Unique Double-Ring Structure of the Peroxisomal Pex1/Pex6 Atpase

Unique double-ring structure of the peroxisomal PNAS PLUS Pex1/Pex6 ATPase complex revealed by cryo-electron microscopy

Neil B. Bloka,b,1, Dongyan Tana,b,1, Ray Yu-Ruei Wangc,d,1, Pawel A. Penczeke, David Bakerc,f, Frank DiMaioc, Tom A. Rapoporta,b,2, and Thomas Walza,b,2

aHoward Hughes Medical Institute, Harvard Medical School, Boston, MA 02115; bDepartment of Cell Biology, Harvard Medical School, Boston, MA 02115; cDepartment of Biochemistry, University of Washington, Seattle, WA 98195; dGraduate Program in Biological Physics, Structure and Design, University of Washington, Seattle, WA 98195; eDepartment of Biochemistry and Molecular Biology, The University of Texas Medical School, Houston, TX 77054; and fHoward Hughes Medical Institute, University of Washington, Seattle, WA 98195

Edited by Wah Chiu, Baylor College of Medicine, Houston, TX, and approved June 15, 2015 (received for review January 6, 2015) Members of the AAA family of ATPases assemble into hexameric short succession (3, 8–10). For double-ring ATPases, the co- double rings and perform vital functions, yet their molecular ordination between ATPase subunits is even more complex, as mechanisms remain poorly understood. Here, we report structures there may be communication both within a given ring and be- of the Pex1/Pex6 complex; mutations in these proteins frequently tween the two rings. It seems that generally most of the ATP cause peroxisomal diseases. The structures were determined in the hydrolysis occurs in only one ring. For example, the N-terminal presence of different nucleotides by cryo-electron microscopy. Models ATPase domains of NSF, which form the D1 ring, hydrolyze were generated using a computational approach that combines ATP much more rapidly than the subunits in the D2 ring (11). Monte Carlo placement of structurally homologous domains into den- The reverse is true for p97, in which the D2 ring performs the sity maps with energy minimization and refinement protocols. Pex1 bulk of ATP hydrolysis (12, 13). Although single-ring AAA and Pex6 alternate in an unprecedented hexameric double ring. Each ATPases seem to move polypeptides through their central pore, protein has two N-terminal domains, N1 and N2, structurally related it is not clear whether this model applies to NSF or p97 (14). to the single N domains in p97 and N-ethylmaleimide sensitive factor More generally, it is unknown why some ATPases even have a BIOCHEMISTRY (NSF); N1 of Pex1 is mobile, but the others are packed against the second ring. In fact, some members of the double-ring ATPase double ring. The N-terminal ATPase domains are inactive, forming a family perform functions similar to their single ring counterparts symmetric D1 ring, whereas the C-terminal domains are active, likely (e.g., ClpAP vs. ClpXP, although ClpAP appears to grip sub- in different nucleotide states, and form an asymmetric D2 ring. These strates more tightly than ClpXP) (15). results suggest how subunit activity is coordinated and indicate strik- Double-ring ATPases pose particular challenges. For single- ing similarities between Pex1/Pex6 and p97, supporting the hypothe- ring ATPases, it was possible to covalently link three or even all sis that the Pex1/Pex6 complex has a role in peroxisomal protein six of the ATPase subunits (16). This linkage enabled muta- import analogous to p97 in ER-associated protein degradation. genesis of individual subunits, which in turn allowed testing the effect of one subunit on the activity of adjacent subunits in the Pex1 | Pex6 | AAA ATPase | cryo-electron microscopy | peroxisome ring. A similar analysis is possible in single-ring hexameric

