Regulation of the transcriptional activator NtrC1: structural studies of the regulatory and AAA⁺ ATPase domains

Abstract

Transcription by54 RNA polymerase depends on activators that contain ATPase domains of the AAA + class. These activators, which are often response regulators of two-component signal transduction systems, remodel the polymerase so that it can form open complexes at promoters. Here, we report the first crystal structures of the ATPase domain of an activator, the NtrC1 protein from the extreme thermophile Aquifex aeolicus. This domain alone, which is active, crystallized as a ring-shaped heptamer. The protein carrying both the ATPase and adjacent receiver domains, which is inactive, crystallized as a dimer. In the inactive dimer, one residue needed for catalysis is far from the active site, and extensive contacts among the domains prevent oligomerization of the ATPase domain. Oligomerization, which completes the active site, depends on surfaces that are buried in the dimer, and hence, on a rearrangement of the receiver domains upon phosphorylation. A motif in the ATPase domain known to be critical for coupling energy to remodeling of polymerase forms a novel loop that projects from the middle of an ␣ helix. The extended, structured loops from the subunits of the heptamer localize to a pore in the center of the ring and form a surface that could contact 54 .

Investigating protein–protein interfaces in bacterial transcription complexes: a fragmentation approach

“…The recent crystal structure of the ADP-bound NtrC1, another bacterial s 54 -dependent EBP, indicates that the GAFTGA motif indeed forms part of a stable loop structure that protrudes from each NtrC1 subunit into the lumen of the central pore of an oligomeric ring of NtrC1 protomers, rather like the iris of a camera. (16) This exciting new high-resolution structure confirms the proposed arrangement of the GAFTGA motif within the PspF model and demonstrates that these sequences are ideally placed to interact with s 54 . However, determining the precise ways in which the s 54 -RNAP and PspF associate for remodelling the closed complexes will require detailed co-complex structural information.…”

“…(11) Characteristically, all AAAþ members have the ability to self-assemble into higher order oligomeric structures, often as hexamers arranged in a ring shape. (13)(14)(15)(16) They also have the conserved functionality of transforming the energy derived from NTP hydrolysis to a mechanical force typically used to remodel protein and protein-nucleic acid complexes. (11,12,17) Sequence alignments have demonstrated that AAAþ proteins share common motifs, namely the AAA minimum consensus, Walker A, Walker B, Sensor I and Sensor II.…”

“…(11) The AAA minimum consensus and Walker motifs mediate NTP binding and hydrolysis, whilst the Sensor motifs are proposed to interact with the g-phosphate of the nucleotide. (12) Interestingly, the nucleotide active site is created between two monomers, (16) highlighting the functional link between nucleotide hydrolysis and oligomeric state of the activator. Therefore, the ATPase activity of these proteins can be regulated by controlling the rate of self-association.…”

Purification and Characterization of the AAA+ Domain ofSinorhizobium melilotiDctD, a σ54-Dependent Transcriptional Activator

“…The ring structures of AAAϩ proteins can be created through the regulated assembly of monomers or dimers, the formation a stable ring upon assembly of the subunits, or the formation of a ring from a single polypeptide chain containing multiple AAAϩ domains (42). Activators of 54 -holoenzyme appear to form ring structures through the regulated assembly of subunits (18). Crystal structures were reported recently for the ADP-bound form of the ATPase domain alone and the ATPase and adjacent regulatory do-mains of Aquifex aeolicus NtrC1, a 54 -dependent activator (18).…”

Meddling Vibrio cholerae Murmurs: A Neoteric Advancement in Cholera Research

“…The LuxO has two separate N-terminal regulatory and C-terminal DNA binding domain where the oligomerization or AAA ? ATPase domain is located between the receiver domain and a helix turn helix (HTH) motif [31,65,66]. The HTH motif are known to recognize enhancer like sequence between 100 and 150 base pairs present in upstream of the promoter of the target genes.…”