Structure of Trypanosoma peroxisomal import complex unveils conformational heterogeneity

Domain: neuroscience
Published: 2025-12-11
Editorial outcome: minor_revision

Abstract
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 cytosolic 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 the process is only partially understood. Here, we characterize the cargo-Pex5-Pex14 NTD ternary complex from Trypanosoma cruzi. Cryo-electron microscopy maps enabled model building for Pex5 (residues 327–462 and 487–653) bound to malate dehydrogenase (MDH; residues 1–323) cargo tetramer and Pex14 NTD (residues 21–85). The model provides insight into conformational heterogeneity and identifies secondary interfaces. Specifically, we observe that orientations of Pex5 relative to MDH span a 17° angle. Additionally, PTS1- and Wxxx(F/Y)-independent contact surfaces are observed at MDH-Pex5 and Pex5-Pex14 NTD interfaces, respectively. Mutational analysis indicates that the non-PTS1 MDH-Pex5 interface does not significantly contribute to the affinity, but limits the conformational heterogeneity of MDH-Pex5 interface. The Pex5-Pex14 NTD interface constitutes an extended binding site of Pex14 NTD over Pex5. We discuss the implications of these findings for understanding peroxisomal import mechanism.

Introduction
Peroxisomes compartmentalize a set of important metabolic processes in eukaryotic cells, including catabolism of fatty acids and polyamines, biosynthesis of ether phospholipids, glyoxylate cycle in germinating seeds, glycolysis in Trypanosoma and many others1,2. Despite lacking genetic material and protein synthesis machinery, peroxisomes efficiently perform their functions by importing enzymes (referred to as cargo) from the cytoplasm3. The import process is orchestrated by a complex network of peroxin (Pex) proteins, numbering over a dozen. Pex proteins recognize specific peroxisomal targeting signal (PTS) sequences present on the folded cargo proteins in the cytoplasm. Subsequently, the cargo is translocated into the peroxisomal lumen, and the Pex system is recycled for a subsequent round of cargo import4. Remarkably, this translocation process occurs without unfolding of the cargo proteins5,6.

The initial step of peroxisomal import involves recognition of a more common PTS1 sequence by soluble receptor Pex5 in the cytoplasm. The recognition of the less common PTS2 sequence requires an additional receptor, Pex7. Pex5 consists of two structural domains: N-terminal unstructured region guiding peroxisomal membrane translocation and C-terminal globular domain adopting tetratricopeptide repeat (TPR) fold and responsible for cargo recognition7,8. The PTS1 signal consists of variants of a Ser-Lys-Leu-COOH consensus sequence at the C-terminus of cargos4. Once bound by Pex5 in the cytosol, the cargo-Pex5 complex is subsequently translocated across the membrane by Pex13, and Pex14 assists in the process9, but the structural details of the interactions and the mechanism of translocation are insufficiently understood.

The primary interaction between cargo and Pex5 is well-characterized through structures of isolated PTS1-peptide binding to the C-terminal TPR domain of Pex57,8. The PTS1 peptide burrows into the central cavity of the Pex5 TPR domain8. Three structures of cargo-bound Pex5 TPR domain are available, which demonstrates secondary, PTS1-independent interactions between the cargo and Pex5 TPR domain. The extent to which secondary interactions contribute to cargo-Pex5 affinity varies significantly, and thus the role of secondary interactions is yet to be completely understood10–14.

The process of translocating the Pex5-cargo complex across the peroxisomal membrane is subject to ongoing debate. What is established is that the import of the Pex5-cargo complex involves assistance from membrane-spanning proteins, Pex14 and Pex139. It has been suggested that Pex5-cargo docking at the membrane is assisted primarily via interaction with Pex14. Different studies propose that cargo-Pex5, in conjunction with Pex14 and Pex13, facilitates the formation of a dynamic pore, or engages a preformed Pex13 pore, enabling translocation of the cargo15–17. N-terminal domain of Pex14 (Pex14 NTD) was additionally implicated in cargo extraction from the translocation pore. The nature and dynamics of the pore and the exact sequence of events require further investigation.

Pex14 consist of three structural regions: N-terminal Pex5-binding domain facing the peroxisomal matrix (Pex14 NTD), cytoplasmic coiled coil domain and C-terminal unstructured region. The interaction between Pex5 and Pex14 is primarily mediated through the recognition of multiple Wxxx(F/Y) motifs on Pex5 by Pex14 NTD 18–21. This interaction has been extensively studied, typically through a system of short peptides containing the Pex5 Wxxx(F/Y) motif binding to the Pex14 NTD 22. However, a comprehensive understanding of the Pex14 binding site on Pex5 is still elusive, as structural data of full-length Pex5 (or extended TPR domain including at least one Wxxx(F/Y) motif) bound to Pex14 are currently unavailable. Earlier studies proposed that the binding of Pex14 to the Pex5-cargo complex triggers cargo release23, but the cooperative role of multiple copies of Pex14 at the Wxxx(F/Y) motifs within Pex5 in binding and/or release of the cargo remains uncharacterized18–21.

In this study, we present the structure of the ternary complex involving cargo-Pex5-Pex14 NTD from Trypanosoma cruzi, the pathogen responsible for human Chagas disease24. Our structural analysis unravels the conformational heterogeneity of interactions of Pex5 with its cargo, and secondary interfaces between cargo-Pex5 and Pex5-Pex14 NTD. The role of the secondary interfaces is investigated by structure-guided mutational analysis. Furthermore, our results show that the Pex14 binding at the Pex5 TPR domain proximal Wxxx(F/Y) site is not sufficient for structural reorganization of the Pex5 TPR domain and cargo release in vitro.

Results
Reconstitution of cargo-Pex5-Pex14 ternary complex
Specialized peroxisomes, known as glycosomes, import glycolysis-associated enzymes (cargo) from the cytosol through the Pex import system and compartmentalize glycolytic reactions in Trypanosoma. The Pex import process is a validated drug target in Chagas disease25–27. After assessing various cargos (glycolytic enzymes) and Pex constructs (Supplementary Table 1), we chose glycosomal malate dehydrogenase (MDH, residues 1–323, UniProt Q4DRD8) as a cargo, and full length Pex5 (residues 1–666, GenBank PBJ69826.1) and N-terminal domain of Pex14 (Pex14 NTD; residues 21–85, GenBank RNC55913.1) for in vitro reconstitution of the ternary complex. MDH is a crucial enzyme for the glycolysis auxiliary pathway, maintaining the pool of malate/oxaloacetate28,29. All three proteins were individually expressed in Escherichia coli and purified to homogeneity. The complex was reconstituted in vitro by mixing purified proteins and separating the complex from non-complexed components using size exclusion chromatography (SEC). The SEC profile of mixed components showed additional peaks at a higher molecular weight, corresponding to the ternary complex (Supplementary Fig. 1). The presence of all three proteins in these peaks was confirmed by the SDS-PAGE (Supplementary Fig. 1).

Cryo-EM reconstructions and heterogeneity of the ternary complex
Upon initial cryo-EM data analysis, the tetrameric arrangement of MDH was evident, yet significant variability in the MDH:Pex5 stoichiometry was noted (Fig. 1). Avoiding chemical crosslinking, which could potentially homogenize the population, we instead extracted information on compositional heterogeneity from the available dataset. Classification efforts yielded 2D class averages representing diverse compositions of complexes, prominently featuring densities corresponding to complexes with one Pex5 molecule (MP1), two Pex5 molecules (MP2), and three (or four; the exact stoichiometry is ambiguous in 2D class average due to the dihedral symmetry) Pex5 molecules (MP3/4) bound to the MDH tetramer (Fig. 1A). The density corresponding to the third Pex5 molecule in MP3/4 was relatively weak (Fig. 1A).

We first attempted to reconstruct high-resolution 3D maps of MP1, MP2 and MP3/4 using relevant particles and data processing procedures not accounting for conformational variability (see Methods). We successfully reconstructed MP1 and MP2 maps showing one and two copies of Pex5 bound to MDH tetramer, respectively (Fig. 1B). Processing of MP3/4 particles did not produce a reasonable quality map, likely due to the low occupancy of the 3rd (and/or 4th) copies of Pex5 and was not analyzed further.

MP1 and MP2 maps show a well-resolved MDH region, but poorly resolved Pex5 region(s), indicating conformational heterogeneity in the latter component (Fig. 1). The resulting maps facilitated de novo model building for MDH, forming a tetramer of ~10 nm diameter. The tetramer is composed of two dimers related by dihedral (D2) symmetry (Fig. 1C). The peroxisomal targeting signal 1 (PTS1) motifs of all MDH subunits are found at the periphery of the tetramer and are equally accessible to the solvent (Fig. 1C).

To elucidate the conformational variability and improve the density of the Pex5 region, we employed three-dimensional variability analysis (3DVA) using cryoSPARC30. This approach improved the density of the Pex5 region and revealed significant heterogeneity in Pex5 orientations relative to MDH, as depicted in Supplementary Movies 1 and 2. The resulting data facilitated reconstruction of distinct 3D volumes representing various orientations of Pex5 relative to MDH within the MP1 and MP2 complexes. We refined and built two representative atomic structures for each complex state, capturing two extreme conformations, namely, MP1-close (MP1-c), MP1-distal (MP1-d), MP2-close (MP2-c), and MP2-distal (MP2-d) (Fig. 2; Supplementary Fig. 2 and Supplementary Movies 3–7). The refined maps facilitated model building of MDH and Pex5 residues 327–462 and 487–653, encompassing the TPR domain and an upstream helix containing the Wxxx(F/Y) motif (residues 327–345) (Supplementary Figs. 3–6). The helix distinguishes our structure from previously available Pex5 TPR domain crystal structures8, in which the Wxxx(F/Y) motif-containing helix is not resolved. The density corresponding to the rest of Pex5 N-terminal domain (NTD; residues 1–326) was not discernible in our structure, indicating its flexible nature even in the presence of Pex14 NTD, consistent with observations from prior studies31.

It is of note that while we observed particles with two Pex5 molecules each bound to the different dimer of the MDH tetramer, we did not observe particles with two Pex5 molecules bound to the same dimer (Fig. 2C). The underlying reason for this specific topology remains unclear as the PTS1 binding sites on all protomers are equally accessible (Fig. 1C). Furthermore, the presence of particles representing the MP3/4 complex (Fig. 1A), where three or four Pex5 molecules are bound to MDH tetramer (two Pex5 on one MDH-dimer and one or two Pex5 on another MDH-dimer), suggests that there are no steric clashes between two Pex5 molecules bound to single MDH-dimer. However, it is uncertain whether the binding of more than one Pex5 is necessary for the import of the oligomeric cargo. The piggy-backing of cargo isoforms lacking the PTS targeting signal within heteromultimeric complexes suggests that the binding of Pex5 to all subunits of the oligomer is not required for translocation5,6. Furthermore, recent findings indicate that predominantly a single Pex5 molecule binds to Eci1 hexameric cargo, implying that the binding of a single Pex5 molecule is sufficient for efficient import14.

