stability and are catalytically active. It is also inferred that these monomeric subunits have flexible loops which rigidify at the dimer interface on dimerization, causing a 1000-fold increase of kcat and a 10-fold decrease of Km.

Protein engineering with monomeric triosephosphate isomerase (monoTIM): the modelling and structure verification of a seven-residue loop.

Protein engineering experiments have been carried out with loop-1 of monomeric triosephosphate isomerase (monoTIM). Loop-1 of monoTIM is disordered in every crystal structure of liganded monoTIM, but in the wild-type TIM it is a very rigid dimer interface loop. This loop connects the first beta-strand with the first alpha-helix of the TIM-barrel scaffold. The first residue of this loop, Lys13, is a conserved catalytic residue. The protein design studies with loop-1 were aimed at rigidifying this loop such that the Lys13 side chain points in the same direction as seen in wild type. The modelling suggested that the loop should be made one residue shorter. With the modelling package ICM the optimal sequence of a new seven-residue loop-1 was determined and its structure was predicted. The new variant could be expressed and purified and has been characterized. The catalytic activity and stability are very similar to those of monoTIM. The crystal structure (at 2.6 A resolution) shows that the experimental loop-1 structure agrees well with the modelled loop-1 structure. The direct superposition of the seven loop residues of the modelled and experimental structures results in an r.m.s. difference of 0.5 A for the 28 main chain atoms. The good agreement between the predicted structure and the crystal structure shows that the described modelling protocol can be used successfully for the reliable prediction of loop structures.

Active site properties of monomeric triosephosphate isomerase (monoTIM) as deduced from mutational and structural studies.

MonoTIM is a stable monomeric variant of the dimeric trypanosomal enzyme triose phosphate isomerase (TIM) with less, but significant, catalytic activity. It is known that in TIM, three residues, Lys 13 (loop 1), His 95 (loop 4), and Glu 167 (loop 6) are the crucial catalytic residues. In the wild-type TIM dimer, loop 1 and loop 4 are very rigid because of tight interactions with residues of the other subunit. Previous structural studies indicate that Lys 13 and His 95 have much increased conformational flexibility in monoTIM. Using site-directed mutagenesis, it is shown here that Lys 13 and His 95 are nevertheless essential for optimal catalysis by monoTIM: monoTIM-K13A is completely inactive, although it can still bind substrate analogues, and monoTIM-H95A is 50 times less active. The best inhibitors of wild-type TIM are phosphoglycolohydroxamate (PGH) and 2-phosphoglycolate (2PG), with KI values of 8 microM and 26 microM, respectively. The affinity of the monoTIM active site for PGH has been reduced approximately 60-fold, whereas for 2PG, only a twofold weakening of affinity is observed. The mode of binding, as determined by protein crystallographic analysis of these substrate analogues, shows that, in particular, 2PG interacts with Lys 13 and His 95 in a way similar but not identical to that observed for the wild-type enzyme. This crystallographic analysis also shows that Glu 167 has the same interactions with the substrate analogues as in the wild type. The data presented suggest that, despite the absence of the second subunit, monoTIM catalyzes the interconversion of D-glyceraldehyde-3-phosphate and dihydroxyacetone phosphate via the same mechanism as in the wild type.

An interface point-mutation variant of triosephosphate isomerase is compactly folded and monomeric at low protein concentrations.

Wild-type trypanosomal triosephosphate isomerase (wtTIM) is a very tight dimer. The interface residue His-47 of wtTIM has been mutated into an asparagine. Ultracentrifugation data show that this variant (H47N) only dimerises at protein concentrations above 3 mg/ml. H47N has been characterised at a protein concentration where it is predominantly a monomer. Circular dichroism measurements in the near-UV and far-UV show that this monomer is a compactly folded protein with secondary structure similar as in wtTIM. The thermal stability of the monomeric H47N is decreased compared to wtTIM: temperature gradient gel electrophoresis (TGGE) measurements give Tm-values of 41 degrees C for wtTIM, whereas the Tm-value for the monomeric form of H47N is approximately 7 degrees C lower.

Three new crystal structures of point mutation variants of monoTIM: conformational flexibility of loop-1, loop-4 and loop-8.

BACKGROUND: Wild-type triosephosphate isomerase (TIM) is a very stable dimeric enzyme. This dimer can be converted into a stable monomeric protein (monoTIM) by replacing the 15-residue interface loop (loop-3) by a shorter, 8-residue, loop. The crystal structure of monoTIM shows that two active-site loops (loop-1 and loop-4), which are at the dimer interface in wild-type TIM, have acquired rather different structural properties. Nevertheless, monoTIM has residual catalytic activity. RESULTS: Three new structures of variants of monoTIM are presented, a double-point mutant crystallized in the presence and absence of bound inhibitor, and a single-point mutant in the presence of a different inhibitor. These new structures show large structural variability for the active-site loops, loop-1, loop-4 and loop-8. In the structures with inhibitor bound, the catalytic lysine (Lys13 in loop-1) and the catalytic histidine (His95 in loop-4) adopt conformations similar to those observed in wild-type TIM, but very different from the monoTIM structure. CONCLUSIONS: The residual catalytic activity of monoTIM can now be rationalized. In the presence of substrate analogues the active-site loops, loop-1, loop-4 and loop-8, as well as the catalytic residues, adopt conformations similar to those seen in the wild-type protein. These loops lack conformational flexibility in wild-type TIM. The data suggest that the rigidity of these loops in wild-type TIM, resulting from subunit-subunit contacts at the dimer interface, is important for optimal catalysis.

Evolutionary conservation of a microbody targeting signal that targets proteins to peroxisomes, glyoxysomes, and glycosomes.

Peroxisomes, glyoxysomes, glycosomes, and hydrogenosomes have each been classified as microbodies, i.e., subcellular organelles with an electron-dense matrix that is bound by a single membrane. We investigated whether these organelles might share a common evolutionary origin by asking if targeting signals used for translocation of proteins into these microbodies are related. A peroxisomal targeting signal (PTS) consisting of the COOH-terminal tripeptide serine-lysine-leucine-COOH has been identified in a number of peroxisomal proteins (Gould, S.J., G.-A. Keller, N. Hosken, J. Wilkinson, and S. Subramani. 1989. J. Cell Biol. 108:1657-1664). Antibodies raised to a peptide ending in this sequence (SKL-COOH) recognize a number of peroxisomal proteins. Immunocryoelectron microscopy experiments using this anti-SKL antibody revealed the presence of proteins containing the PTS within glyoxysomes of cells from Pichia pastoris, germinating castor bean seeds, and Neurospora crassa, as well as within the glycosomes of Trypanosoma brucei. Western blot analysis of purified organelle fractions revealed the presence of many proteins containing this PTS in both glyoxysomes and glycosomes. These results indicate that at least one of the signals, and therefore the mechanism, for protein translocation into peroxisomes, glyoxysomes, and glycosomes has been conserved, lending support to a common evolutionary origin for these microbodies. Hydrogenosomes, the fourth type of microbody, did not contain proteins that cross-reacted with the anti-PTS antibody, suggesting that this organelle is unrelated to microbodies.