he AAA (ATPases associated with diverse cellular activities) Significance Tfamily of ATPases contains a large number of proteins that perform important functions in cells (1). Many members of this Pex1 and Pex6 are members of the AAA family of ATPases, which family form hexamers. These hexamers consist either of a single contain two ATPase domains in a single polypeptide chain and ring of ATPase domains or of stacked rings formed by tandem form hexameric double rings. These two Pex proteins are involved ATPase domains in a single polypeptide chain (type I and II in the biogenesis of peroxisomes, and mutations in them fre- ATPases, respectively). Both classes of AAA ATPases often use quently cause diseases. Here, we determined structures of the the energy of ATP hydrolysis to move macromolecules. Ex- Pex1/Pex6 complex by cryo-electron microscopy. Novel computa- amples of single-ring ATPases include the F1 ATPase (2), which tional modeling methods allowed placement of Pex1/Pex6 do- rotates a polypeptide inside its central pore, ClpX (3), which mains into subnanometer density maps. Our results show that moves polypeptides into the proteolytic chamber of ClpP, and the peroxisomal Pex1/Pex6 ATPases form a unique double-ring the helicase gp4 of T7 phage (4), which pulls a DNA strand structure in which the two proteins alternate around the ring. Our through its center. Double-ring ATPases generally act on poly- data shed light on the mechanism and function of this ATPase and peptides. Prominent members of this family include N-ethyl- suggest a role in peroxisomal protein import similar to that of p97 maleimide sensitive factor (NSF), p97/VCP (valosin-containing in ER-associated protein degradation. protein), and Hsp104 in eukaryotes and ClpB in bacteria (5–7). NSF dissociates SNARE complexes generated during membrane Author contributions: D.B., F.D., T.A.R., and T.W. designed research; N.B.B., D.T., R.Y.-R.W., fusion, p97/VCP plays a role in many processes, including ER- P.A.P., and T.W. performed research; N.B.B., D.T., and R.Y.-R.W. analyzed data; and N.B.B. associated protein degradation (ERAD), and Hsp104 and ClpB and T.A.R. wrote the paper. disassemble protein aggregates. The authors declare no conflict of interest. The molecular mechanism of many hexameric AAA ATPases This article is a PNAS Direct Submission. is only poorly understood, particularly for those that move Data deposition: The EM 3D maps of Pex1/Pex6 without imposed symmetry have been polypeptide chains. In addition, the mechanism appears to differ deposited in the EMDatabank, www.emdatabank.org (accession codes EMD-6359 and EMD-6360, for maps of complexes with ATPγS or ADP, respectively). among the known examples. For the F1 ATPase, it has been 1N.B.B., D.T., and R.Y.-R.W. contributed equally to this work. established that ATPase domains hydrolyze ATP continuously 2To whom correspondence may be addressed. Email: [email protected] or in a strictly consecutive manner around the ring (2). In contrast, [email protected]. in another single-ring ATPase, ClpX, several ATPase domains This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. hydrolyze ATP in sporadic bursts, either simultaneously or in 1073/pnas.1500257112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1500257112 PNAS Early Edition | 1of9 Downloaded by guest on September 29, 2021 ATPases that consist of different subunits, such as the six Rpt over X-ray crystallography, as flexible particles can be analyzed subunits that form an ATPase ring in the 19S regulatory sub- and asymmetry is not limiting. Until recently, EM structures, unit of the proteasome (17) or the mitochondrial AAA pro- including those of p97, NSF, and ClpB, were of relatively low teases composed of two alternating subunits (18). For double- resolution (>15 Å) (20, 22–25). Recent developments in cryo- ring ATPases, covalent linkage of the subunits is impractical. electron microscopy (cryo-EM) single-particle analysis, including However, double-ring ATPases consisting of different subunits the use of direct electron detectors and novel image analysis al- are conceivable and would offer the opportunity to mutate gorithms, have paved the way to obtain higher resolution. Indeed, specific subunits. while this manuscript was under review, subnanometer-resolution A deeper understanding of the mechanism of polypeptide- structures of NSF were reported (26). moving hexameric ATPases requires structural information. We are interested in understanding the structure and function Unfortunately, only a few full-length structures are available at a of the Pex1/Pex6 complex, as this complex is important for hu- resolution that allows fitting of the polypeptide chain (10, 19– man health and is a potential example of a double-ring ATPase 21). Crystallography has proven difficult, particularly because consisting of two different polypeptide chains. Both proteins are crystallization is favored by symmetric structures in which all predicted to contain two ATPase domains (D1 and D2; Fig. 1A) subunits are in the same nucleotide state. In addition, some and are known to associate with one another (27, 28). Pex1 and domains in ATPases seem to be rather flexible. In such cases, Pex6 are peroxisome-associated proteins that are conserved in all electron microscopy (EM) analysis offers significant advantages eukaryotic cells. They were identified in genetic screens for

A B C

MW LysateNi-NTAStreptavidin El.Gel Filtration El. 170 kDa 130 kDa 100 kDa

55 kDa

35 kDa

15 kDa 50 nm D Top ediS mottoB

D2 Ring

1 E 2

3 3 1 2 1

Top Oblique

Fig. 1. Structure determination of the Pex1/Pex6 complex. (A) Domain structures of Pex1, Pex6, p97, and NSF. Shown are the N-terminal domains (N1, N2, N) and the ATPase domains (D1 and D2). (B) The Pex1/Pex6 complex was expressed in yeast cells and purified in different steps. Samples were analyzed by SDS/PAGE and Coomassie blue staining. Shown are samples from the crude lysate, after elution from Ni-NTA beads, after elution from streptavidin beads, and after gel filtration. MW, molecular weight markers. (C) Negative-stain EM image of the purified Pex1/Pex6 complex in the presence of ATPγS. (D) The Pex1/ Pex6 complex in ATPγS was analyzed by cryo-EM. Shown is the density map in different views, calculated without imposition of symmetry. (E) Different views showing the three visible N-terminal domains of the Pex1/Pex6 complex. The numbers in the circles indicate different N domains.

2of9 | www.pnas.org/cgi/doi/10.1073/pnas.1500257112 Blok et al. Downloaded by guest on September 29, 2021 peroxisome function in various organisms. Mutations in human (SI Appendix,Fig.S1C). These class averages were used to cal- PNAS PLUS Pex1 and Pex6 are by far the most frequent cause of peroxisome culate an initial 3D model using the stochastic hill-climbing (SHC) biogenesis disorders, such as Zellweger syndrome (29, 30). In algorithm implemented in SPARX (40). This model was then used these diseases, proteins fail to be imported from the cytosol into for 3D clustering and refinement of ∼47,000 particle images, ei- peroxisomes. Affected children have neurodevelopmental defects ther with or without imposition of threefold symmetry. The final and multisystemic symptoms, including hepatic dysfunction, and symmetrized and unsymmetrized maps had resolutions of 6.2 and they often die before puberty (31). Pex1 and Pex6 have also been 7.3 Å, respectively (the unsymmetrized map is shown in Fig. 1D,a implicated in the fusion of peroxisome precursor vesicles (32–34). comparison of the two maps in SI Appendix,Fig.S1D and E,and Interestingly, the proposed role for Pex1 and Pex6 in peroxisomal Fourier shell correlation curves in SI Appendix,Fig.S1F). protein import is similar to that of p97 in ERAD (35). In ERAD, The Pex1/Pex6 complex consists of two stacked hexameric p97 cooperates with the cofactors Ufd1 and Npl4 to extract ubiq- rings, D1 and D2, in which Pex1 and Pex6 alternate around the uitinated proteins from the ER membrane (36, 37), and similarly, ring (Fig. 1 D and E). Three distinct regions of density corre- Pex1 and Pex6 are thought to recycle ubiquitinated import receptor sponding to the N-terminal domains are visible. They allow the Pex5 by extracting it from the peroxisomal membrane (35). identification of the D1 ring and give the molecule a unique Here, we used single-particle cryo-EM to provide a sub- triangular shape with side lengths of ∼165 Å. Each of the D1 and nanometer-resolution EM structure of a double-ring AAA D2 rings has a height of ∼35 Å and a diameter of ∼120 Å, similar ATPase, the structure of the Saccharomyces cerevisiae Pex1/Pex6 to the dimensions of the ATPase rings in p97. The D1 ring has a complex. We were able to derive models for the Pex1/Pex6 relatively narrow triangular pore (side length of ∼20 Å), whereas complex in different nucleotide states, using a hybrid method the D2 ring has a wider pore with an annular diameter of ∼30 Å. that combines the placement of domains from structural ho- Continuous density is seen for the D1 ring even at higher threshold mologs into the density map with model-building and refinement levels, whereas the density of the D2 ring becomes fragmented. Even protocols implemented in Rosetta (38). Pex1 and Pex6 alternate without symmetry imposed, the N domains and D1 ring show a near- around a double ring, forming an unprecedented arrangement. perfect threefold symmetry, whereas the D2 ring is asymmetric. With Each of the two proteins has tandem N-terminal domains, N1 symmetry imposed, the density in the D2 ring worsened, further and N2, which are structurally related to the single N domains in supporting its native asymmetry. Density connecting the two rings is p97 and NSF. Both N2 domains and the N1 domain of Pex6 are visible only at lower threshold levels. In the unsymmetrized map, packed against the double ring, whereas the N1 domain of Pex1 these connections are asymmetric; they are strong on one side of the