Pex5-binding on MDH is characterized by conformational heterogeneity
Recognition of the MDH by Pex5 is mediated via PTS1 motif at the central cavity of Pex5 TPR domain (Fig. 2B). The interaction is stabilized by a network of hydrogen bonds involving the PTS1 C-terminal carboxylate group, main chain acceptors, and lysine side chain amine, as observed previously in different species8 (Supplementary Fig. 7). Some rearrangement of this network is evident between the distal and close states. For instance, the side chain of MDH(K322) forms an H-bond with Pex5(E407) in the distal state, while in the close state, it forms an H-bond with Pex5(N439) (Supplementary Fig. 7). Similarly, H-bonds involving the C-terminus of MDH(L323) are partly rearranged between the distal and close states (Supplementary Fig. 7).

The overall geometry of the PTS1-bound Pex5 TPR domain in T. cruzi is compact compared to the structure of the apo-Pex5 TPR domain32. Similar compaction was also observed for Trypanosoma brucei and the human TPR domain of Pex57,8,10,12,32, suggesting that TPR domain compaction upon PTS1 binding is a general phenomenon across different organisms.

The 3D structural variability (3DVA) analysis reveals that Pex5, when bound to PTS1, exhibits conformation variability on the surface of MDH. We categorized the raw particles into 20 clusters, each representing varying tilt covering the range of orientations between distal and close conformations. The 3DVA results reveal the presence of particles across the whole range of the conformational space between close and distal states (Supplementary Fig. 2). This conformational heterogeneity can be characterized by two components: tilt and twist. Pex5 TPR domain tilts nearly as a rigid body between close and distal conformations with a maximum tilt angle of 17° relative to the PTS1 (Fig. 2A; and Supplementary Movies 1–7). Additionally, Pex5 TPR domain twists up to 13° relative to the axis roughly parallel to the TPR3 motif (Supplementary Movie 8). The combined tilting and twisting result in a radial displacement of Pex5 TPR domain ranging from 3 to 8 Å (Fig. 2). Specifically, TPR motifs 1, 2, 6, and 7 (residues 330–425, 527–640) show displacements of 5–8 Å, which is greater than the displacement of the central TPR motifs 3, 4, and 5 (residues 428–525) that is ~3.5 Å (Figs. 2B, C and Supplementary Movies 1–8).

Further, 3DVA results suggest that there is no coordination between the variability of two Pex5 molecules in the MP2 complex (Supplementary Fig. 9).

Secondary MDH-Pex5 interfaces do not significantly contribute to affinity
Along PTS1-mediated interaction, two additional interfaces between MDH and Pex5 TPR domain are identified by PISA analysis (Fig. 3A). Interface 1 involves MDH residues 62–70 and Pex5 residues 439–444, forming a loop connecting α6 and α7. Notably, MDH residues 62–70 form a loop (MDH 62–70) unique to the glycosomal isoform and absent in the cytosolic/mitochondrial isoforms and homologs from other organisms (Supplementary Fig. 10), suggesting its possible specific role in glycosomal import33. Interface 2 involves MDH residues 142–145 and Pex5 helix α15 made up of residues 625–633 (Fig. 3A).

Because close analysis of interfaces 1 and 2 does not reveal classical features of protein-protein interaction34 and because identified conformational heterogeneity affects the extent of the interfaces, we investigated if the identified interfaces contribute to affinity. Mutants were generated targeting residues at the MDH-Pex5 interface, and their affinities were determined by isothermal titration calorimetry (ITC) (Table 1). The mutants were evaluated in the context of Pex5 eTPR (residues 314–666; Supplementary Table 4), a truncated Pex5 construct slightly exceeding the region defined by density in our structure (residues 327–462 and 487–653). The affinity of Pex5 eTPR and MDH was estimated at Kd ~435 nM (Table 1). To disrupt interface 1, we replaced the MDH 62–70 loop with a GS-linker, resulting in MDH (Δ62–70). This resulted in a moderate decrease (~2.3-fold) (p<0.001) in affinity towards Pex5 eTPR, indicating little contribution of the interface to affinity. Interface 1 at the Pex5 side is composed of backbone atoms and thus not directly amenable to mutational analysis. To assess the contribution of interface 2, a double mutant of Pex5 eTPR (R625A, D629A) was constructed. The mutant exhibited an affinity comparable to the wild type Pex5 eTPR (p=0.5), indicating insignificant contribution of interface 2 to MDH-Pex5 affinity. The MDH side of the interface was composed of backbone atoms and thus was not analyzed by mutagenesis. To further explore the collective impact of secondary MDH-Pex5 interfaces, a linker (GSGS) was introduced between PTS1 and MDH to physically separate the binding partners, thus disrupting all secondary interactions. The MDH (GSGS) linker variant demonstrated nearly three times higher affinity compared to the wild type MDH (p<0.001) (Table 1). The change in affinity was entropy-driven, suggesting that the secondary interactions have an overall negative effect on affinity by restricting the conformational heterogeneity of the wild-type interface. The affinity of MDH (GSGS) is comparable to that of a short peptide encompassing PTS1 of MDH (residues 319-ARSKL-323; p>0.1) (Table 1), again suggesting that the introduction of GSGS linker indeed abolished all secondary interactions at the Pex5-MDH interface.

The above findings collectively demonstrate that neither of the identified secondary interfaces significantly contributes to the MDH-Pex5 affinity and only restrict the conformational heterogeneity of Pex5 TPR domain binding at the surface of MDH.

Transient interaction of Pex5 457–489 loop with MDH
The maps accounting for the distal states of MDH-Pex5 complexes (MP1-d and MP2-d) show additional density next to Pex5 residue Ser491 and facing MDH 62–70 loop (Fig. 3B; Supplementary Fig. 11). Notably, the Pex5 457–489 loop (linking TPR motifs 3 and 4) is unresolved in all existing crystal structures, indicating its flexible nature. The additional density is observed only at sites occupied by Pex5, and not at the vacant PTS1 sites, indicating that it corresponds to a fragment of the Pex5 457–489 loop (Fig. 3B; Supplementary Fig. 11). Additionally, the density was absent in the map of MDH tetramers solved in this study (Supplementary Fig. 11), further suggesting it originates from Pex5. These results indicate restriction of conformational flexibility of the Pex5 457–489 loop in the distal state of the MDH-Pex5 complex.

The identified additional density was insufficient to construct an atomic model of the Pex5 loop, but indicates that the loop 457–489 extends into the groove between MDH subunits (Fig. 3B). In glycosomal MDH, the groove is notably deeper (volume ~4900 Å3) compared to non-glycosomal MDH (PDB id: 5ZI4) (volume ~2500 Å3) owing to the insertion of MDH 62–70 loop, characteristic only for the glycosomal MDH. To determine the significance of the identified interaction, we tested the binding of Pex5 mutant with a partial deletion of the loop [Pex5 eTPR (Δ470–480)] with MDH. Pex5 eTPR (Δ470–480) retained the ability to recognize MDH-derived PTS1 peptide, indicating the deletion had no significant impact on the structure of the mutant, though the affinity was moderately (~3.1×) decreased compared to the wild type (p<0.001; Table 1). At the same time, the mutant exhibited more than four times higher affinity for MDH compared to wild-type Pex5 eTPR (p<0.001). This suggests that the Pex5 470–480 loop sterically clashes with MDH in the distal state of the complex, limiting the available tilt and affinity of the interaction. To further test the hypothesis, we mutated the Pro490 residue, which breaks the TPR4 helix at the onset of the Pex5 457–489 loop into arginine, a residue which we expected to support the expansion of the TPR4 helix and thus limit the loop flexibility. The affinity of Pex5 eTPR (P490R) mutant was compromised more than 3-fold compared to the wild type (p<0.001), and the change in affinity was primarily driven by the entropic component, again demonstrating that the interactions of Pex5 457–489 loop at the intersubunit groove of MDH limit the conformational variability available for the complex.

Pex14 NTD binding site and Wxxx(F/Y) independent interactions with Pex5
We observed weak density beyond the Pex5 TPR domain, which was difficult to interpret in any of the 3DVA-derived maps and was not examined for the purpose of variability analysis described in prior paragraphs. The density could have corresponded to either a fragment of the Pex5 N-terminal domain or Pex14 NTD. To clarify the ambiguity, we reconstituted in vitro and solved the structure of the MDH-Pex5 binary complex devoid of Pex14 NTD (see Methods). The map for the binary complex revealed no density beyond the MDH and Pex5 TPR domain (Supplementary Fig. 13), indicating that the additional density observed in the ternary complex originates from Pex14 NTD. In the ternary complex map, we improved this density by focused refinement using all collected data. Such data treatment did not account for the conformational heterogeneity of the MDH-Pex5 interface, but resolved the density features in the Pex region and enabled the unambiguous atomic model building of Pex14 NTD located on the Pex5 TPR proximal Wxxx(F/Y) motif. The final atomic model of the ternary complex (designated as MP1P) was built in a composite map comprising the consensus MDH density map derived as described earlier in the text (Fig. 1) and the Pex5-Pex14 NTD density map derived from focused refinement. The MP1P model consists of the MDH tetramer, Pex5 (residues 327–462, 487–653), and Pex14 NTD (residues 21–85) (Fig. 4; Supplementary Fig. 14).

The ternary complex structure constructed in MP1P composite map reveals an extended Pex14 NTD-Pex5 binding site involving two distinct interfaces (Fig. 4). The first interface encompasses Pex5 Wxxx(F/Y) motif22 (328-WVREY-332) constituting a part of an α-helix (residues 327–345) extending beyond the globular structure of the TPR domain of Pex5 (Fig. 4). The core interactions in this region are contributed by bulky sidechains within the 328-WVREY-332 motif (underlined) which bury in a hydrophobic groove on the surface of Pex14 NTD. The Wxxx(F/Y) interface resembles closely that observed in previously solved structures of short peptides encompassing the Wxxx(F/Y) motif in complex with Pex14 NTD 22 (Supplementary Fig. 15). The second, non-Wxxx(F/Y) interface has not been observed before and involves Pex14 NTD helix α3 and 353-NNPYMY-358 motif of Pex5 (Fig. 4 and Supplementary Figs. 16, 17). The non-Wxxx(F/Y) interface contributes ~800 Å² of buried surface area out of a total ~1300 Å² comprising the Pex14 NTD-Pex5 interface. The remaining ~500 Å² are contributed by the Wxxx(F/Y) site. Helix α3 of Pex14 NTD (Supplementary Fig. 17) and two amino acids (underlined) in 353-NNPYMY-358 motif of Pex5 are conserved (Supplementary Fig. 16). The 353–NNPYMY-358 region forms an extended interhelix linker separated from surrounding helical regions by helix-breaking P352 and P355. The resolution limits detailed analysis of interactions, but CB atoms are clearly discernable, allowing to roughly predict the position of the sidechains. Sidechains of Pex5 N353 and Y358 extend in the direction of Pex14, but seem not to be involved in any specific interactions. The sidechains of A48 and Q52 of Pex14 extend in the direction of Pex5, but again, no specific interactions can be predicted in the region. Overall, the interface does not resemble protein-protein interaction interfaces34, but rather a proximity assembly.

