Crystal structure of the ATPase domain of the human AAA+ protein paraplegin/SPG7

Abstract

Paraplegin is an m-AAA protease of the mitochondrial inner membrane that is linked to hereditary spastic paraplegias. The gene encodes an FtsH-homology protease domain in tandem with an AAA+ homology ATPase domain. The protein is believed to form a hexamer that uses ATPase-driven conformational changes in its AAA-domain to deliver substrate peptides to its protease domain. We present the crystal structure of the AAA-domain of human paraplegin bound to ADP at 2.2 A. This enables assignment of the roles of specific side chains within the catalytic cycle, and provides the structural basis for understanding the mechanism of disease mutations.

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Figure 1. Domain arrangement and sequence comparison of paraplegin/SPG7 and related AAA+ proteins.

A. Domain arrangement of paraplegin, FtsH, VCP, and spastin. Homologies included are the FtsH extracellular (Pfam entry PF36480), AAA+ (PF00004), metallopeptidase M41 (PF01434), Cell division protein-48 (CDC48) N-terminal (PF02359), CDC48-2 (PF02933), and microtubule interacting and transport (MIT; PF04212) domains. B. Sequence alignment of the ATPase domains of paraplegin and related proteins to illustrate the positions of conserved residues. Residue numbering and secondary structural elements are indicated for paraplegin (PDB entry 2qz4) above the alignment. Walker A and B, and Sensor 1 and 2 motifs, the arginine residue predicted to act as an arginine finger, as well as the pore loop are indicated below the alignment. Indicated by green asterisks are HSP disease related positions. Sequences shown are human paraplegin/SPG7 (residues 305–565; PDB entry 2qz4; gene identification code 116242796). Thermus thermophilus FtsH (126–624; 2dhr; gi:8051696), Thermotoga maritima FtsH (147–610; 2cea; gi:15643346), human p97/VCP (116–417; gi:112818458), and human spastin/SPG4 (114–437; gi:11875211).

Figure 2. Purification and crystallization of paraplegin^305–565.

A. Coomassie-stained SDS-polyacrylamide gel showing the purity of crude paraplegin^305–565 after TEV-cleavage (left lane; red asterisk, hexahistidine-tagged protein; black asterisk, cleaved protein), and after the final purification step (right lane). B. Example of crystals grown under the conditions that yielded diffraction data.

Figure 3. Overview of the paraplegin ATPase domain structure.

A. Schematic representation of the crystal structure of a monomer of paraplegin^305–565 with bound ADP. Sequence motifs indicated in the sequence alignment in Figure 1 have been mapped onto the structure. The positions of disease-related residues are labeled in blue. B. Electrostatic surface representation of paraplegin^305–565 illustrating the nucleotide binding cleft.

Figure 4. The nucleotide binding site of paraplegin.

Details of side chain interactions with ADP.

Figure 5. Model of the paraplegin hexamer.

A. The hexameric structure of paraplegin^305–565 was modelled by aligning our crystal structure (blue) with each monomer within the T. thermophilus FtsH hexamer crystal structure (2dhr; orange). The outline of one monomer is indicated by grey shading, and the N- and C-termini of another monomer are indicated. The boxed area is expanded in panel B. B. Close-up of the region around the pore loops and the monomer interface around the nucleotide binding site. The hydrophobic pore loop residue Phe228 of FtsH, implicated in substrate binding, is shown in green, and the corresponding paraplegin residue Ile832 is shown in purple. Paraplegin Arg470, shown in red, is a putative arginine finger that activates ATP hydrolysis in the neighbor monomer following a conformational change in the ring structure.