is mobile. Our data also indicate that the D1 subunits are in- double ring and weak elsewhere (see below). BIOCHEMISTRY active and form a symmetric ring, whereas the D2 subunits hydrolyze ATP and form an asymmetric ring, in which the subunits Model Building into the EM Density Maps. The HHsearch algorithm are likely in different nucleotide states. The structural similarity (41) predicted that both Pex1 and Pex6 have two N-terminal to p97 supports a role for the Pex1/Pex6 complex in peroxisomal domains, designated N1 and N2, which have a double Ψ-β barrel protein import that is analogous to that of p97 in ERAD. fold, followed by tandem AAA ATPase domains, D1 and D2. The D2 domains of Pex1 and Pex6 have structural homologs of Results high sequence identity (∼40%), whereas the N and D1 domains Cryo-EM Density Map of the Pex1/Pex6 Complex in the Presence of have only low sequence identity templates (<20%). However, ATPγS. To obtain purified S. cerevisiae Pex1/Pex6 complex for even for the least conserved domain by sequence identity, the N2 structural analysis, we coexpressed both proteins in yeast cells. of Pex1, the identified structural homologs all share the same Pex1 was expressed under the GAL1 promoter with an N-terminal overall fold (SI Appendix,Fig.S6), although these templates are as streptavidin-binding peptide (SBP) tag. Pex6 was also expressed different from one another by sequence identity as they are from under the GAL1 promoter with an N-terminal His14 tag. The the Pex1 domain. HHsearch identified the crystal structures of p97 protein complex was purified from the soluble fraction by con- and NSF as the top-ranked templates for the domains of Pex1 and secutive affinity purification on Ni-NTA and streptavidin resins Pex6 (SI Appendix, Figs. S2–S5 and Fig. 1A). (Fig. 1B). The complex was finally subjected to gel-filtration To place the domains of Pex1 and Pex6 into the density map, chromatography in the presence of ATPγS (Fig. 1B), a nucleotide we developed a method for the assembly of protein complex that is only poorly hydrolyzed. The purified complex contained models. We first used a known representative Ψ-β barrel and a only Pex1 and Pex6. They were present in stoichiometric amounts, known AAA domain structure from the Protein Data Bank as judged from the intensities of the bands in Coomassie blue- (PDB) to identify the location of these domains in the density stained SDS gels. When Pex1 or Pex6 were overexpressed in- map. To this end, a full rotation and translation search in the dividually, the proteins were insoluble, suggesting that they need symmetrized cryo-EM map was performed, identifying candidate each other for proper folding. placements for each fold (see SI Appendix for further details). In negative-stain EM images, the Pex1/Pex6 sample had a This preliminary positioning into the cryo-EM density map distinctly triangular shape (Fig. 1C), suggesting that Pex1 and resulted in an average real-space correlation of 0.7, calculated Pex6 alternate around the ring. Essentially no side views were per domain with the “fit to density” tool in UCSF Chimera. The visible, indicating that the particles preferentially adsorbed to the agreement to the density map suggests that the domain fold grid in a face-on orientation. identification results from HHsearch are reliable. To obtain higher resolution, samples were vitrified by plunge- Next, we replaced these representative placements with freezing in liquid ethane for imaging by cryo-EM. The images (SI the actual, unique domains of Pex1 and Pex6. We used the Appendix, Fig. S1A) and subsequent 2D class averages (SI Ap- RosettaCM pipeline (38) to generate ∼25 structural homology pendix, Fig. S1B), calculated with the iterative stable alignment models for each domain. In each case, nonaligned residues were and clustering (ISAC) procedure (39), showed that the complex excluded, resulting in partial models. We then used combina- adopted the same face-on view in vitrified ice as in negative-stain tions of these models to identify mutually compatible domain EM images. The preferred orientation in vitrified ice is likely due placements. Compatibility was assessed with a scoring function to the complexes aligning at the air/water interface. To increase that evaluates the quality of the fit into the density map, the the number of orientations of the Pex1/Pex6 complex, we reduced degree of clashes between domains, and consistency with known the surface tension by adding a low concentration of detergent linker lengths between adjacent domains in a polypeptide chain. immediately before freezing. In these images, additional views of Four possible domain assignments emerged. To distinguish the complex were present; this was confirmed by 2D class averages among these, we added the unaligned residues originally omitted