To evaluate the contribution of the non-Wxxx(F/Y) interface to Pex5-Pex14 NTD affinity, we designed and tested the affinity of relevant mutants in the context of Pex5 eTPR. Affinities were determined in the presence of the PTS1 peptide to maintain a cargo-bound-like conformation of Pex5 eTPR, presumably a physiologically relevant binding conformation (affinities in the absence of PTS1 are presented for reference in Supplementary Table 2). The affinity (Kd) of the wild-type Pex14 NTD for Pex5 eTPR was estimated at ~53 nM by ITC (Table 2). The affinity of Pex14 NTD (Q52A) mutant was comparable (p>0.8) to the wild type, indicating that Q52 does not contribute specific interactions with Pex5 eTPR. The affinity of Pex14 NTD (A48F) decreased more than two times compared to the wild type (p<0.001). This demonstrates that the sidechain of A48 indeed points in the direction of Pex5 eTPR as predicted, but the interface is flexible enough to accommodate a bulky sidechain in place of a short sidechain of Ala48 with only insignificant steric hinderance. Mutating Y358 of Pex5 eTPR to alanine decreased the affinity of the mutant by ~1.5 times compared to the wild type, again as predicted in terms of involvement of Y358 in the interface, though the effect has not reached statistical significance (p=0.07). Minor influence of Y358A residue substitution again suggests that the interface is flexible to accommodate significant changes in side chain properties without a large effect on affinity. Pex5 eTPR (N353E) had almost no effect on affinity (p>0.4), demonstrating that hydrogen bonding is not involved in the interaction. To further substantiate the above findings, we introduced a GSGS-spacer between the Pex5 TPR domain and Wxxx(F/Y) motif to retain the primary interface, but physically separate the secondary interaction surface. The mutation resulted in a roughly 3.5-fold decrease in the affinity (p<0.001), demonstrating that both the Wxxx(F/Y) and non-Wxxx(F/Y) sites contribute to the Pex14 NTD-Pex5 eTPR affinity, with the former site having a major contribution.

Structural rearrangement within the TPR domain of Pex5 was demonstrated upon PTS1 binding12. Since we demonstrated that Pex14 NTD interacts directly with the TPR domain, we tested the potential influence of Pex14 NTD on Pex5 eTPR affinity for PTS1. The affinity of the fluorescently labeled PTS1 peptide for Pex5 eTPR was determined at Kd = 254 ± 9 nM by fluorescence polarization. The affinity was ~3.2 times higher in presence of excess Pex14 NTD (Kd = 80 ± 5 nM; p<0.001) (Supplementary Fig. 18). Comparable increase in affinity of full length Pex5 (containing three Wxxx(F/Y) motifs) for PTS1 in the presence of Pex14 NTD was observed (Kd = 88 ± 5 nM and Kd = 37 ± 2 in the absence and presence of Pex14 NTD, respectively; p<0.001). These data indicate that at the tested conditions, Pex14 NTD binding does not release the cargo from Pex5 (or Pex5 eTPR). In contrast, it slightly increases the affinity of Pex5 (and consistently of Pex5 eTPR) for the cargo. In turn, the affinity of Pex14 NTD for Pex5 eTPR determined by ITC in the presence of PTS1 peptide (Kd = 53 ± 12; Table 2) is lower (p<0.001) compared to the affinity determined in the absence of PTS1 peptide (Kd = 35 ± 5; Supplementary Table 2).

Methods
Protein expression and purification
Genes encoding Trypanosoma cruzi MDH, full-length Pex5 (residues 1–666, GenBank PBJ69826.1), truncated Pex5 containing an extended TPR domain containing a single Wxxx(F/Y) motif (residues 314–666; Pex5 eTPR), and Pex14 NTD (residues 21–85) were synthesized de novo with N-terminal 6×His, 6×His and 6×His-TEV site tag, respectively. Genes were cloned in pET24(+) vector using NdeI and XhoI restriction sites. Recombinant vectors were transformed individually into E. coli BL21 (DE3) for overexpression. Proteins were purified by Ni-affinity (Ni-NTA) and size exclusion chromatography (SEC) (Supplementary Methods). Purified Pex14 NTD was subjected to TEV protease treatment to remove the 6×His-tag. Mutations were introduced by site-directed mutagenesis, and the mutants were purified as wild-type proteins. Details of all the protein constructs used in this study are provided in Supplementary Table 4.

In vitro reconstitution and purification of the ternary complex
Purified MDH (tetramer), full-length Pex5 and Pex14 NTD were mixed at 1:4:12 molar stoichiometry and incubated overnight at 4 °C for ternary complex formation. The formed complex was separated from the remaining individual components by size exclusion chromatography (SEC) using Superose 6 10/300.

Analysis of the complex formation
The highest molecular weight peak after the void volume of Superose 6 10/300 was collected as the potential ternary complex sample and analyzed by SDS-PAGE. 10 µL of the sample was mixed with 2x SDS-PAGE sample loading buffer and run on the 15% SDS-PAGE gel along with the molecular weight marker in Tris-Glycine running buffer, followed by the Coomassie blue staining. The presence of all components was confirmed from the resultant band pattern (Supplementary Fig. 1).

Cryo-EM of MDH-Pex5-Pex14 ternary complex
Cryo-EM data collection: Purified ternary complex (~0.75 mg/mL) was vitrified on holey-carbon grids (Quantifoil R 2/1) using FEI Vitrobot Mark IV. Grids were glow-discharged at 8 mA for 70 s using the LEICA EM ACE200 glow-discharger. Glow-discharged grids were loaded with 3 µL of sample, blotted (blot time: 5 s; blot force: 5 units) and flash-frozen by plunging into liquid ethane. Frozen grids were screened for the quality of ice and particle density on 200 kV Glacios microscope equipped with Falcon 4 detector prior to data collection on 300 kV Titan Krios G3i Cryo-TEM microscope equipped with Falcon 4 detector at a magnification of ×105 k. Each exposure was recorded into 40 dose-fractioned frames with a total electron dose of 42.25 e−/Å2 with a pixel size of 0.86 Å/px. Screening and data collection were performed at the cryo-EM facility at the Solaris synchrotron in Krakow, Poland.

Cryo-EM data processing: Data were processed using cryoSPARC45. Original pixel size 0.86 Å/px was used throughout the processing. Micrographs were motion corrected and subjected to contrast transfer function (CTF) estimation using patch motion correction and patch CTF estimation jobs, respectively. Protein particles were picked on a subset of micrographs using blob-picker. Picked particles were subjected to template-free 2D classification to generate initial 2D templates. Generated 2D templates were used to pick particles from the whole dataset by template-picker with parameters: particle diameter, 150 Å; Low pass filter to apply (template/micrograph), 20 Å/20 Å; Angular sampling, 5⁰; Minimum separation distance, 75 Å; Maximum number of picks to consider, 2000. Bad particles were removed by iterative rounds of 2D classification. We first followed the data processing procedure not accounting for conformational variability. Particles in selected classes representing MP1, MP2, and MP3/4 complex were used for initial model generation using ab-initio 3D reconstruction and subsequent refinement using homogenous and heterogeneous refinement jobs in cryoSPARC. This resulted in the maps for MP1 and MP2 showing satisfactory density for MDH, but blurred density for the Pex region. The 3D-variability analysis (3DVA)30 was used to classify the particles based on heterogeneity. Selected particle clusters and associated volume maps were individually subjected to non-uniform homogenous refinement in order to reconstitute individual 3D maps representing different compositional and conformational states of the complex, i.e., MP1-c, MP1-d, MP2-c, and MP2-d (see Results for the definition of states). Local refinement using the local mask on the Pex region was performed to improve the map within the Pex region. Local mask was generated to include the density of a single copy of the Pex region in both distal and close states of MP1-c, MP1-d, MP2-c, and MP2-d complexes. All particles, including particles in the class representing MP3/4, were used for the local refinement using this mask, resulting in well-resolved density for Pex14 NTD. Locally refined Pex region map was combined with the consensus map using Combine map tool in Phenix to generate the final map of MP1P complex46. Density maps were further sharpened by B-factor-based sharpening in cryoSPARC and using EMReady47. Overall quality of cryo-EM data and data-processing workflow is depicted in Supplementary Figs. 2–6, 14 and Supplementary Table 3.

Model building and refinement: Crystal structure of T. cruzi MDH (PDB id: 7QOZ) and Pex5 TPR domain (PDB id: 8OS1) were used to fit in MP1 and MP2 maps using Dock in map program of Phenix suite46. Docked models were subjected to flexible fitting into the map using the namdinator web-server48. Additional residues were manually added to the Pex5 TPR model at the N-termini as guided by the density map. The procedure resulted in the final model of Pex5 comprising residues 327–462 and 487–653. Fitted models were refined against the map by iterative cycles of automatic refinement using phenix real-space refinement49 and interactive refinement using COOT50 until reasonable geometry and map-to-model correlation were achieved. For the MP1P structure, the refined models of MDH and Pex5 from the above structures were rigid-body fitted in the map and further refined. The Pex14 NTD was then de novo built into the map to complete the MDH-Pex5-Pex14 NTD model. For Pex14 NTD de novo model building, its alphafold51-predicted structure was docked within the map. The model was trimmed and refined as needed according to the map by COOT50, phenix real space refine46 and ISOLDE52. Validations of model and model vs. data for all structures are summarized in Supplementary Figs. 3–6, 14 and Supplementary Table 3.

Cryo-EM structure determination of MDH-Pex5 binary complex and MDH alone
The MDH-Pex5 binary complex was in vitro reconstituted similarly to the MDH-Pex5-Pex14 NTD ternary complex, but without Pex14 NTD. The cryo-EM structure of MDH alone was solved from a dataset containing MDH-Pex5-Pex14 NTD by selection of particles that contained only the MDH tetramer core. The atomic structures were derived as described for the ternary complex. The MDH-Pex5 structure was used to verify the assignment of the density to the Pex14 NTD in the MP1P map. The MDH structure was used to verify the assignment of the density to the Pex5 457–489 loop in the MP1-d and MP2-d maps.

Data analysis and representation
The analysis, manipulation and representation of cryo-EM maps and 3D structures were performed using COOT50, PyMOL (Schrödinger, LLC), UCSF Chimera53 and chimeraX54. The twist and tilt angles describing the movement of Pex5 relative to MDH were estimated for the close model with reference to the distal model, while both models were aligned on the MDH structure. The twist angle was determined by the measure rotation tool in chimeraX54, which reports the rotation between the coordinate systems of the two models with respect to the imaginary axis shown in Supplementary movie 8. We define the tilt angle as the angle between the imaginary 2D planes encompassing selected atoms from Wxxx(F/Y) containing helix and α15 helix of Pex5 in distal and close states54. Sequence alignments were performed and presented using MEGAX software and ESpript server55, respectively. The statistical significance of differences in compared values was evaluated using Welch’s t-test (two-tailed). The p value of below 0.05 was considered significant.

