Trapping of a transcription complex using a new nucleotide analogue: AMP aluminium fluoride

Mechanochemical proteins rely on ATP hydrolysis to establish the different functional states required for their biological output. Studying the transient functional intermediate states these proteins adopt as they progress through the ATP hydrolysis cycle is key to understanding the molecular basis of their mechanism. Many of these intermediates have been successfully ‘trapped’ and functionally characterised using ATP analogues. Here, we present a new nucleotide analogue, AMP-AlF_x, which traps PspF, a bacterial enhancer binding protein, in a stable complex with the σ⁵⁴-RNA polymerase holoenzyme. The crystal structure of AMP-AlF_x•PspF_1-275 provides new information on protein-nucleotide interactions and suggests that the β and γ phosphates are more important than the α phosphate in terms of sensing nucleotide bound states. In addition, functional data obtained with AMP-AlF_x establish distinct roles for the conserved catalytic AAA⁺ (ATPases associated with various cellular activities) residues, suggesting that AMP-AlF_x is a powerful new tool to study AAA⁺ protein family members and, more generally, Walker motif ATPases.     

Asymmetric nucleotide transactions of the HslUV protease

ATP binding and hydrolysis are critical for protein degradation by HslUV, a AAA ⁺ machine containing one or two HslU₆ ATPases and the HslV₁₂ peptidase. Although each HslU homohexamer has six potential ATP-binding sites, we show that only three or four ATP molecules bind at saturation and present evidence for three functional subunit classes. These results imply that only a subset of HslU and HslUV crystal structures represents functional enzyme conformations. Our results support an asymmetric mechanism of ATP binding and hydrolysis, and suggest that molecular contacts between HslU and HslV vary dynamically throughout the ATPase cycle. Nucleotide binding controls HslUV assembly and activity. Binding of a single ATP allows HslU to bind HslV, whereas additional ATPs must bind HslU to support substrate recognition and to activate ATP hydrolysis, which powers substrate unfolding and translocation. Thus, a simple thermodynamic hierarchy ensures that substrates bind to functional HslUV complexes, that ATP hydrolysis is efficiently coupled to protein degradation, and that working HslUV does not dissociate, allowing highly processive degradation.     

Heterogeneous nucleotide occupancy stimulates functionality of phage shock protein F, an AAA+ transcriptional activator

The catalytic AAA+ domain (PspF1-275) of an enhancer-binding protein is necessary and sufficient to contact sigma54-RNA polymerase holoenzyme (Esigma54), remodel it, and in so doing catalyze open promoter complex formation. Whether ATP binding and hydrolysis is coordinated between subunits of PspF and the precise nature of the nucleotide(s) bound to the oligomeric forms responsible for substrate remodeling are unknown. We demonstrate that ADP stimulates the intrinsic ATPase activity of PspF1-275 and propose that this heterogeneous nucleotide occupancy in a PspF1-275 hexamer is functionally important for specific activity. Binding of ADP and ATP triggers the formation of functional PspF1-275 hexamers as shown by a gain of specific activity. Furthermore, ATP concentrations congruent with stoichiometric ATP binding to PspF1-275 inhibit ATP hydrolysis and Esigma54-promoter open complex formation. Demonstration of a heterogeneous nucleotide-bound state of a functional PspF1-275.Esigma54 complex provides clear biochemical evidence for heterogeneous nucleotide occupancy in this AAA+ protein. Based on our data, we propose a stochastic nucleotide binding and a coordinated hydrolysis mechanism in PspF1-275 hexamers.     