Blok et al. PNAS Early Edition | 3of9 Downloaded by guest on September 29, 2021 in the partial models and used RosettaCM to build and refine model for the entire complex. This final model fits well into the each domain to better fit the EM density map. After further entire unsymmetrized EM density map (Fig. 2). Although we have optimization of domain compatibility, a single solution emerged refined all-atom models, we have refrained from drawing conclu- in which the N1 domain of Pex1 was excluded from the map. sions based on the modeled positions of amino acid side chains. Although the same domain placement emerged as the best solution using partial and complete amino acid sequences, the Confirmation of the Assignment of Domains in Pex1 and Pex6. Given score difference from alternative solutions was significantly in- the sequence similarities of Pex1 and Pex6 and the resolution of creased (SI Appendix, Table S1). This domain placement is dis- the density map, it was important to experimentally validate the tinguished from the next best solutions by a large score gap (SI assignment of the polypeptide chains. We noticed that treatment Appendix, Fig. S6 and Tables S1 and S2). Furthermore, the top of the Pex1/Pex6 complex with elastase or trypsin truncated solution fits the density map better than the next best (SI Ap- Pex1, but left Pex6 intact (Fig. 3A). Mass spectrometry (MS) of pendix, Fig. S7). Even if we consider fit-to-density or geometry fragments generated with these proteases showed that Pex1 was criteria individually, the identified model has the best score (SI truncated at the N terminus, before residue Ile173 (SI Appendix, Appendix, Fig. S8). When the fit to the experimental density map Fig. S10 A–C), resulting in a fragment that lacks only the N1 was used as the sole criterion, the two best alternative solutions domain. We therefore purified the elastase-treated Pex1/Pex6 have the polypeptide chain termini in consecutive domains so far complex by gel filtration (Fig. 3B) and used it for cryo-EM apart that they cannot be connected by the corresponding linker; analysis. A low-resolution map, generated from 5,000 particles, they are therefore highly disfavored by the geometry terms (SI was completely superimposable onto a map of the full-length Appendix, Fig. S9). Taken together, these results show that the complex low-pass filtered to the same resolution (Fig. 3C). The domains can be unambiguously placed into the density map. similarity of the structures of the proteolyzed and nonproteolyzed We completed the structure by building the linkers between samples provides strong evidence for the correct assignment of the domains using RosettaCM and performing density-guided all- domains of Pex1 and Pex6 in our model. atom refinement in the context of the full assembly (42). The Both the proteolysis and the modeling suggest that the N1 do- resulting symmetric model of the Pex1/Pex6 complex showed good main of Pex1 is connected to the rest of the protein through a agreement with the density map, particularly in the N and the D1 flexible linker. The N1 domain per se is likely folded, as a crystal domains. To improve the model for the D2 ring, we used the structure of the isolated domain has been reported (43). To con- unsymmetrized density map, which contains better resolved density firm the presence of a flexibly attached domain in the full-length in the D2 ring than the symmetrized map. Further refinement of Pex1/Pex6 complex, we analyzed negative-stain EM images in more individual D2 domains, followed by reassembly into the full-length detail. 2D clustering with ISAC was performed using a relatively proteins and refinement of the full-length proteins, resulted in a large window around each of the particles (SI Appendix,Fig.S10D).

A Pex6 Side Bottom Pex1 Top Pex6 Pex1

Pex1

Pex6

Pex1 N2 B Pex1 N2 Oblique D1 Pore

Pex6 N2 Pex1 N2 Pex6 N2 Pex6 N2

D1 Ring

Pex6 N1 D2 Ring Pex6 N1

Pex6 N1 N Domains

Fig. 2. Fit of molecular models for Pex1 and Pex6 into the density map. (A) Models of Pex1 and Pex6 (red and dark blue, respectively) were fitted into the unsymmetrized density map (light blue envelope) determined in the presence of ATPγS. The model was derived as described in the text. Shown are different views. (B) Magnified views illustrating the quality of the fit. The different domains of Pex1 and Pex6 are indicated.

4of9 | www.pnas.org/cgi/doi/10.1073/pnas.1500257112 Blok et al. Downloaded by guest on September 29, 2021 Consistent with the presence of a flexible linker to the N1 domain of PNAS PLUS ABPex1, many of the classes displayed additional density at varying locations on the side of the triangular complex (Fig. 3D). Although flexibility likely precluded visualization of all three Pex1 N1 domains simultaneously, a number of 2D class averages showed two domains (Fig. 3E). The position of the domain is consistent with the expected location of the linker to the N2 domain of Pex1. These results, the cryo-EM structures of the nonproteolyzed and proteolyzed Pex1/Pex6 complex, and the modeling all support the conclusion that the N1 domain of Pex1 is attached through a flexible linker to C the rest of the complex. Our assignment of Pex1 and Pex6 is also in agreement with a recent low-resolution structure determined by negative-stain EM, in which the position of Pex6 was determined by fusion to the maltose-binding protein (44). Finally, the experimental data provide strong validation for the computational domain- placement procedure.