Isothermal titration calorimetry (ITC)
ITC measurements were performed in 20 mM sodium phosphate buffer containing 100 mM NaCl at pH 6.5 (Pex5 eTPR–Pex14 NTD interaction) or 100 mM Hepes buffer containing 150 mM NaCl and 2 mM β-mercaptoethanol at pH 7.5 (MDH-Pex5 eTPR interaction) using a Nano ITC 2G (TA Instruments). Before the experiments, proteins were extensively dialyzed against the relevant buffer. Where relevant, the complex was prepared post-dialysis by mixing Pex5 eTPR (20 μM) with the PTS1 peptide (NH2-ARSKL-COOH; 50 μM) and the mixture was used for ITC. The binding experiment involved 5 μL injections of Pex14 NTD (175 μM) or Pex5 eTPR solution (250 μM) into the calorimeter cell containing Pex5 eTPR + PTS1 (20 μM) or MDH (25 μM), respectively. Injections were performed at 300 s intervals. All experiments were conducted at 25 °C with a stirring rate of 250 rpm. The heat of dilution was determined by titration of Pex14 NTD or Pex5 eTPR to protein-free buffer. The data were analyzed using NanoAnalyze software (TA instruments). The N value (defined as N=St*(AFcell/AFsyr), where St reflects stoichiometry [number of binding sites], and AF represents active fractions in cell and syringe), the dissociation constant (Kd) and the enthalpy change (ΔH) were obtained from fitting the data with a binding model. The Gibbs free energy (ΔG) and change in entropy (ΔS) were calculated using −RT ln Kd = ΔG = ΔH − TΔS, where R is the gas constant and T is the absolute temperature.

Fluorescence polarization assay
Fluorescence polarization (FP) was determined using Tecan Infinite Pro F200 plate reader with the 485 nm excitation and 535 nm emission filters. The fluorescence intensities parallel and perpendicular to the plane of excitation were determined in Corning black 96-well NBS assay plates at room temperature. Fluorescence polarization values were expressed in millipolarization units (mP).

