Nucleotide-induced asymmetry within ATPase activator ring drives σ54–RNAP interaction and ATP hydrolysis

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 s54-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 s54 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.

Binding of Substrates to the Central Pore of the Vps4 ATPase Is Autoinhibited by the Microtubule Interacting and Trafficking (MIT) Domain and Activated by MIT Interacting Motifs (MIMs)

“…This follows from our observations that a single peptide binds per hexamer, which necessarily requires an asymmetric interface, and is dependent upon the presence of ADP⅐AlF x or ADP⅐BeF x , which allow the ATPase to adopt multiple nucleotide-bound states. This is consistent with models of other ring-like ATPases, including F 1 ATPase (63), E1 helicase (64), Rho helicase (59), the 26S proteasome (65,66), the bacterial enhancer-binding protein NtrC1 (58), and N-ethylmaleimide-sensitive factor (NSF) (67), which all display asymmetry of the subunits that results in a spiral staircase arrangement of the pore loops. An attractive model is that Vps4 translocates substrate, at least partially, through the central pore, driven by changes in pore loop conformations that propagate around the hexamer ring in concert with the ATP hydrolysis cycle.…”

Unique ATPase Site Architecture Triggers cis-Mediated Synchronized ATP Binding in Heptameric AAA+-ATPase Domain of Flagellar Regulatory Protein FlrC

“…This is not a very rare observation because Lee and Huber (49) reported that Rhizobium meliloti C4-dicarboxylic acid transport protein D cross-links with 54 even without Nt binding. In FlrC, the small asymmetry created in the central pore and their elevated thermal vibration upon AMP-PNP binding probably point toward the generation of local asymmetry upon ATP binding, which may enhance during ATP hydrolysis, as observed previously for the other bEBPs (8,9), although the extent of asymmetry may differ in this case. Dimension of the central pore and disposition of the L1 loops around the central pore would seemingly play a crucial role in causing asymmetry (8,9).…”

“…Cross-linking and EM reconstruction studies have recently shown that only one oligomeric assembly of bEBP is sufficient enough to simultaneously bind RNAP-54 and the upstream promoter region using varying numbers of participating L1 loops (2,9,47,48). Notably, 54 binds bEBP and RNAP through its highly conserved regions I and III.…”

“…The ATPbound AAA ϩ domain of A. aeolicus NtrC1 E239A , revealed a heptameric assembly with asymmetry in the central channel (8). Recently, the AAA ϩ domain of NtrC1 is found to form a split ring hexamer in the presence of ADP ϩ BeF 3 Ϫ that argues for flexibility in packing stoichiometry and interface angles of the constituting monomers (9). ESI-MS results of A. aeolicus NtrC4 showed that, although the full-length and activated R-AAA ϩ proteins form hexamers, the isolated AAA ϩ , unactivated R-AAA ϩ , and AAA ϩ -DNA binding domains form heptamers (10).…”

Mechanistic insights into c-di-GMP–dependent control of the biofilm regulator FleQ fromPseudomonas aeruginosa

“…PspF ring oligomers are believed to undergo sequential hydrolysis where asymmetry in the intersubunit interactions confers cooperativity in nucleotide binding and catalysis (30). In addition, solution structural studies on ring hexamers of another nonhelical bEBP, NtrC1, revealed that one of the intersubunit interfaces is significantly less tight, which led to a mechanistic model where bEBP planar ring split is necessary to drive interactions with the σ 54 -RNAP holoenzyme and ATPdependent remodeling (31).…”