Structural Features of the Pex1/Pex6 Complex. The density map shows the N domains of Pex1 and Pex6 in positions above and at the side of the D1 ring (Fig. 2). In our model, the N2 domain of Pex1 is positioned above the plane of the D1 ring, whereas the N1 domain of Pex6 is located at the side of the D1 ring and reaches down to the D2 ring, without actually contacting it. The N2 domain of Pex6 is at an intermediate position with respect to the plane of the D1 ring. The N1 domain of Pex6 is larger than the N2 domains, as it contains two additional short helices. It is in direct contact with the N2 domain of Pex6. Pex1 and Pex6 are structurally quite similar to each other. The BIOCHEMISTRY N1 domain of Pex6 is readily superimposed on that of Pex1 (SI Appendix, Fig. S11A), the structure of which was previously determined by X-ray crystallography as an isolated domain (43). The N2 domains of the two proteins also have the same fold, but the differences are slightly more pronounced than for the N1 domains (SI Appendix, Fig. S11B). The individual D1 and D2 domains of Pex1 and Pex6 can also easily be superimposed (SI Appendix, Fig. S11 C and D). The six D1 ATPase domains form a symmetric ring with all subunits in the same plane (Fig. 4 A and B). Thus, in the D1 ring, Pex1 and Pex6 are essentially equivalent, arranged as in other hexameric AAA ATPase complexes that consist of only one polypeptide chain (19, 45, 46). Nevertheless, there are subtle differences between Pex1 and Pex6. For example, each subunit contributes three helices that project slightly differently into the D central pore (Fig. 2 A, Left and B, Right). The D2 ring is asymmetric, but all six subunits are approxi- mately in the same plane (Fig. 4 C and D). Within the ring, three pairs of closely apposed Pex1/Pex6 ATPase domains are clearly visible. At these Pex1→Pex6 interfaces, the second region of E homology (SRH), containing conserved Arg residues of Pex1, comes close to residues in the nucleotide-binding pocket of Pex6. In contrast, the distances between the Pex6→Pex1 interfaces are different around the ring. At one interface, there is close contact Fig. 3. The N1 domain of Pex6 is not visible in the cryo-EM density map of (∼8–9 Å distance between the second conserved Arg in the SRH the Pex1/Pex6 complex. (A) Purified Pex1/Pex6 complex was incubated and the Walker A Lys in the nucleotide-binding pocket), whereas μ with increasing concentrations of elastase or trypsin (given in g/mL) for there are significant gaps at the other two (∼15 and ∼22 Å; Fig. 20 min at 25 °C. The samples were analyzed by SDS/PAGE and Coomassie blue staining. The arrows indicate a protected fragment of Pex1 lacking 4C). At least at the widest junctions, strong binding of the nu- the N1 domain, as determined by MS. MW, molecular weight markers. cleotide between neighboring ATPase subunits seems unlikely. (B) Purification of a Pex1/Pex6 complex lacking the N1 domain of Pex1. These results suggest that not all subunits in the D2 ring are in The purified complex was treated with elastase and subjected to gel fil- the same nucleotide-bound state. Because the Pex1/Pex6 com- tration. (C) Comparison of the density maps of the nonproteolyzed (blue) plex was saturated with ATPγS during purification, it is likely and proteolyzed (purple) Pex1/Pex6 complex, both determined in ATPγS. that some sites are in the ATP-bound state. The map for the nonproteolyzed complex was filtered to about the same Interestingly, the D1 and D2 rings interact most strongly close ∼ resolution as that for the proteolyzed complex ( 14 Å). The maps are to the location of the most widely spaced ATPase subunits in the shown individually in different views as well as after superposition. (D) Selected 2D class averages of negatively stained full-length Pex1/Pex6 D2 ring (Fig. 4 C, arrow, and E, Left). At the contact sites, the complex. The brackets indicate a mobile domain peripheral to the tri- D2 subunits of both Pex1 and Pex6 are rotated within the ring angular complex. (E)AsinD, but class averages showing multiple mobile plane such that the helical fingers pointing into the pore are domains simultaneously. moved downward (Fig. 4D, arrows). Little contact between D1