In FP binding experiments, Pex5 eTPR (9.5 µM) and Pex5 (2.8 µM) were serially diluted in the presence and absence of Pex14 NTD (34 µM, constant concentration). 6-FAM labeled PTS1 peptide was added at 10 nM concentration. The FP values were determined and plotted against the log of Pex5 concentration, and the dissociation constant (Kd) was obtained by fitting the experimental data using the following equation:
FP = FPmin + (FPmax − FPmin) * c / (Kd + c) (1)
where, FP is the experimentally determined fluorescence polarization, FPmax represents maximum value of fluorescence polarization (PTS1 peptide saturated with Pex5/Pex5 eTPR), FPmin represents a baseline level (PTS1 peptide alone), Kd is the dissociation constant, and c is the protein concentration. All experiments were repeated three times, Kds were determined independently for each repeat and averaged.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This review is on a revised version of the manuscript entitled “Structure of Trypanosoma peroxisomal import complex unveils conformational dynamics”, which includes a file on the main manuscript, another one on Supplementary Material, and an extensive rebuttal letter. As it turned out to be impossible to track back various changes mentioned in the rebuttal letter, the reviewer asked for a version of the manuscript with the changes highlighted (it would have been appreciated if such version had been provided without explicit request). As it became evident that large parts of the manuscript were completely rewritten (including the title), the reviewer decided to use this new version as an independent one and to review it from scratch. I will also respond to some comments made in the rebuttal letter.\n\nA major point the authors are aiming for is a claim of distinct swinging motions of the Pex5 receptor upon binding to the cargo used in this contribution (MDH). An alternative interpretation would be to consider a quasi-continuous ensemble of slightly different conformational states, within the limits found in the total of 20 particle clusters (Supplementary Figure 2). The authors suggest the second scenario in their own words by stating: “The random distribution of particles among the clusters indicates a continuous spectrum of Pex5 swinging motion relative to MDH with no preferential orientations (Supplementary Figure 2).” Unfortunately, there is no further characterisation of the 20 clusters, e.g. in terms of the tilt/twist angles of Pex5/MDH arrangements. Is there any particular reason why the second and third most populated clusters (16, 20) were not used for structural interpretation? It the scenario of a conformational ensemble model is true (which appears to be more likely in the author’s own words, see above), the proposed swinging model is either an inaccurate description or is not existent, unless experimental evidence for swinging is provided. Morph videos are illustrative but do not prove swinging. Clarification of this is crucial to avoid a misconception of the data presented. Please note that there are other systems in biology with distinct swinging motions, based on experimental evidence (for some examples, see review PMID: 34547238). A similar argument applies to a frequently used statement of “conformational dynamics”. It remains unclear what the authors exactly mean with it: if it is conformational dynamics in a sense of change from state A to state B, there is no experimental evidence for it. If it refers to conformational wobbliness of the CTD of the Pex5 receptor, it is probably closer to the truth, but this is a well-established concept for many years, referring to some of the literature cited by the authors.\n\nThe authors make excessive use of statements about “canonical” versus “non-canonical” interfaces, addressing the Pex5/MDH interactions and Pex5/Pex14 interactions. As “non-canonical” is used for the description of different, unrelated interfaces, it would be advantageous to simply refrain from such terminology and define them in a more defined way, i.e. PTS1 / non-PTS1 for Pex5/cargo and Wxxx(F/Y) / non-Wxxx(F/Y) for Pex5/Pex14. Simplified versions of this would be fine too.\n\nThere is still a lack of clarity on Pex5 constructs used in different experiments. While in the rebuttal letter under point 2 it is stated: “All the binding data reported in the revised version is derived at conditions which make the ITC data interpretation in the context of cryo-EM structure more relevant.” In the HIGHLIGHTS section of the manuscript, however, it is stated “The structure of the peroxisomal import ternary complex (MDH-Pex5TPR-Pex14NTD) is determined using cryo-EM.” I remain confused as to what construct was used for different types of experiments. Therefore, I would like to request to use a non-ambiguous nomenclature throughout the experimental results text, providing clarity which Pex5 constructs were used, such as Pex5(FL) and Pex5(CTD), for instance. The use of the term Pex5TPR is also suboptimal, as this construct exceeds the well-established TPR array in Pex5. Finally, what was the reason to use different Pex5 constructs? Insight into this needs to be transparent.\n\nAre there binding data for the ternary Pex5/Pex14/MDH complex (point 8 of the rebuttal letter)? This is confirmed in the rebuttal letter. In Table 2 and in the text it is mentioned that a “short peptide encompassing PTS1 of MDH (residues 319-ARSKL-323)” was used. Why was this peptide, lacking any secondary binding sites, used instead of MDH? I also could not find any description in the Methods section how this peptide complex was prepared. How was it purified, characterised and what was its concentration, when used in experiments? Is there any difference in Pex14 binding in the presence/absence of the peptide? What would be the difference in the presence/s absence of MDH?\n\nAny statistical significance analysis of the binding data (Tables 1 and 2) is missing. In the absence of this, the interpretation of the observed changes is more a matter of belief than based on statistical evidence. Such analysis needs to be added to allow their quantitative interpretation. This is essential as quite a number of binding data are either unexpected or inconclusive. In general, these data need to be better explained. In the absence of estimated statistical significance, they may put the correctness of some of the structural data into question.\n\nAlthough publishers are consistently request to refrain from superiority claims, both in a sense of superiority of own data and inferiority of others, the manuscript repeats inferiority claims multiple times by classifying previous findings as “poor”, directly or indirectly suggesting superiority of their own data (see e.g. reply to point 13 in the rebuttal letter). The data presented do not explain why Pex5-cargo binding facilitates translocation into the peroxisomal lumen (abstract), a “process poorly understood”. Therefore, despite very interesting structural insights, I do have doubts that they really facilitate an improved understanding of the peroxisomal cargo translocation process.\n\nSupplementary items are not cited in sequential order. This needs to be corrected.\n\nAdditional comments:\nPlease remove the statement “the causative agent of human Chagas disease” from the abstract. This is repeated three times in the manuscript. The data presented neither link to nor explain why Trypanosoma cruzi is the causative agent of human Chagas disease.\n\nIntroduction, p. 4: “What is established is that the import of the Pex5-cargo complex involves assistance from membrane-spanning receptors, Pex14 and Pex13(5).” Neither Pex14 nor Pex13 are described as “receptors” in reference 5 nor, to the best of my knowledge, in other relevant literature. Pex5 is the receptor.\n\nResults, section “Cryo-EM reconstructions and heterogeneity of the ternary complex”, page 7: “It is of note that although we observed particles with two Pex5 molecules bound on the same side of the MDH tetramer, we did not observe particles with two Pex5 molecules bound to diagonally opposite MDH subunits (Figure 2B).” Without further explanation, this sentence does not make sense. The inherent MDH symmetry needs to be explained or cited somewhere. Assuming that it is 222, there is no preferred side of the tetramer. Unless the symmetry is different, there is a requirement for two Pex5 molecules in “diagonal” positions in the MP3 complex, even if one of the two is not interpretable in molecular detail.\n\nResults, section “Cryo-EM reconstructions and heterogeneity of the ternary complex”, page 7: “The piggybacking of cargo isoforms within heteromultimeric complexes suggests that saturation of all Pex5 interaction sites is not required for translocation29,30.” I could not find any discussion on the lack of requirement for saturated binding of cargos in references 29+30.\n\nResults, section “Pex5-binding on MDH is of dynamic nature”, page 7: “This interaction is stabilized by a network of hydrogen bonds involving the PTS1 C-terminal carboxylate group, mainchain donors and acceptors, and lysine sidechain amine, as observed previously in different species7.” Could this network please be shown for the structure presented here? Is this network around the PTS1 motif preserved in different binding conformations or is it different?\n\nResults, section “Pex5-binding on MDH is of dynamic nature”, page 7: “Similar compaction is also observed for Trypanosoma brucei and human Pex56-8,31, suggesting that TPR domain compaction upon PTS1 binding is general across species.” To the best of my knowledge, the concept of binding site compaction was introduced by Fodor et al., (2015, PMID: 25369882), and confirmed in reference 31, from some of the authors of this paper. Unfortunately, previous work was also not be adequately cited in reference 31.\n\nResults, section “Secondary MDH-Pex5 interfaces does not significantly contribute to affinity”, page 9: “Because close analysis of interfaces 1 and 2 does not reveal classical features of protein-protein interaction and because identified swinging motion affects the extent of the interfaces, we wanted to test if the identified interfaces contribute to affinity.” What are \"classical features of protein-protein interaction\"? Please define.\n\nFigure 1:\nWhat is the \"standard data processing pipeline\"? Please reference.\nA. Assuming 222 MDH symmetry (further comments elsewhere), how could it be excluded that a 4th Pex5 component is simply hidden behind MDH because of symmetry?\nB. Pex5 and MDH are clearly separated by different colors. There is no need of further encircling.\nC, What is the message of this panel? I don’t get it.\n\nFigure 2:\nA. How was the angular movement (tilt) measured? This needs to be described in the methods section.\nB. Is this a movement (translation) or rotation, or a combination of both? Could the movements please be plotted against the visible Pex5 sequence, in an additional supplement figure? Given that the color code of the second Pex5 molecule in MP2 is limited to green/yellow, are the authors suggesting that the flexibility of Pex5 binding of the second Pex5 molecules in MP2 is more restricted than for the first Pex5 molecule?\n\nFigure 3:\nA. The mixture of cartoon (MDH) and surface (Pex5) is more confusing than clarifying. Could any specific interactions (hydrogen bonds, salt bridges) be identified, and if so, please indicated? To what extent are the interfaces shown in the different binding conformations (close, distal, intermediate, as explained in S Figure 8) are preserved or different?\nB. I don't get the sentence \"The density is not seen in a groove distal form Pex5 occupied site (red).\" Isn't this logical, if no Pex5 is bound?\n\nFigure 4 (and corresponding text)\nIt is surprising that mutagenesis of residues, based on the structural model of the interaction by Pex5 with Pex14, shows only little or no effect (for statistical assessment, see comments elsewhere): how sure are the authors about a correct sequence register of Pex14? Is this model supported by AF3 predictions and previous structural data of the Pex14-NTD with a Pex5 peptide. Please elaborate on this, possibly by adding figures in the supplement.\n\nSupplementary Figure 1A: Please characterise all three major peaks of the ternary complex by SDS-PAGE. This is important for clarification of incomplete occupancy of Pex5 in MDH/Pex5 complexes.\n\nI will not comment on all replies of the rebuttal letter here, as the review would become too excessive.", "reviewer_2": "With the better resolution structure of the MDH:PEX5:PEX14 NTD complex and additional ITC data, the authors have significantly improved the quality of the presented data; however, I still have reservations about whether the data presented advances our understanding of peroxisome import. The importance of the dynamics at the PEX5:MDH interface is limited by the lack of a functional import assay to test these hypotheses. The authors’ claim that binding of PEX14 to the proximal WXXXF motif is insufficient to cause cargo release could be an interesting result for the understanding peroxisome import, but the authors do not have quantitative data, for example of PEX5 and PTS1 peptide affinity with and without PEX14 to conclusively make their claim. In summary, I agree that the author’s data now support their claims (1 - Cryo-EM structure of Pex5:Pex14 NTD:MDH, 2 - Pex5 – MDH interaction is dynamic, and 3-Pex5-Pex14 NTD site extends beyond canonical site), but I am not convinced these claims advance our understanding of peroxisome import.\n\nSeveral suggestions that I think the authors could address with changes to the text:\n\n“Furthermore, our results show that the Pex14NTD binding at Pex5 proximal Wxxx(F/Y) site is not enough for structural reorganization of the Pex5 NTD and cargo release in vitro.” The reader is not given enough context in the introduction to understand the importance of this statement. It would be helpful to talk about domain architecture of Pex14, topology at the membrane, the need for release of cargo in the peroxisome in the intro. This is well covered in the discussion, but could be discussed more in the introduction.\n\n“The density is present only at sites occupied by Pex5 and not empty PTS1 sites indicating it corresponds to a fragment of Pex5457-489 loop (Figure 3B; Supplementary Figure 12).” It seems possible this density could be a loop of MDH stabilized by the presence of Pex5 instead of a loop of Pex5 stabilized by MDH. Can the authors more clearly state why they think this density can definitively be assigned to PEX5 (ie rule out missing loops in MDH)?\n\nThe authors have chosen to exclude their previous ITC data of PEX5 and PEX14 interactions without PTS1 peptide present. This data could be useful for interpreting how PTS1 binding alters PEX5 affinity for PEX14, which is relevant to the authors discussion of PEX14 causing PEX5 to release its cargo.\n\nThe authors could discuss the conservation of the non-canonical interfaces for PEX14 NTD and PEX5.