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 3 1 2 1 Top Oblique 2 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.