Receiver domains control the active-state stoichiometry of Aquifex aeolicus sigma54 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. sigma(54) activators are proteins that undergo regulated assembly to form an active ATPase ring that enables transcription by sigma(54)-polymerase. Previous studies of AAA(+) ATPase domains from sigma(54) activators have shown that some are heptamers, while others are hexamers. Because active oligomers assemble from off-state dimers, it was thought that even-numbered oligomers should dominate, and that heptamer formation would occur when individual domains of the activators, rather than the intact proteins, were studied. Here we present results from electrospray ionization mass spectrometry experiments characterizing the assembly states of intact NtrC4 (a sigma(54) activator from Aquifex aeolicus, an extreme thermophile), as well as its ATPase domain alone, and regulatory-ATPase 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.

Figure 1. NtrC4 and NtrC1 structures

σ⁵⁴ activators exist as inactive dimers and active hexamers and heptamers. NtrC4 has dimeric DNA binding domains and dimeric activated receiver domains and NtrC1 crystallized in a heptameric active state, while PspF and ZraR crystallized as hexamers. PDB IDs 3DZD (1), 3E7L (2), 2JRL (3), 1NY6 (4), and 2VII (5).

Figure 2. Mass spectra of (a) unactivated and (b) activated NtrC4-RCD

In the presence of MgCl₂, BeF₃⁻, and ADP, and heated to ~62 °C, the NtrC4-RCD protein forms hexamers. The tetramer observed in both spectra may be a non-specific aggregate of the dimer.

Figure 3. Mass spectra of (a) unactivated and (b) activated NtrC4-RC

Multiple higher-order oligomers are seen for unactivated NtrC4-RC, while only hexamer is observed when the protein is activated with MgCl₂, BeF₃⁻, and ADP, and heated to ~62 °C.

Figure 4. Mass spectra of (a) NtrC4-C and (b) NtrC4-CD

Both the NtrC4-C and -CD constructs assemble to multiple oligomers at 29 ºC without activation. Increasing the solution temperature results only in a decrease in signal-to-noise for the higher-order oligomers. Adding MgCl₂, BeF₃⁻, and ADP to the solutions did not change the observed stoichiometries.

Figure 5

Tandem mass spectra resulting from collisionally activated dissociation (argon target gas) of the 34+ ion isolated from the heptamer charge state distribution as a function of (a) low (30 V), (b) intermediate (50 V), and (c) higher (70 V) acceleration voltage.