\n\n“Proteins were purified by Ni-affinity (Ni-NTA) and size exclusion chromatography (SEC).” Authors should describe buffers, lysis, concentration methods for purification and EM grid preparation.\n\nI have minor comments on typos/phrasing below, if it helps the authors.\n\nMinor comments:\n“Pex14 was additionally implemented in cargo extraction from the translocation pore.” Phrasing is off - did you mean implicated instead of implemented?\n\n“The maps accounting for the distal states of MDH-Pex5 complex (MP1-d and MP2-d) show additional density proceeding Pex5 residue Ser491 and facing MDH62-70 loop (Figure 3B; Supplementary Figure 12).” Did you mean preceding instead of proceeding?\n\n“To furhter test the hypothesis, we mutated Pro490 residue” – typo in further\n\n“The density is not seen in a groove distal form Pex5 occupied site (red).” – typo in form\n\n“Prior studies involving ATG and SCP2” – typo in AGT\n\n“again demonstrating that the intractions at the intersubuint groove of MDH unfavorably affect the complex dynamics and affinity.” – typos in interactions and intersubunit"}, "author_response": "Attachment 1 – Responses to the reviewer’s comments (R2) NCOMMS-23-36304A\nPoint-by-point responses to the reviewers’ comments (R2)\nReviewer #1\n“This review is on a revised version of the manuscript entitled “Structure of Trypanosoma\nperoxisomal import complex unveils conformational dynamics””\nMajor comments:\n1. “A major point the authors are aiming for is a claim of distinct swinging motions of the Pex5\nreceptor upon binding to the cargo used in this contribution (MDH). An alternative interpretation\nwould be to consider a quasi-continuous ensemble of slightly different conformational states,\nwithin the limits found in the total of 20 particle clusters (Supplementary Figure 2). The authors\nsuggest the second scenario in their own words by stating: “The random distribution of particles\namong the clusters indicates a continuous spectrum of Pex5 swinging motion relative to MDH\nwith no preferential orientations (Supplementary Figure 2).” Unfortunately, there is no further\ncharacterisation of the 20 clusters, e.g. in terms of the tilt/twist angles of Pex5/MDH\narrangements. Is there any particular reason why the second and third most populated clusters\n(16, 20) were not used for structural interpretation? It the scenario of a conformational\nensemble model is true (which appears to be more likely in the author’s own words, see\nabove), the proposed swinging model is either an inaccurate description or is not existent,\nunless experimental evidence for swinging is provided. Morph videos are illustrative but do not\nprove swinging. Clarification of this is crucial to avoid a misconception of the data presented.\nPlease note that there are other systems in biology with distinct swinging motions, based on\nexperimental evidence (for some examples, see review PMID: 34547238). A similar argument\napplies to a frequently used statement of “conformational dynamics”. It remains unclear what\nthe authors exactly mean with it: if it is conformational dynamics in a sense of change from\nstate A to state B, there is no experimental evidence for it. If it refers to conformational\nwobbliness of the CTD of the Pex5 receptor, it is probably closer to the truth, but this is a well-\nestablished concept for many years, referring to some of the literature cited by the authors.”\nOur data documents the conformational heterogeneity of Pex5 (abbreviation refers to the\nhTPR\nresolved part of Pex5 in cryo-EM map) orientations relative to MDH cargo in the structure of\nMDH/Pex5 /Pex14 complex. In further top-down analysis the heterogeneity could be\nhTPR NTD\ninterpreted as snapshots of a continuous motion between boundary conditions or a “quasi-\ncontinuous enesmble of slightly different conformational states”, but our data does not allow to\ndistinguish between the two models. What our 3DVA results show, is the presence of particles across\nthe whole conformation space between the close and distal states of the complex (Supplementary\nFigure 2). This is consistent with a relatively low energy contribution of PTS1-independent interactions.\nHowever, the number of clusters resulting from 3DVA analysis is shaped by input parameters and\nshould not be interpreted as a number of discrete states. Our data does not distinguish between\ncontinuous, quasi-continuous or discrete distribution of Pex5 orientations relative to MDH. The best\nhTPR\nmodel explaining the data assumes ensemble of states that are in dynamic equilibrium with\nlow-energy barrier between the states and we refer to this model as “conformational dynamics”.\nThis model does not conclude on the lifetime or relative population of states. We now clearly\nintroduce the term and discuss the above considerations in the revised manuscript (R2).\nFor detailed characterization we chose the boundary conditions (the most “distal” and most\n“close” conformations) and not “second and third most populated clusters (16, 20)”. The choice\nwas deliberate and made to delineate Pex5 /MDH interactions which limit further\nhTPR\nAttachment 1 – Responses to the reviewer’s comments (R2) NCOMMS-23-36304A\nreorientation of Pex5 relative to MDH. The populations of the clusters given in\nhTPR\nSupplementary Figure 2 are based on a single experiment (3DVA of a single cryo-EM dataset\ncollected from a single grid). The figure is presented to illustrate the distribution in this single\nexperiment only, but because the data lacks estimation of interexperiment variation it should\nnot be used to conclude on quantitative population differences (if any) between clusters.\nResults shown in Supplementary Figure 2 only allow to conclude that particles are present\nacross the whole range of the conformational space between close and distal states.\nThe morph video shows continuous morphing through all identified clusters and not just\nbetween boundary conditions. We agree with the reviewer that this does not “prove swinging”,\nbut we do not make such claim. The video demonstrates that no significant outliers are present\namong clusters. Because clusters provide continuous / quasi-continuous representation of the\nintermediates between boundary states and because clusters are closely interspersed, the\ndetailed analysis of intermediate clusters does not add significant information and only the\nboundary conditions were analyzed.\n2. “The authors make excessive use of statements about “canonical” versus “non-canonical”\ninterfaces, addressing the Pex5/MDH interactions and Pex5/Pex14 interactions. As “non-\ncanonical” is used for the description of different, unrelated interfaces, it would be\nadvantageous to simply refrain from such terminology and define them in a more defined way,\ni.e. PTS1 / non-PTS1 for Pex5/cargo and Wxxx(F/Y) / non-Wxxx(F/Y) for Pex5/Pex14.\nSimplified versions of this would be fine too.”\nThe suggested changes in terminology were introduced in the revised version according to the\nreviewer’s suggestion to clearly define the described interfaces. “Noncanonical” is now only\nused in the abstract and at the conclusion of the discussion as a concise general term to\ncollectively distinguish previously described interactions from those described in this study.\nThe term is not used in the manuscript text with reference to particular Pex5 /MDH or\nhTPR\nPex5 /Pex14 interactions.\nhTPR\n3. “There is still a lack of clarity on Pex5 constructs used in different experiments. While in the\nrebuttal letter under point 2 it is stated: “All the binding data reported in the revised version is\nderived at conditions which make the ITC data interpretation in the context of cryo-EM structure\nmore relevant.” In the HIGHLIGHTS section of the manuscript, however, it is stated “The\nstructure of the peroxisomal import ternary complex (MDH-Pex5TPR-Pex14NTD) is\ndetermined using cryo-EM.” I remain confused as to what construct was used for different\ntypes of experiments. Therefore, I would like to request to use a non-ambiguous nomenclature\nthroughout the experimental results text, providing clarity which Pex5 constructs were used,\nsuch as Pex5(FL) and Pex5(CTD), for instance. The use of the term Pex5TPR is also\nsuboptimal, as this construct exceeds the well-established TPR array in Pex5. Finally, what\nwas the reason to use different Pex5 constructs? Insight into this needs to be transparent.”\nA non-ambiguous nomenclature of constructs was introduced and consistently used in the\nrevised version of the manuscript (R2) including the highlights. In addition, supplementary table\n4 is introduced to summarize the nomenclature. Suboptimal overlap with terminology used in\nprevious literature for the description of slightly different constructs was avoided. The reasons\nfor using truncated Pex5 construct is stated in R2 version.\n4. “ (a) Are there binding data for the ternary Pex5/Pex14/MDH complex (point 8 of the rebuttal\nletter)? This is confirmed in the rebuttal letter. (b) In Table 2 and in the text it is mentioned that\na “short peptide encompassing PTS1 of MDH (residues 319-ARSKL-323)” was used. Why was\nthis peptide, lacking any secondary binding sites, used instead of MDH? I also could not find\nany description in the Methods section how this peptide complex was prepared. How was it\nAttachment 1 – Responses to the reviewer’s comments (R2) NCOMMS-23-36304A\npurified, characterised and what was its concentration, when used in experiments? (c) Is there\nany difference in Pex14 binding in the presence/absence of the peptide? What would be the\ndifference in the presence/s absence of MDH?”\n(a) We provide binding data for Pex5 (residues 314-666, equivalent to Pex5 ; contains\neTPR hTPR\nthe proximal WxxxF/Y motif and TPR domain) and wild type MDH (Table 1). We also provide\nbinding data for Pex5 and Pex14 in the presence of PTS1 peptide (Table 2). The rebuttal\neTPR NTD\nletter confirms that the latter binding affinity is determined in the presence of PTS1. Such\nexperimental setup was requested by Reviewer #2 in comment #2 of the prior round of reviews\n(R1) to ensure the closed conformation of TPR domain of Pex5. It was not our intention to\nclaim anything other than the above in the prior rebuttal or here.\n(b) The PTS1 peptide was chosen according to the suggestion of Reviewer #2 from comment\n#2 of the prior round of reviews. The particular NH2-ARSKL-COOH peptide was chosen\nbecause of satisfactory solubility according to our prior experience. The complex was\ncharacterized by ITC (Table 1). The concentration of the complex used in experiments was\nstated in materials and methods in section pertaining to ITC. In the revised version of the\nmanuscript we have modified the earlier description of ITC experiment to explicitly state how\nwas the Pex5-PTS1 complex created.\n(c) Affinity of Pex14 for Pex5 determined by ITC in the presence of PTS1 peptide\nNTD eTPR\n(K =53±12) is lower (p<0.001) compared to affinity determined in the absence of PTS1 peptide\nd\n(K =35±5). The statistical significance was determined using the Welch’s t-test. The tetrameric\nd\nform of MDH significantly complicates the model and we have not attempted to determine the\naffinity of Pex14 for (Pex5 ) (MDH tetramer). We however demonstrated that interaction\nNTD eTPR 4\nwith Pex14 modulates the PTS1 affinity of Pex5 (K =254±9 nM and K =80±5 nM; p <\nNTD eTPR d d\n0.001) in the absence and presence of Pex14NTD, respectively) and Pex5 (Kd=88±5 nM and\nKd=37±2; p <0.001), consistent with direct interaction of Pex14 with TPR domain of Pex5.\nNTD\nThis data is included and discussed in the revised version of the manuscript.\n5. “Any statistical significance analysis of the binding data (Tables 1 and 2) is missing. In the\nabsence of this, the interpretation of the observed changes is more a matter of belief than\nbased on statistical evidence. Such analysis needs to be added to allow their quantitative\ninterpretation. This is essential as quite a number of binding data are either unexpected or\ninconclusive. In general, these data need to be better explained. In the absence of estimated\nstatistical significance, they may put the correctness of some of the structural data into\nquestion.”\nAnalysis of statistical significance of differences in binding data among tested constructs has\nbeen introduced in the revised version of the manuscript. p-values have been reported to\nsupport the described quantitative interpretations.\n6. “Although publishers are consistently request to refrain from superiority claims, both in a\nsense of superiority of own data and inferiority of others, the manuscript repeats inferiority\nclaims multiple times by classifying previous findings as “poor”, directly or indirectly suggesting\nsuperiority of their own data (see e.g. reply to point 13 in the rebuttal letter). The data presented\ndo not explain why Pex5-cargo binding facilitates translocation into the peroxisomal lumen\n(abstract), a “process poorly understood”. Therefore, despite very interesting structural\ninsights, I do have doubts that they really facilitate an improved understanding of the\nperoxisomal cargo translocation process.”\nAttachment 1 – Responses to the reviewer’s comments (R2) NCOMMS-23-36304A\nWe did not intend to claim superiority/inferiority of any our/prior data. What we intended was\nto state that some of the processes still require further clarification which would substantiate\nthe results of prior research while building on those results. The issues referred to by the\nreviewer are our unintentional linguistic errors which we have removed from the revised\nversion. Alongside we have toned down the too broadly defined claims referred to by the\nreviewer.\n7. “Supplementary items are not cited in sequential order. This needs to be corrected.”\nThe supplementary items are now cited in sequential order in the revised version of the\nmanuscript (R2).\nAdditional comments:\na. “Please remove the statement “the causative agent of human Chagas disease” from the\nabstract. This is repeated three times in the manuscript. The data presented neither link to nor\nexplain why Trypanosoma cruzi is the causative agent of human Chagas disease.”\nThe statement was removed from the revised abstract.\nb. “Introduction, p. 4: “What is established is that the import of the Pex5-cargo complex involves\nassistance from membrane-spanning receptors, Pex14 and Pex13 (5).” Neither Pex14 nor\nPex13 are described as “receptors” in reference 5 nor, to the best of my knowledge, in other\nrelevant literature. Pex5 is the receptor.”\nReference to ‘receptors’ was replaced by ‘proteins’ in the revised manuscript.\nc. “Results, section “Cryo-EM reconstructions and heterogeneity of the ternary complex”, page\n7: “It is of note that although we observed particles with two Pex5 molecules bound on the\nsame side of the MDH tetramer, we did not observe particles with two Pex5 molecules bound\nto diagonally opposite MDH subunits (Figure 2B).” Without further explanation, this sentence\ndoes not make sense. The inherent MDH symmetry needs to be explained or cited somewhere.