Blok et al. PNAS Early Edition | 5of9 Downloaded by guest on September 29, 2021 AC changed to Asp-Gln in Pex1 and to Ala-His in Pex6 (Fig. 6A). Both mutations are known to severely affect ATP hydrolysis in related ATPases (48). In addition, the SRH domains of both proteins lack the Arg residues (Arg finger) that are required for ATP hydrolysis in the neighboring ATPase subunit. These features indicate that the ATPase subunits in the D1 ring can bind, but not hydrolyze, ATP. The tight spacing of the subunits and the rotational symmetry of the D1 ring suggest that all six sites are occupied by nucleotide. Because the Pex1/Pex6 complex disas- sembles in the absence of added nucleotide (47), it seems likely that nucleotide binding to the D1 ATPase subunits is required BD for the stability of the double-ring structure. Similar conclusions have been reached for p97 and NSF, in which the ring that is less active in ATP hydrolysis (D1 and D2, respectively) is thought to stabilize the overall complex (11–13). The D2 subunits of both Pex1 and Pex6 contain the typical E Walker A and B motifs, the two conserved Arg residues in the SRH, and other features characteristic of active ATPases (Fig. 6A). To test whether the D2 subunits in Pex1 and Pex6 are equivalent for overall ATP hydrolysis, we generated mutants with inactivated Walker A and B motifs and expressed and purified various combinations of these proteins. As expected, a complex in which the Walker B motifs of both Pex1 and Pex6 were mutated had essentially no ATPase activity (Fig. 6B). Similarly, complete inactivation was observed when only Pex6 lacked a functional Fig. 4. Subunit arrangement in the ATPase rings of the Pex1/Pex6 complex. Walker B motif, whereas in the reverse situation, ATPase activity (A) Slice through the D1 ring. Note that the ATPase subunits are arranged in a was only reduced by 40%. Thus, the D2 ATPase domains of Pex1 symmetric manner. (B) To demonstrate the planar character of the D1 ring, the and Pex6 are not equivalent. Interestingly, the essential Walker same helix of each subunit is displayed. The rest of the protein was masked. → (C) Slice through the D2 ring. Note that the subunits are unequally spaced with B motif of Pex6 is located at the tight Pex1 Pex6 interface in our Pex1 and Pex6 arranged in three pairs. The approximate distances between structure (Fig. 4C), whereas the nonessential Walker B motif of the subunits are indicated. The distances across the Pex6→Pex1 interfaces are Pex1 is located at the variably spaced Pex6→Pex1 interfaces. The shown in red. Distances were determined between the Arg finger in one correlation between ATPase activity and structural features also subunit and the conserved residues of the Walker A motif in the other. The supports the assignment of Pex1 and Pex6 in our model. Mutations arrows indicate the sites of strong density connecting the D1 and D2 rings. in the Walker A motifs did not completely eliminate ATP hydro- (D)AsinB, but for the D2 ring. The arrows indicate the helices that are most lysis, even when present in both proteins (Fig. 6B), presumably tilted toward the central pore. They belong to the subunits indicated with an because they did not entirely abolish ATP binding. arrow in C.(E, Left) Strong connections between the D1 and D2 rings at the sites indicated by arrows in C and D.(Right) Equivalent view demonstrating Discussion weak connections at another point of the ring. Here we report EM structures of the peroxisomal AAA ATPase Pex1/Pex6. Our results show that the ATPase forms a unique and D2 is seen on the other side of the D2 ring, where the double-ring structure in which the two proteins alternate around subunits contact each other (Fig. 4E, Right). the ring. Both Pex1 and Pex6 have previously unrecognized tandem N-terminal domains that are structurally related to the Structure of the Pex1/Pex6 Complex in the Presence of ADP. We next single N domains in p97 and NSF. The D1 ring is symmetric, in determined a structure of the Pex1/Pex6 complex in the presence contrast to the D2 ring. Like in p97, the D2 ring subunits conduct of ADP. The complex was purified as before, except that ADP was the bulk of ATP hydrolysis. These results are based on sequence present during gel filtration. The purified complex again consisted homology and experimental data alone. However, a novel compu- of stoichiometric quantities of Pex1 and Pex6, but it was less stable tational method was required to assign Pex1 and Pex6 into the than in ATPγS, as assessed by negative-stain EM and as reported density map and to realize that the N1 domain of Pex1 is flexibly previously (47). However, the complex was stable at lower salt and attached to the complex. These model predictions could be exper- higher nucleotide concentrations. We used cryo-EM analysis to imentally confirmed by proteolysis and negative-stain EM experi- generate density maps with and without threefold symmetry ments and are in agreement with the positions of Pex1 and Pex6 imposed at resolutions of 7.4 and 8.8 Å, respectively (SI Appendix, reported by others (44). Although the computational approach Fig. S1G). The unsymmetrized map (Fig. 5A) was used to fit a resulted in a molecular model of the Pex1/Pex6 complex, we have model of the Pex1/Pex6 complex, using the structure in ATPγSas been careful not to interpret the data beyond the constraints of the a starting point for refinement in RosettaCM. density map resolution. Nevertheless, our results give insight into The density maps and models were surprisingly similar for the the mechanism and function of this double-ring ATPase. structures in ATPγS and ADP (Fig. 5B). The N and D1 domain The division of labor between the D1 and D2 rings of Pex1/ regions were completely superimposable, and only small, if any, Pex6 is similar to that in p97. However, the ATPases in the D1 changes were seen in the D2 ring (Fig. 5C). The D2 ring was ring of p97 have the canonical Walker motifs and retain a low again asymmetric, suggesting that the differences between the ATPase activity (12), whereas the ones in the Pex1/Pex6 complex ATPase subunits are caused by nucleotide occupancy rather than lack the typical motifs and seem to be inactive. The D2 ring is by the presence of the γ-phosphate of the nucleotide. almost symmetric in all p97 structures, regardless of the nucleotide present, but the symmetry may be caused by crystallization. Nucleotide Hydrolysis by the ATPase Domains of Pex1 and Pex6. Se- Asymmetry in an ATP-hydrolyzing ring, as in the D2 ring of the quence analysis of Pex1 and Pex6 shows that the D1 domains Pex1/Pex6 complex, has also been observed for other poly- lack residues required for ATP hydrolysis in related ATPases. peptide-moving ATPases, such as ClpX, MecA/ClpC, and F1 Specifically, the conserved Walker B motif residues Asp-Glu are ATPase (10, 21, 49). As demonstrated for ClpX and F1 ATPase,

6of9 | www.pnas.org/cgi/doi/10.1073/pnas.1500257112 Blok et al. Downloaded by guest on September 29, 2021 A In vivo experiments with Walker B mutants show that ATP hy- PNAS PLUS drolysis at both sites is needed for cell viability (51, 52). Thus, even the less important sites may hydrolyze ATP. We speculate that ATP hydrolysis occurs in a unidirectional manner around the ring, with one subunit primarily affecting ATP hydrolysis of only one of the neighboring subunits. ATP binding would be initiated on one side of the ring at the nucleotide-free sites where the subunits are most widely spaced, a model reminiscent of the rotary mechanism proposed for the F1 ATPase (49). In- BC terestingly, in the case of ClpX, there may also be one or two nucleotide-free sites in the ring (10). There might also be communication between the D1 and D2 rings during the functional cycle of the Pex1/Pex6 ATPase. Such communication has been suggested for other double-ring ATPases (53), but the density between the D1 and D2 rings observed on one side of the Pex1/Pex6 complex is stronger than previously observed. Interestingly, this strong connection between the rings occurs at the point of biggest gaps between D2 ring subunits. In addition, these subunits undergo a rotation toward the center of the D2 ring. The intriguing coincidence of these features could mean that either the D1 ring interaction affects the nucleotide state of subunits in the D2 ring or vice versa. The differences between the structures determined in ATPγS and ADP are surprisingly small and may not even be significant at the resolution of our cryo-EM maps. However, similarly small differences have been seen in the F1 ATPase (54) or in p97 (55) crystallized with different nucleotides. In contrast, surprisingly

