Receiver Domains Control the Active-State Stoichiometry of Aquifex aeolicus σ54 Activator NtrC4, as Revealed by Electrospray Ionization Mass Spectrometry

Abstract

A common challenge with studies of proteins in vitro is determining which constructs and conditions are most physiologically relevant. σ 54 activators are proteins that undergo regulated assembly to form an active ATPase ring that enables transcription by σ 54 -polymerase. Previous studies of the AAA+ ATPase domains from σ 54 activators have shown that some are heptamers, while others are hexamers. Because the active oligomers assemble from off-state dimers, it was thought that even-numbered oligomers should dominate, and that heptamer formation might occur when individual domains of the activators were studied rather than the intact proteins. Here we present results from electrospray mass spectrometry experiments that characterize the assembly states of intact NtrC4 (a σ 54 activator from Aquifex aeolicus, an extreme thermophile) as well as its ATPase domain alone, and regulatoryATPase and ATPase-DNA binding domain combinations. We show that the full-length and activated regulatory-ATPase proteins form hexamers, whereas the isolated ATPase domain, unactivated regulatory-ATPase and ATPase-DNA binding domain form heptamers. Activation of the N-terminal regulatory domain is the key factor stabilizing the hexamer form of the ATPase, relative to the heptamer.

Molecular Mechanisms of Two-Component Signal Transduction

“…Upon phosphorylation/activation they form higher-order oligomeric states (hexamer or heptamer) triggered by oligomerization of a central σ54 binding ATPase domain. These ring-like structures induce open complex formation of RNA polymerase [166]. …”

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

“…In full-length NtrC1, the equilibrium between heptamer and hexamer rings may be influenced by the presence of the N-terminal two-component receiver and C-terminal domains. Mass spectroscopy data showed that hexamer is favored over heptamer in fulllength constructs of NtrC4, an EBP related to NtrC1 (Batchelor et al 2009). The receiver domain forms stable homodimers, and the C-terminal DNA-binding domain directs assembly of four protomers in a tandem array on ''enhancers'' (Doucleff et al 2005;Vidangos et al 2013).…”

Desalting protein ions in native mass spectrometry using supercharging reagents

“…For example, NtrC4 (a σ activator protein from Aquifex aeolicus ) requires millimolar concentrations of certain salts, e.g., Mg 2+ , BeF 3 − , and ADP, in order to assemble into an active hexamer. 35, 36 Several techniques for reducing sodium ion adduction to proteins in ESI have been developed that do not require removal of the salts from solution prior to ion formation. Buffer loading, 21, 22 in which high concentrations of a volatile buffer, typically ammonium acetate, is added to solution, reduces sodium ion adduction to proteins and reduces the number of salt ion clusters formed.…”