\nAssuming that it is 222, there is no preferred side of the tetramer. Unless the symmetry is\ndifferent, there is a requirement for two Pex5 molecules in “diagonal” positions in the MP3\ncomplex, even if one of the two is not interpretable in molecular detail.”\nWe appreciate the reviewer pointing to our invalid description. We have accordingly modified\nthe text in the revised manuscript (including explicitly citing the symmetry of the complex) and\nmade relevant modifications to Figure 1 to clearly illustrate the related manuscript text.\nd. “Results, section “Cryo-EM reconstructions and heterogeneity of the ternary complex”, page\n7: “The piggybacking of cargo isoforms within heteromultimeric complexes suggests that\nsaturation of all Pex5 interaction sites is not required for translocation 29,30.” I could not find\nany discussion on the lack of requirement for saturated binding of cargos in references 29+30.”\nOur description was unclear at that point. What we meant was that it is uncertain whether\nbinding of more than one Pex5 molecule is necessary for translocation of the oligomeric cargo.\nThe piggybacking of cargo isoforms lacking PTS1 within heteromultimeric complexes29,30\nsuggests that the binding of Pex5 to all subunits of the oligomer is not required for\ntranslocation.\nAttachment 1 – Responses to the reviewer’s comments (R2) NCOMMS-23-36304A\nThe cited literature refers to piggybacking only, while the discussion on the required number of\nPex5 molecules is specific for this manuscript.\nThe description is now modified accordingly in the revised version of the manuscript.\ne. “Results, section “Pex5-binding on MDH is of dynamic nature”, page 7: “This interaction is\nstabilized by a network of hydrogen bonds involving the PTS1 C-terminal carboxylate group,\nmainchain donors and acceptors, and lysine sidechain amine, as observed previously in\ndifferent species7.” Could this network please be shown for the structure presented here? Is\nthis network around the PTS1 motif preserved in different binding conformations or is it\ndifferent?”\nThe referenced hydrogen bond network has been depicted in Supplementary Figure 7 for both\nthe distal and closed states. The rearrangement of this network is evident between the distal\nand close states and now described in the revised manuscript text with depiction in added\nSupplementary Figure 7.\nf. “Results, section “Pex5-binding on MDH is of dynamic nature”, page 7: “Similar compaction\nis also observed for Trypanosoma brucei and human Pex5 6-8,31, suggesting that TPR domain\ncompaction upon PTS1 binding is general across species.” To the best of my knowledge, the\nconcept of binding site compaction was introduced by Fodor et al., (2015, PMID: 25369882),\nand confirmed in reference 31, from some of the authors of this paper. Unfortunately, previous\nwork was also not be adequately cited in reference 31.”\nThe reference Fodor et al. (2015, PMID: 25369882) was accidentally omitted in the referenced\nfragment of the text (the work was cited elsewhere in this manuscript in mentioned context).\nThe reference is now included in the revised version of the manuscript in the context of the\nbinding site compaction in the place indicated by the reviewer.\ng. “Results, section “Secondary MDH-Pex5 interfaces does not significantly contribute to\naffinity”, page 9: “Because close analysis of interfaces 1 and 2 does not reveal classical\nfeatures of protein-protein interaction and because identified swinging motion affects the extent\nof the interfaces, we wanted to test if the identified interfaces contribute to affinity.” What are\n\"classical features of protein-protein interaction\"? Please define.”\nIn a simplified view, but still sufficient for the purpose of the discussion, protein-protein\ninteractions are driven largely by hydrophobic effect which requires certain surface\ncomplementarity. Often, the more dynamic the interaction, the higher the modulation by buried\ncharged and polar residues. For a detailed discussion the reader is directed in the revised\nversion of the manuscript to a review by Keskin at al. (Chem. Rev. 2008, 108, 1225-1244).\nThe MDH-Pex5 interfaces 1 and 2 show neither significant surface complementarity,\nhTPR\nhydrophobicity nor charged or polar residues aligned for mutual interactions. Therefore, they\ndo not resemble protein-protein interaction sites, and are rather defined in this work as\nproximity regions.\nh. Figure 1\n(i) “What is the \"standard data processing pipeline\"? Please reference.”\nThe term has been removed in the revised version of the manuscript. We now clearly\nrefer to two different data treatments used, that is “data processing procedure not\naccounting for conformational variability” and “3D-variability analysis”. The approaches\nare described in details in the revised Methods section.\nAttachment 1 – Responses to the reviewer’s comments (R2) NCOMMS-23-36304A\n(i) “Panel A: Assuming 222 MDH symmetry (further comments elsewhere), how could it\nbe excluded that a 4th Pex5 component is simply hidden behind MDH because of\nsymmetry?”\nThank you for pointing out our inconsistent reasoning. The third and fourth copies of\nPex5 can be superimposed in 2D class average and thus cannot be differentiated. We\nhave now named the third subset as MP3/4 in the revised manuscript to reflect the\nindistinguishable character of the two configurations.\n(ii) “Panel B: Pex5 and MDH are clearly separated by different colors. There is no need of\nfurther encircling.”\nThe circles are removed in the revised manuscript.\n(iii) “Panel C: What is the message of this panel? I don’t get it.”\nThe panel shows that all 4 PTS1 sites within MDH tetramer are equally accessible, that\nis, no clear steric occlusion is present at any of the sites which could preclude Pex5\nbinding. The figure caption was revised accordingly to clearly point to the message of\nthe panel.\ni. Figure 2\n(i) “How was the angular movement (tilt) measured? This needs to be described in the\nmethods section.”\nRelevant description was included in the revised methods section.\n(ii) ”(a) Is this a movement (translation) or rotation, or a combination of both? (b) Could the\nmovements please be plotted against the visible Pex5 sequence, in an additional\nsupplement figure? (c) Given that the color code of the second Pex5 molecule in MP2\nis limited to green/yellow, are the authors suggesting that the flexibility of Pex5 binding\nof the second Pex5 molecules in MP2 is more restricted than for the first Pex5\nmolecule?”\n(a) The movement is described in detail in section “Pex5-binding on MDH is of dynamic\nnature”: “(…) This swinging motion can be characterized by two components: tilting\nand twisting. Pex5hTPR, tilts nearly as a rigid body, oscillating between close and\ndistal conformations with a maximum tilt angle of 17° relative to the PTS1 (Figure\n2A; Supplementary Movies 1-7). Additionally, during this tilting motion, Pex5hTPR\ntwists up to 13° relative to the axis roughly parallel to the TPR3 motif\n(Supplementary Movie 8). The combined tilting and twisting motions result in a\nradial displacement of Pex5hTPR ranging between 3 to 8 Å (Figure 2).\n(b) The supplementary figure suggested by the reviewer and depicting the residue-by-\nresidue movements plotted against the Pex5 sequence was included as\nSupplementary figure 8.\n(c) The fact that the flexibility of the second Pex5hTPR molecule in MP2 is more\nrestricted compared to the first Pex5hTPR molecule stems directly from the\nexperimental data. The color coding in panels B and C reflects comparison of\nclosed and distal conformations without any assumptions or data manipulation.\nClosed and distal conformations are overlayed based on MDH and colors represent\nthe difference in position of corresponding C atoms between the two states.\nα\nj. Figure 3\nAttachment 1 – Responses to the reviewer’s comments (R2) NCOMMS-23-36304A\n(i) “The mixture of cartoon (MDH) and surface (Pex5) is more confusing than clarifying.\nCould any specific interactions (hydrogen bonds, salt bridges) be identified, and if so,\nplease indicated? To what extent are the interfaces shown in the different binding\nconformations (close, distal, intermediate, as explained in S Figure 8) are preserved or\ndifferent?”\nThe figure was modified in line with the reviewer comment. We have created renderings\nwith and without the surface representation and decided that to best incorporate the\nreviewer request and retain figure clarity a semi-transparent surface model with\nselected residues in stick model is most relevant to depict all the details discussed in\nthe text. The interface is described in the section “Pex14 binding site and\nNTD\nnoncanonical interactions with Pex5” and the involved residues are now depicted and\nlabeled in the revised version of the figure, but no specific interactions are involved in\nthese proximity assemblies as discussed in the text (see also response to additional\ncomment “g” above). Corresponding figure for proximal conformation is provided in\nrevised Supporting Information (Supplementary figure 12). The interfaces are largely\npreserved in close and distal conformations.\n(ii) “I don't get the sentence \"The density is not seen in a groove distal form Pex5 occupied site (red).\"\nIsn't this logical, if no Pex5 is bound?”\nThe sentence was introduced in response to comment #10 of reviewer #1 in the prior\n(R1) round of reviews. The reviewer requested supporting evidence that the density is\nindeed a fragment of Pex5 loop. Apart from additional experimental evidence (MDH\nstructure in the absence of Pex5) the revised text refers to the fact that the density is\npresent only in the presence of Pex5 and not the unoccupied sites. The conclusion is\n“logical”, but is stated there to attract readers attention to the fact which otherwise might\nwent overlooked.\nk. “Figure 4 (and corresponding text): It is surprising that mutagenesis of residues, based on\nthe structural model of the interaction by Pex5 with Pex14, shows only little or no effect (for\nstatistical assessment, see comments elsewhere): how sure are the authors about a correct\nsequence register of Pex14? Is this model supported by AF3 predictions and previous\nstructural data of the Pex14-NTD with a Pex5 peptide. Please elaborate on this, possibly by\nadding figures in the supplement.”\nThe fit of Pex14 into the density is unambiguous in terms of sequence register. This is\nNTD\nbecause the Pex5hTPR is rigid and well defined by density and the Wxxx(F/Y) motif interaction\nwith C-terminal domain of Pex14 is well defined by prior structures. These two facts, even\nNTD\nin absence of any density accounting for Pex14 would position Pex14 exactly in the place in\nwhich it is found in our structure. Our structure, however, has a clear density to account for the\nhelices comprising Pex14 . Because the helices are not parallel, but located at various\nNTD\nangles to each other, it is impossible to shift the rigid body of Pex14 within the density derived\nfrom cryo-EM without violating the fit of some helices. The above, together with availability of\nprevious structures of Pex14 makes the sequence register assignment unambiguous.\nNTD\nThe resolution in the referenced region does not allow to see particular sidechains, but rigid\nnature of Pex14 and available structures allow to predict with high confidence the direction\nNTD\nat which the sidechains point.\nAlphaFold3 places the entire Pex5 helix containing the Wxxx(F/Y) motif in position which is not\nsupported by experimental data. The helix is clearly defined by density in our structure and\nAttachment 1 – Responses to the reviewer’s comments (R2) NCOMMS-23-36304A\nextends from the TPR domain in a different orientation than suggested by AlphaFold3.\nAccordingly, Pex14 is positioned in a completely different place than seen in experimental\nNTD\ndata. This requires a conclusion that in this particular case AlphaFold3 fails to predict the\ncorrect structure with regard to the orientation of Pex14 relative to Pex5.\nNTD\nAs suggested by reviewer, we compared the Pex14 structure bound to the Pex5 Wxxx(F/Y)\nNTD\ncontaining motif helix (part of MP1P complex structure PDB id: 8gi0 of the present study) with\nthe previously described Pex14-Pex5 structure (PDB id: 2w84). The RMSD between these two\nstructures is minimal (~1.0 Å between equivalent 46 Cα atoms) suggesting the significant\nsimilarity between the two and thus indicating the correctness of Pex14 assignment to the\nNTD\ndensity. A supplementary Figure 15 is added to show this comparison.\nl. “Supplementary Figure 1A: Please characterise all three major peaks of the ternary complex\nby SDS-PAGE. This is important for clarification of incomplete occupancy of Pex5 in\nMDH/Pex5 complexes.”\nThe requested data is provided in the revised Supplementary Figure 1.\nReviewer #2\nMajor comments:\n1. “With the better resolution structure of the MDH:PEX5:PEX14 NTD complex and additional\nITC data, the authors have significantly improved the quality of the presented data; however, I\nstill have reservations about whether the data presented advances our understanding of\nperoxisome import. The importance of the dynamics at the PEX5:MDH interface is limited by\nthe lack of a functional import assay to test these hypotheses. The authors’ claim that binding\nof PEX14 to the proximal WXXXF motif is insufficient to cause cargo release could be an\ninteresting result for the understanding peroxisome import, but the authors do not have\nquantitative data, for example of PEX5 and PTS1 peptide affinity with and without PEX14 to\nconclusively make their claim. In summary, I agree that the author’s data now support their\nclaims (1 - Cryo-EM structure of Pex5:Pex14 NTD:MDH, 2 - Pex5 – MDH interaction is\ndynamic, and 3-Pex5-Pex14 NTD site extends beyond canonical site), but I am not convinced\nthese claims advance our understanding of peroxisome import.”\nThe data presented in our work supports the three claims enumerated by the reviewer:\n(1) Structure of MDH:Pex5:Pex14 assembly demonstrates how the three components\nNTD\ninteract together forming the translocation/post-post translocation complex\n(2) The extent of the dynamics of the Pex5-cargo interaction, a characteristic unknown\nfrom prior crystallographic structures of Pex5-cargo complexes\n(3) Pex5-Pex14 interface extending beyond the canonical site\nNTD\nWe do not make any claims beyond the above conclusions in our manuscript. The findings are\nsupported by mutational analysis of the influence of identified interfaces on binding. Data\nsupporting dynamics of Pex5-cargo interaction reported by others subsequently to our study\nin different organism (bioRxiv 2024.06.02.597006) suggests that dynamics may be common\nto the Pex5-cargo interface. These considerations were highlighted in the manusctipt text in\nresponse to the first round of comments of the reviewers (R1) and are now strengthened in\nresponse to the above comment.