large differences have recently been reported for NSF (26). BIOCHEMISTRY It is interesting to compare the position of the two N domains of Pex1 and Pex6 with that of the single N domain of p97. The N domain of p97 is in a “down conformation” relative to the plane of the D1 ring, regardless of the nucleotide state (19) (SI Ap- pendix, Fig. S12). It displays an “up conformation” in p97 disease mutants crystallized with ATPγS (45) (SI Appendix, Fig. S12). In the structures of the Pex1/Pex6 complex, the N domains vary substantially in their positions relative to the D1 ring. In our model, the N2 domain of Pex1 is in the most extreme up conformation, even more above the D1 ring than the N domain in Fig. 5. Comparison of the structures of the Pex1/Pex6 complex determined in the p97 disease mutants. On the other hand, the N1 domain of ATPγS and ADP. (A) Density map of the Pex1/Pex6 complex obtained in ADP, Pex6 is in a down conformation similar to the situation in WT calculated without imposing symmetry. Shown are different views. (B) Com- p97 (SI Appendix, Fig. S12). parison of models of the Pex1/Pex6 complex in ATPγS and ADP (blue and orange, The exact function and the substrates of the Pex1/Pex6 com- respectively). (C)AsinB, but with magnified views of the individual domains. plex are currently unknown. However, the structural similarity of the Pex1/Pex6 complex to p97 supports a model in which the at any given moment, these ATPases may have only a subset complex functions in peroxisomal protein import (35). In this model, the import receptor Pex5 delivers the substrate to the of their subunits occupied with nucleotide. The variation in peroxisomal membrane, is ubiquitinated, and is then returned to distances between D2 subunits of the Pex1/Pex6 complex is the cytosol by the Pex1/Pex6 complex. Thus, the Pex1/Pex6 more pronounced than seen with F1 ATPase or MecA/ClpC and complex would function analogously to p97 in ERAD, in which is equivalent to that of ClpX. Because the distances across the → the ATPase extracts poly-ubiquitinated proteins from the ER Pex1 Pex6 interfaces are small and constant, it seems that ei- membrane. As proposed for p97, the Pex1/Pex6 complex may ther the occupancy of the Pex6 subunits does not affect subunit move substrate using the ATPase activity of the D2 ring do- spacing or these subunits are mostly in a nucleotide-occupied → mains. In ERAD, poly-ubiquitin chains are recognized by p97 state. In contrast, two of the Pex6 Pex1 distances are too large and its cofactor Ufd1/Npl4 (13). Interestingly, these chains bind to coordinate nucleotide by the neighboring subunits (Fig. 4C). to N domains in both p97 and Ufd1. These domains share the We therefore assume that these two sites are unoccupied and same fold as the tandem N domains in Pex1 and Pex6 (19, 56). It that nucleotide occupancy of the Pex1 subunits has a major effect is thus possible that the Pex1/Pex6 complex functionally corre- on subunit spacing. A correlation between subunit spacing and sponds to the p97/Ufd1/Npl4 complex, such that the peroxisomal nucleotide occupancy has also been observed in recent structures ATPase would contain built-in cofactor domains. It is, however, of dynein (50). In our ATPγS structure, some of the occupied conceivable that an additional unidentified cofactor is required. sites might contain ADP, as the differences from the ADP It should be noted that the Pex1/Pex6 complex has also been structure are only small and ATPγS can slowly be hydrolyzed or proposed to function in the fusion of peroxisomal precursor vesicles be contaminated with ADP. (32–34), a role reminiscent of NSF in vesicular transport. However, Our Walker B mutations show that all subunits in the D2 ring no substrate or cofactor analogous to SNAREs or α-SNAP has hydrolyze ATP, but one interface between Pex1 and Pex6 is more been identified for the Pex1/Pex6 complex, and the activities of the important for overall ATP hydrolysis than the other. Specifically, ATPase domains D1 and D2 are reversed relative to NSF. Pex1 subunits cannot hydrolyze ATP when Pex6 subunits are in Our work establishes a precedent for generating a molecular the ATP-bound state, whereas the reverse is not true (Fig. 6B). model of a protein complex on the basis of an EM map of

Blok et al. PNAS Early Edition | 7of9 Downloaded by guest on September 29, 2021 subjected to gel filtration on a Superose 6 column in the presence of 0.3 mM A ATPγS or 4 mM ADP.

Proteolysis Experiments. Purified Pex1/Pex6 complex (0.9 mg/mL) was incubated with 0–100 μg/mL elastase or trypsin for 20 min at 25°C. For EM experiments, 1.7 mg/mL Pex1/Pex6 complex was incubated with 35 μg/mL elastase at 4°C for 10 min. The reaction was quenched with diisopropyl fluorophosphate before isolation by gel filtration.