Coupling σ Factor Conformation to RNA Polymerase Reorganisation for DNA Melting

ATP-driven remodelling of initial RNA polymerase (RNAP) promoter complexes occurs as a major post recruitment strategy used to control gene expression. Using a model-enhancer-dependent bacterial system (σ⁵⁴-RNAP, Eσ⁵⁴) and a slowly hydrolysed ATP analogue (ATPγS), we provide evidence for a nucleotide-dependent temporal pathway leading to DNA melting involving a small set of σ⁵⁴-DNA conformational states. We demonstrate that the ATP hydrolysis-dependent remodelling of Eσ⁵⁴ occurs in at least two distinct temporal steps. The first detected remodelling phase results in changes in the interactions between the promoter specificity σ⁵⁴ factor and the promoter DNA. The second detected remodelling phase causes changes in the relationship between the promoter DNA and the core RNAP catalytic β/β′ subunits, correlating with the loading of template DNA into the catalytic cleft of RNAP. It would appear that, for Eσ⁵⁴ promoters, loading of template DNA within the catalytic cleft of RNAP is dependent on fast ATP hydrolysis steps that trigger changes in the β′ jaw domain, thereby allowing acquisition of the open complex status.     

Regulation of type VI secretion gene clusters by sigma54 and cognate enhancer binding proteins

Type VI secretion systems (T6SS) are bacteriophage-derived macromolecular machines responsible for the release of at least two proteins in the milieu, which are thought to form an extracellular appendage. Although several T6SS have been shown to be involved in the virulence of animal and plant pathogens, clusters encoding these machines are found in the genomes of most species of gram-negative bacteria, including soil, marine, and environmental isolates. T6SS have been associated with several phenotypes, ranging from virulence to biofilm formation or stress sensing. Their various environmental niches and large diversity of functions are correlated with their broad variety of regulatory mechanisms. Using a bioinformatic approach, we identified several clusters, including those of Vibrio cholerae, Aeromonas hydrophila, Pectobacterium atrosepticum, Pseudomonas aeruginosa, Pseudomonas syringae pv. tomato, and a Marinomonas sp., which possess typical -24/-12 sequences, recognized by the alternate sigma factor sigma 54 (σ(54) or σ(N)). σ(54), which directs the RNA polymerase to these promoters, requires the action of a bacterial enhancer binding protein (bEBP), which binds to cis-acting upstream activating sequences. Putative bEBPs are encoded within the T6SS gene clusters possessing σ(54) boxes. Using in vitro binding experiments and in vivo reporter fusion assays, we showed that the expression of these clusters is dependent on both σ(54) and bEBPs.     

Novel essential residues of Hda for interaction with DnaA in the regulatory inactivation of DnaA: unique roles for Hda AAA+ Box VI and VII motifs

Escherichia coli ATP–DnaA initiates chromosomal replication. For preventing extra‐initiations, a complex of ADP–Hda and the DNA‐loaded replicase clamp promotes DnaA‐ATP hydrolysis, yielding inactive ADP–DnaA. However, the Hda–DnaA interaction mode remains unclear except that the Hda Box VII Arg finger (Arg‐153) and DnaA sensor II Arg‐334 within each AAA⁺ domain are crucial for the DnaA‐ATP hydrolysis. Here, we demonstrate that direct and functional interaction of ADP–Hda with DnaA requires the Hda residues Ser‐152, Phe‐118 and Asn‐122 as well as Hda Arg‐153 and DnaA Arg‐334. Structural analyses suggest intermolecular interactions between Hda Ser‐152 and DnaA Arg‐334 and between Hda Phe‐118 and the DnaA Walker B motif region, in addition to an intramolecular interaction between Hda Asn‐122 and Arg‐153. These interactions likely sustain a specific association of ADP–Hda and DnaA, promoting DnaA‐ATP hydrolysis. Consistently, ATP–DnaA and ADP–DnaA interact with the ADP–Hda‐DNA–clamp complex with similar affinities. Hda Phe‐118 and Asn‐122 are contained in the Box VI region, and their hydrophobic and electrostatic features are basically conserved in the corresponding residues of other AAA⁺ proteins, suggesting a conserved role for Box VI. These findings indicate novel interaction mechanisms for Hda–DnaA as well as a potentially fundamental mechanism in AAA⁺ protein interactions.