Mechanism of Homotropic Control to Coordinate Hydrolysis in a Hexameric AAA+ Ring ATPase

Division of Biology, Imperial College London, London SW7 2AZ, UK.
Journal of Molecular Biology (Impact Factor: 4.33). 09/2008; 381(1):1-12. DOI: 10.1016/j.jmb.2008.05.075
Source: PubMed


AAA(+) proteins are ubiquitous mechanochemical ATPases that use energy from ATP hydrolysis to remodel their versatile substrates. The AAA(+) characteristic hexameric ring assemblies raise important questions about if and how six often identical subunits coordinate hydrolysis and associated motions. The PspF AAA(+) domain, PspF(1-275), remodels the bacterial sigma(54)-RNA polymerase to activate transcription. Analysis of ATP substrate inhibition kinetics on ATP hydrolysis in hexameric PspF(1-275) indicates negative homotropic effects between subunits. Functional determinants required for allosteric control identify: (i) an important link between the ATP bound ribose moiety and the SensorII motif that would allow nucleotide-dependent *-helical */beta subdomain dynamics; and (ii) establishes a novel regulatory role for the SensorII helix in PspF, which may apply to other AAA(+) proteins. Consistent with functional data, homotropic control appears to depend on nucleotide state-dependent subdomain angles imposing dynamic symmetry constraints in the AAA(+) ring. Homotropic coordination is functionally important to remodel the sigma(54) promoter. We propose a structural symmetry-based model for homotropic control in the AAA(+) characteristic ring architecture.

Download full-text


Available from: Nicolas Joly,
  • Source
    • "It was also shown that beginning near 1 mM concentration, ATP inhibits ATPase activity of the s54-dependent activators . For NtrC1, this inhibition is only partial (Chen et al. 2010); for PspF, it is more complete (Joly et al. 2006; Schumacher et al. 2008). Therefore, it appears that the activator is highly sensitive to the nucleotide concentration , and thus a more detailed study of structural changes induced by ATP is needed to understand how the bEBP motor functions. "
    [Show abstract] [Hide abstract]
    ABSTRACT: It is largely unknown how the typical homomeric ring geometry of ATPases associated with various cellular activities enables them to perform mechanical work. Small-angle solution X-ray scattering, crystallography, and electron microscopy (EM) reconstructions revealed that partial ATP occupancy caused the heptameric closed ring of the bacterial enhancer-binding protein (bEBP) NtrC1 to rearrange into a hexameric split ring of striking asymmetry. The highly conserved and functionally crucial GAFTGA loops responsible for interacting with σ54-RNA polymerase formed a spiral staircase. We propose that splitting of the ensemble directs ATP hydrolysis within the oligomer, and the ring's asymmetry guides interaction between ATPase and the complex of σ54 and promoter DNA. Similarity between the structure of the transcriptional activator NtrC1 and those of distantly related helicases Rho and E1 reveals a general mechanism in homomeric ATPases whereby complex allostery within the ring geometry forms asymmetric functional states that allow these biological motors to exert directional forces on their target macromolecules.
    Genes & development 11/2013; 27(22):2500-11. DOI:10.1101/gad.229385.113 · 10.80 Impact Factor
  • Source
    • "The crystal structure of mutated, linked ClpX also shows a similar dimer of trimers structure (Glynn et al., 2009). Though similar nucleotide exchange reactions have been suggested by others (Hersch et al., 2005; Schumacher et al., 2008; Singleton et al., 2000), albeit without evidence of distinct functional consequences, the crystal structures of some AAA ATPases (e.g., HslU [Bochtler et al., 2000; Sousa et al., 2000; Yakamavich et al., 2008]) revealed seemingly promiscuous binding patterns for ATP analogs or ADP. An unambiguous elucidation of the binding exchange reactions for those ATPases has proven difficult because the number of nucleotides bound per hexamer has rarely been determined to a definite integer value (i.e., prior results could not distinguish between three or four nucleotides per hexamer). "
    [Show abstract] [Hide abstract]
    ABSTRACT: In the eukaryotic 26S proteasome, the 20S particle is regulated by six AAA ATPase subunits and, in archaea, by a homologous ring complex, PAN. To clarify the role of ATP in proteolysis, we studied how nucleotides bind to PAN. Although PAN has six identical subunits, it binds ATPs in pairs, and its subunits exhibit three conformational states with high, low, or no affinity for ATP. When PAN binds two ATPγS molecules or two ATPγS plus two ADP molecules, it is maximally active in binding protein substrates, associating with the 20S particle, and promoting 20S gate opening. However, binding of four ATPγS molecules reduces these functions. The 26S proteasome shows similar nucleotide dependence. These findings imply an ordered cyclical mechanism in which two ATPase subunits bind ATP simultaneously and dock into the 20S. These results can explain how these hexameric ATPases interact with and "wobble" on top of the heptameric 20S proteasome.
    Cell 02/2011; 144(4):526-38. DOI:10.1016/j.cell.2011.02.005 · 32.24 Impact Factor
  • Source
    • "In bEBPs the ATP hydrolysis site is configured through interactions between adjacent AAA+ protomers in the hexameric ring (Schumacher et al., 2008). Since the GAFTGA motif relays nucleotide-dependent interactions at this site to enable contact with s 54 , we were interested to examine if the G266 substitutions influence ATPase activity. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Bacterial enhancer binding proteins (bEBPs) are specialized transcriptional activators that assemble as hexameric rings in their active forms and utilize ATP hydrolysis to remodel the conformation of RNA polymerase containing the alternative sigma factor σ(54). Transcriptional activation by the NorR bEBP is controlled by a regulatory GAF domain that represses the ATPase activity of the central AAA+ domain in the absence of nitric oxide. Here, we investigate the mechanism of interdomain repression in NorR by characterizing substitutions in the AAA+ domain that bypass repression by the regulatory domain. Most of these substitutions are located in the vicinity of the surface-exposed loops that engage σ(54) during the ATP hydrolysis cycle or in the highly conserved GAFTGA motif that directly contacts σ(54). Biochemical studies suggest that the bypass mutations in the GAFTGA loop do not influence the DNA binding properties of NorR or the assembly of higher order oligomers in the presence of enhancer DNA, and as expected these variants retain the ability to activate open complex formation in vitro. We identify a crucial arginine residue in the GAF domain that is essential for interdomain repression and demonstrate that hydrophobic substitutions at this position suppress the bypass phenotype of the GAFTGA substitutions. These observations suggest a novel mechanism for negative regulation in bEBPs in which the GAF domain targets the σ(54)-interaction surface to prevent access of the AAA+ domain to the sigma factor.
    Molecular Microbiology 09/2010; 77(5):1278-88. DOI:10.1111/j.1365-2958.2010.07290.x · 4.42 Impact Factor
Show more