ATPase Assays. Pex1/Pex6 complex was incubated with 1 mM ATP at 25°C. Phosphate release was followed over time using the EnzChek assay (Life Technologies). All mutants were stable and assembled into hexamers, as assessed by negative-stain EM. B Electron Microscopy and Image Processing. Specimens were negatively stained with uranyl formate as previously described (58). Samples were imaged using a4K× 4K CCD camera (Gatan) on a Tecnai 12 electron microscope (FEI) operated at 120 kV. The particles were interactively selected with Boxer (59), windowed with SPIDER (60), and subjected to ISAC (39) implemented in SPARX (40). The final stable classes include ∼51% of the entire dataset. For cryo-EM, vitrified specimens were prepared on a glow-discharged holey carbon grid by plunge-freezing in liquid ethane. In some cases, 0.018% Triton (wt/vol) was added to the protein solution just before freezing. Cryo-EM data were collected at liquid nitrogen temperature using a K2 Summit direct electron detector camera (Gatan) on a Tecnai F20 electron microscope operated at 200 kV. Dose-fractionated image stacks were recorded with UCSF Image 4 (61) in superresolution counting mode. Dose-fractionated image stacks of vitrified Pex1/Pex6 complex were 2× binned and motion corrected, as previously described (61). The defocus was determined with CTFFIND3 (62). Particles were selected interactively (59). The particle images were subjected to ISAC (39), and resulting 2D class averages were used to calculate an initial 3D model with SPARX for 3D particle classification in RELION (63). Particles were then subjected to 3D auto- refinement in RELION. Where indicated, threefold symmetry was imposed during 3D classification and refinement. The resolutions of the final density maps were estimated by refining two half datasets independently and using the Fourier shell correlation (FSC) = 0.143 criterion (SI Appendix, Fig. S1 F and G). The maps were postprocessed in RELION and are shown after B-factor sharpening. Figures were prepared with UCSF Chimera (64). Fig. 6. ATPase activity of Pex1 and Pex6 mutants. (A) Sequences of the Modeling Polypeptide Chains into Density Maps. Sequence alignment with Walker A and B motifs and of the relevant segment of the SRH in the several different programs identified the domain organization of Pex1 and ATPase domains of Pex1 and Pex6. The first line shows the canonical se- Pex6, with the D1 and D2 domains having AAA ATPase folds and the N1 and quences seen in AAA ATPases. Amino acids highlighted in red in the D1 N2 domains having Ψ-β barrel folds. Rotational and translational searches and D2 domains of the Pex proteins are identical to the canonical residues. with representative models for these domains resulted in a number of high- Residues in blue are different. Mutations made in the Walker A and B confidence placements. Next, individual homology models for the domains motifs are indicated. (B) ATPase activity of WT and mutant Pex1/Pex6 of Pex1 and Pex6 were placed into the preliminary positions of the Ψ-β barrel complex. Complexes containing either WT Pex1 or Pex6, or proteins with or AAA ATPase domain folds. The RosettaCM comparative modeling pipe- mutations in the D2 Walker A or B motifs, were purified, and equal con- line (38) was used to generate threaded templates (partial threads that in- centrations were tested for ATPase activity. The activity is given relative to clude only the aligned residues for each template) for each of the domains that of a complex in which both proteins are WT. Shown are means and of Pex1 and Pex6. Each of the partial threads was superimposed on the SDsofthreeexperiments. corresponding topology placements from the previous step. Each placement was further refined using rigid body minimization into the local density. intermediate resolution. Whereas previous modeling approaches Next, we placed all eight domains of Pex1 and Pex6 into the density map simultaneously. Placement was done with a simulated annealing Monte placed known crystal structures into the density map and used Carlo (MC) sampling procedure, in which each combination of putative do- flexible fitting to improve the fit (57), our methodology uses main placements is assigned a score (SI Appendix, SI Materials and Methods). density-guided comparative modeling of putative domain place- This score assesses (i) the agreement of an assignment to the density data, ments, followed by Monte Carlo sampling to identify the correct (ii) whether neighboring domains clash with one another, and (iii) whether configuration of domains. This procedure allows modeling in cases they can be connected with the known linker lengths. The first iteration of where only structures of distantly related proteins are known or MC assembly was carried out using partial threads of individual domains as where domains of similar, but nonidentical, structure need to be input and converged to four possible domain assignments. Next, we used placed into the density map. We anticipate that this approach will the full sequences of the individual domains (not just the partial threads) to also be applicable to many other cases in which atomic resolution rebuild unaligned residues and to refine each domain into the density map (modeling was done per domain for tractability). The resulting models were then has been impossible to achieve. used as input for a second iteration of MC assembly. This second iteration converged at a single solution of domain placements, in which the N1 domain of Materials and Methods Pex1 is unassigned into the map. The score of this solution was much better Expanded details of experimental procedures are described in the SI Ap- separated from the next best solution than after the first round of MC assembly pendix, SI Materials and Methods. (SI Appendix,TableS1). The best domain placement also gave a much better fit into the density map than the top incorrect solution (SI Appendix,Fig.S7). Expression and Purification of the Pex1/Pex6 Complex. S. cerevisiae Pex1 and Finally, full-length models were constructed in RosettaCM by rebuilding Pex6 were coexpressed in yeast cells with an N-terminal SBP tag on Pex1 and the linkers connecting individual domains and refining the symmetric assembly

an N-terminal His14 tag on Pex6, and purified by sequential affinity steps on into the symmetrized density map. Although this gave good agreement over Ni-NTA and streptavidin agarose resins. The protein complex was then most of the structure, the D2 domains poorly fit the symmetrized map. We then

8of9 | www.pnas.org/cgi/doi/10.1073/pnas.1500257112 Blok et al. Downloaded by guest on September 29, 2021 further refined the D2 domains into the unsymmetrized map of the Pex1/Pex6 ACKNOWLEDGMENTS. We thank Nicholas Bodnar, Ryan Baldridge, and PNAS PLUS complex in ATPγS, using the Rosetta relax protocol augmented with a term Kelly Kim for critical reading of the manuscript. T.A.R., P.A.P., and D.B. assessing agreement to density. The linkers connecting the D1 and D2 domains are supported by National Institutes of Health (NIH) Grants GM05586, were rebuilt, and the entire structure was refined against the unsymmetrized GM60635, and GM092802, respectively. N.B.B. was supported by NIH map. This model was used as a starting point for refinement of the Pex1/Pex6 Grant GM007753. D.B., T.W., and T.A.R. are Howard Hughes Medical structure in ADP, using the Rosetta relax protocol. Institute Investigators.

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