\nAttachment 1 – Responses to the reviewer’s comments (R2) NCOMMS-23-36304A\nRegarding the claims pertaining to the influence (or lack of influence) of Pex14 binding on\nNTD\nPex5-PTS1 interaction - in line with the reviewer suggestion, in the revised version of the\nmanuscript we provide additional data demonstrating that the affinity of Pex5 for PTS1 peptide\nis moderately potentiated by Pex14 binding. This conclusion is consistent with the observation\ndocumented in this study that Pex14 interacts directly with Pex5TPR domain while binding\nNTD\nat the proximal Wxxx(F/Y) motif.\n2. “Several suggestions that I think the authors could address with changes to the text:”\na. “Furthermore, our results show that the Pex14NTD binding at Pex5 proximal Wxxx(F/Y) site\nis not enough for structural reorganization of the Pex5 NTD and cargo release in vitro.” The\nreader is not given enough context in the introduction to understand the importance of this\nstatement. It would be helpful to talk about domain architecture of Pex14, topology at the\nmembrane, the need for release of cargo in the peroxisome in the intro. This is well covered in\nthe discussion, but could be discussed more in the introduction.”\nFollowing the reviewer suggestion we have added the description of Pex14 architecture and\ntopology in the introduction. The extended information supports the earlier present fragment\non the postulated role of Pex14 in cargo release. We have also included additional data on the\ninfluence of Pex14 on the Pex5-PTS1 affinity as described in more detail in response to\nNTD\ncomment 1 of this reviewer.\nb. “The density is present only at sites occupied by Pex5 and not empty PTS1 sites indicating\nit corresponds to a fragment of Pex5457-489 loop (Figure 3B; Supplementary Figure 12).” It\nseems possible this density could be a loop of MDH stabilized by the presence of Pex5 instead\nof a loop of Pex5 stabilized by MDH. Can the authors more clearly state why they think this\ndensity can definitively be assigned to PEX5 (ie rule out missing loops in MDH)?”\nThe possibility that the density accounts for MDH loop stabilized in the presence of Pex5 is\nimprobable as the MDH structure is well defined by electron density including almost all\nresidues irrespective if Pex5 is present or absent at PTS1 sites. There is no “missing loop” (a\nfragment not accounted for by electron density) in apo-MDH which could rigidify upon\ninteraction with Pex5 and that is why we conclude it is the flexible loop of Pex5 which rigidifies\nupon interaction with MDH.\nc. “The authors have chosen to exclude their previous ITC data of PEX5 and PEX14\ninteractions without PTS1 peptide present. This data could be useful for interpreting how PTS1\nbinding alters PEX5 affinity for PEX14, which is relevant to the authors discussion of PEX14\ncausing PEX5 to release its cargo.”\nITC data of Pex5 (TPR domain containing the proximal WXXXF/Y motif) and Pex14\neTPR NTD\ninteraction in the absence of PTS1 is included in the supplementary material and referenced\nin the revised manuscript.\nd. “The authors could discuss the conservation of the non-canonical interfaces for PEX14 NTD\nand PEX5.”\nThe conservation of non-canonical interfaces of Pex14 and Pex5 is now discussed in the\nNTD\nrevised version.\ne. “Proteins were purified by Ni-affinity (Ni-NTA) and size exclusion chromatography (SEC).”\nAuthors should describe buffers, lysis, concentration methods for purification and EM grid preparation.”\nAttachment 1 – Responses to the reviewer’s comments (R2) NCOMMS-23-36304A\nDetailed information is now provided in supplementary material under the title ‘supplementary\nmethod”.\nMinor comments:\n(i) ““Pex14 was additionally implemented in cargo extraction from the translocation pore.”\nPhrasing is off - did you mean implicated instead of implemented?”\nYes. The spelling is now corrected in the revised version.\n(ii) ““The maps accounting for the distal states of MDH-Pex5 complex (MP1-d and MP2-d)\nshow additional density proceeding Pex5 residue Ser491 and facing MDH62-70 loop (Figure\n3B; Supplementary Figure 12).” Did you mean preceding instead of proceeding?”\nYes. This is now corrected in the revised version.\n(iii) ““To furhter test the hypothesis, we mutated Pro490 residue” – typo in further”\nThe typo is corrected in the revised version.\n.(iv) ““The density is not seen in a groove distal form Pex5 occupied site (red).” – typo in form”\nThe typo has been corrected.\n(v) ““Prior studies involving ATG and SCP2” – typo in AGT”\nThe typo has been corrected in the revised version.\n(vi) ““again demonstrating that the intractions at the intersubuint groove of MDH unfavorably\naffect the complex dynamics and affinity.” – typos in interactions and intersubunit”\nBoth typos were corrected in the revised version.\nNCOMMS-23-36304B\nPoint-by-point responses to the reviewers’ comments (R3)\nReviewer #1\nOverall assessment:\n“The changes in response to previous referee comments in the current revised version\n(NCOMMS-23-36304B) are appreciated. Overall, the manuscript has been improved.”\nMajor comments:\n1. “I am still not really convinced by the concept of swinging motions, as swinging implies\ndirectivity, for which there is no evidence. At this point, I think this needs to be left for\nfuture discussion, when this paper is published.”\nFollowing the reservation expressed by the reviewer and related suggestion of the editor to tone\ndown the claims on conformational dynamics, we have removed the terms “swinging motion”\nand “conformational dynamics” from the revised manuscript. We now use the term\n“conformational heterogeneity” to refer to the variability observed in the MDH/Pex5/Pex14\nNTD\ncomplex as generally accepted in the field (Punjani, A. and Fleet, D.J. 3D Variability Analysis:\nResolving continuous flexibility and discrete heterogeneity from single particle cryo-EM\nimages. Journal of Structural Biology, Volume 213, Issue 2, 2021).\nAdditional comments:\n2. “Statistical significance tests are now been mentioned in the text but are still not\nsystematically applied to estimate the significance of the quantitative measurements in\nTables 1 and 2. Mentioning the statistical significance in the text narrative is fine but\ncannot replace a systematic analysis where quantitative data are shown, i.e. Tables 1 and\n2.”\nResults of statistical significance test (p-values) have been incorporated in the Tables 1 and 2 to\nfacilitate easier comparisons of reported values.\n3. Referring to comment 2 above: “This is of particular importance, as surprisingly a Pex5\nversion with a GSGS insert shows an approximately threefold improvement in binding to\nMDH. The statement \"The change in affinity was entropy driven indicating that the\nsecondary interactions have overall negative effect on affinity by restricting the\nconformational dynamics of the wild type interface\", implying that it relieves the restriction\non the conformational dynamics of wt Pex5 remains unproven by any complementary\nexperiments. The next sentence in the text \"the affinity of MDH(GSGS) is comparable to\nthat of a short peptide encompassing PTS1 of MDH (residues 319-ARSKL-323; p>0.1)\n(Table 1) indicating that the introduction of the GSGS linker has indeed abolished all\nsecondary interactions at the Pex5-MDH interface\" even implies that the presence of the\nsecond site in the wild-type context has a negative effect on overall binding. So what is the\nfunctional impact of this second site? This remains an open question.”\nThe interpretation of entropy driven change in affinity of GSGS variant was toned down by\nchanging the verb “indicating” into “suggesting”, as indeed the conclusion is not proven by\ncomplementary evidence other than described in the paragraph referred to by the reviewer.\nIdentical change was introduced to the second sentence cited by the reviewer (on comparison of\naffinity of MDH(GSGS) and PTS1).\nWith the mutagenesis we were asking if the secondary sites contribute to the affinity of Pex5\nand MDH. In other words, if the Pex5-MDH interaction is autonomous or non-autonomous as\ndefined earlier by others and detailed in the first paragraph of the discussion. Our results\nsuggest the autonomous-like model modulated by secondary interactions which have moderate\nnegative effect on affinity and possibly affect the conformational heterogeneity of the complex\nas discussed in the “Discussion” section.\n4. Please remove the term \"noncanonical\" from the abstract, as the term will not be defined\nlater on in the manuscript.\nThe term “noncanonical” was removed from the abstract.\n5. “Unfortunately, the revised nomenclature for Pex5 constructs is still quite confusing. I\ncould not find a definition for the acronym Pex5eTPR. Does the \"e\" stand for \"extended\"?\nAccording to Supplementary Table 4 Pex5hTPR \"Refers to residues within full length Pex5\ndefined by density in the ternary MDH-Pex5-Pex14NTD complex structure\", so the\nconstruct behind it is actually full-length Pex5. So, it's full-length Pex5 that was used for\nexperiments, with limited structural visibility to Pex5hTPR. A statement on p. 10 about\nPex5eTPR as \"a construct roughly equivalent to Pex5hTPR\" adds to further confusion.\nPlease make a clear distinction between expressed and purified constructs, and expressing\nvisibilities in structures.”\n(i) Pex5 was defined in Supplementary Table 4. The table was cited in the main text in the\neTPR\nMaterials and Methods section, but was not cited at the first appearance of the\nabbreviation. This is corrected in the revised version. “e” stands for “extended” as now\ndefined in Supplementary Table 4.\n(ii) Pex5 TPR was not a separate construct, but an acronym referring to the residues defined by\nh\ndensity in the structure reported. This abbreviation was removed in the revised version to\navoid confusion with expressed and purified constructs. Visibilities in structures are\ndescribed by defining the residue ranges in the revised version.\n(iii) There are only two Pex5 constructs used in the entire study. Unless directly indicated\notherwise in the manuscript text, the text full length Pex5 was used for structural studies\nand is referred to as Pex5. Truncated Pex5 was used for affinity studies and is referred to as\nPex5 (extended TPR domain). All the mutants were prepared in Pex5 construct. No\neTPR eTPR\nother acronyms are used in the revised version. If particular domains are discussed, they\nare referred to as such (eg. TPR domain; N-terminal domain).\n6. “I have noted that the new version of the manuscript contains various new changes that\nwere not requested (or requested by the other referee, at least to some extent). In going\nthrough these changes, I have noticed that some new unclear statements have been\nintroduced. At this point, I believe it would be beyond the role of this referee to comment\non them in detail, to avoid an unintended role as advisor rather than referee. However, I\nfeel it is important to flag this up to ensure that the final version to be eventually published\nis free of major deficiencies, ambiguities and/or errors.\nJust one example, to illustrate of the problem: Supplementary Figure 18 contains an\nacronym KdAV. I could not find an explanation for it. The measured values have a\ndimension, nM**-1 I think. However, no dimension is given. These things needs to be\ncorrected.”\nIntroduced changes were made either directly in response to comments of one of the reviewers\nor as a secondary result of requested changes where the manuscript required additional\nadjustment to primary changes.\nData shown in Supplementary Figure 18 was introduced in response to one of the comments in\nR2 round of review (see “Overall assessment” section below where reviewer #2 refers to data\nshown in Supplementary Figure 18). Kd is an average K from free independent\nAV d\nmeasurements. The acronym is now defined in the figure legend. Dimension of the value is\nprovided in the revised version of the manuscript.\nReviewer #2\nOverall assessment:\n“The reviewers have addressed my concerns, and I appreciate their added fluorescence\nanisotropy experiments to assess how PEX14 NTD alters PEX5:PTS1 affinity.”\nAdditional comments:\n1. “The Pex5hTPR is used in the abstract and intro, but isn’t clearly defined until the results\nsection. Perhaps in the other locations hTPR could be replaced by the residue numbers or\nhTPR should be defined earlier.”\nhTPR was removed altogether in the revised version as per suggestion of reviewer #1 and to\naddress the concern of this reviewer. Constructs are now defined as specified in Supplementary\nTable 4 and regions defined by density in determined structures are described as residue\nnumbers in the revised version. (See also response to comment 5 of Reviewer #1)\n2. “Supplementary Figure 7 – coloring the shown sidechains by atom (ie red = oxygen)\nwould help clarify the H-bond network.”\nThe requested coloring scheme was introduced in the revised figure to increase clarity.\n3. “Figure 2 legend – typo in “confirmations” which should be conformations.”\nThe specified typo was corrected.\n4. “In Results section: \"Pex5eTPR (residues 314-666), a construct roughly equivalent to\nPex5hTPR\"\n- Please define hTPR by residue numbers here\n- eTPR notation doesn’t match Supplementary Table 2: Pex5eTPR (residue 316-666)”\nhTPR acronym was removed altogether in the revised version (see response to comment 1 by\nthis reviewer). The residue numbering of eTPR in Supplementary Table 1 was corrected\n(Supplementary table 2 does not contain reference to eTPR residue numbering).\n5. “Authors state: \"To disrupt interface 1, we replaced the MDH62-70 loop with GS-linker.\nThis resulted in a moderate decrease (~2.3-fold) (p<0.001) in affinity towards Pex5eTPR\nindicating little contribution of the interface to affinity.\" Please state the Kd of the GS\nlinker mutant here or indicate that the MDH62-70 loop with GS-linker is (Delta62-70) in\nTable 1. When I look at Table 1, it is not immediately clear if the authors are referring to\nthe mutant GSGS or Delta62-70.”\nThe nomenclature referring to MDH variant with 62-70 loop replaced with GS-linker was made\nconsistent in the main text and Table 1.\n6. “The authors discuss the comparison of the affinity between Pex5 and Pex14 NTD in the\npresence and absence of the PTS1 peptide in the rebuttal letter (“Affinity of Pex14NTD for\nPex5eTPR determined by ITC in the presence of PTS1 peptide (Kd=53±12) is lower\n(p<0.001) compared to affinity determined in the absence of PTS1 peptide (Kd=35±5). The\nstatistical significance was determined using the Welch’s t-test.”, but it isn’t discussed\nin the manuscript (that I could find). I personally find it interesting that the presence of the\nPTS1 peptide increases the Kd between Pex5eTPR and Pex14 NTD, and would encourage\nthe authors to include the statement from their rebuttal in the manuscript. It is interesting\nthat N353E and Y358A Pex5 mutants do not have the same change in Kd with the addition\nof PTS1, suggesting that these mutants might decouple PTS1 binding and PEX14 NTD\nbinding.”\nThe relevant statements discussing the effect of PTS1 peptide on the affinity of Pex14 for\nNTD\nPex5 are included in the revised manuscript according to the reviewer recommendation.\neTPR"}]

Decision letter