ABSTRACT
How AAA+ chaperones conformationally remodel specific target proteins in an ATP-dependent manner is not well understood. Here, we investigated the mechanism of the AAA+ protein Rubisco activase (Rca) in metabolic repair of the photosynthetic enzyme Rubisco, a complex of eight large (RbcL) and eight small (RbcS) subunits containing eight catalytic sites. Rubisco is prone to inhibition by tight-binding sugar phosphates, whose removal is catalyzed by Rca. We engineered a stable Rca hexamer ring and analyzed its functional interaction with Rubisco. Hydrogen/deuterium exchange and chemical crosslinking showed that Rca structurally destabilizes elements of the Rubisco active site with remarkable selectivity. Cryo-electron microscopy revealed that Rca docks onto Rubisco over one active site at a time, positioning the C-terminal strand of RbcL, which stabilizes the catalytic center, for access to the Rca hexamer pore. The pulling force of Rca is fine-tuned to avoid global destabilization and allow for precise enzyme repair.
Subject(s)
Bacterial Proteins/metabolism , Molecular Chaperones/metabolism , Plant Proteins/metabolism , Rhodobacter sphaeroides/enzymology , Ribulose-Bisphosphate Carboxylase/metabolism , Tissue Plasminogen Activator/metabolism , Adenosine Triphosphate/metabolism , Allosteric Regulation , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Binding Sites , Catalytic Domain , Cross-Linking Reagents/chemistry , Deuterium Exchange Measurement , Enzyme Stability , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Molecular Docking Simulation , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Quaternary , Protein Subunits , Rhodobacter sphaeroides/genetics , Ribulose-Bisphosphate Carboxylase/chemistry , Ribulose-Bisphosphate Carboxylase/genetics , Structure-Activity Relationship , Time Factors , Tissue Plasminogen Activator/chemistry , Tissue Plasminogen Activator/geneticsABSTRACT
The peroxisomal proteins Pex1 and Pex6 form a heterohexameric type II AAA+ ATPase complex, which fuels essential protein transport across peroxisomal membranes. Mutations in either ATPase in humans can lead to severe peroxisomal disorders and early death. We present an extensive structural and biochemical analysis of the yeast Pex1/6 complex. The heterohexamer forms a trimer of Pex1/6 dimers with a triangular geometry that is atypical for AAA+ complexes. While the C-terminal nucleotide-binding domains (D2) of Pex6 constitute the main ATPase activity of the complex, both D2 harbour essential substrate-binding motifs. ATP hydrolysis results in a pumping motion of the complex, suggesting that Pex1/6 function involves substrate translocation through its central channel. Mutation of the Walker B motif in one D2 domain leads to ATP hydrolysis in the neighbouring domain, giving structural insights into inter-domain communication of these unique heterohexameric AAA+ assemblies.
Subject(s)
Adenosine Triphosphatases/metabolism , Membrane Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , ATPases Associated with Diverse Cellular Activities , Adenosine Triphosphate/metabolism , Dimerization , Hydrolysis , Protein Binding , Protein TransportABSTRACT
Members of the diverse superfamily of AAA+ proteins are molecular machines responsible for a wide range of essential cellular processes. In this review we summarise structural and functional data surrounding the nucleotide binding pocket of these versatile complexes. Protein Data Bank (PDB) structures of closely related AAA+ ATPase are overlaid and biologically relevant motifs are displayed. Interactions between protomers are illustrated on the basis of oligomeric structures of each AAA+ subgroup. The possible role of conserved motifs in the nucleotide binding pocket is assessed with regard to ATP binding and hydrolysis, oligomerisation and inter-subunit communication. Our comparison indicates that in particular the roles of the arginine finger and sensor 2 residues differ subtly between AAA+ subgroups, potentially providing a means for functional diversification.
Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/classification , Amino Acid Motifs , Animals , Binding Sites , Catalytic Domain , Conserved Sequence , Humans , Hydrogen Bonding , Nucleotides/chemistry , Protein Binding , Protein Interaction Domains and Motifs , Structural Homology, ProteinABSTRACT
Rubisco, the enzyme that catalyzes the fixation of atmospheric CO(2) in photosynthesis, is subject to inactivation by inhibitory sugar phosphates. Here we report the 2.95-Å crystal structure of Nicotiana tabacum Rubisco activase (Rca), the enzyme that facilitates the removal of these inhibitors. Rca from tobacco has a classical AAA(+)-protein domain architecture. Although Rca populates a range of oligomeric states when in solution, it forms a helical arrangement with six subunits per turn when in the crystal. However, negative-stain electron microscopy of the active mutant R294V suggests that Rca functions as a hexamer. The residues determining species specificity for Rubisco are located in a helical insertion of the C-terminal domain and probably function in conjunction with the N-domain in Rubisco recognition. Loop segments exposed toward the central pore of the hexamer are required for the ATP-dependent remodeling of Rubisco, resulting in the release of inhibitory sugar.
