Unique ATPase site architecture triggers cis-mediated synchronized ATP binding in heptameric AAA+ ATPase domain of flagellar regulatory protein FlrC [Microbiology]

February 16th, 2015 by Dey, S., Biswas, M., Sen, U., Dasgupta, J.

Bacterial enhancer binding proteins (bEBPs) oligomerize through AAA+ domains and use ATP hydrolysis driven energy to isomerise RNAP-σ54 complex during transcription initiation. Here we describe the first structure of the central AAA+ domain of the flagellar regulatory protein FlrC (FlrCC), a bEBP that controls flagellar synthesis in Vibrio cholerae. Our results showed that FlrCC forms heptamer both in Nucleotide (Nt) free and bound states without ATP dependent subunit remodelling. Unlike the bEBPs such as NtrC1 or PspF, a novel cis-mediated 'all or none' ATP binding occurs in the heptameric FlrCC, since constriction at the ATPase site, caused by loop L3 and helix α7, restricts the proximity of trans-protomer required for Nt-binding. A unique 'closed to open' movement of Walker A, assisted by trans-acting 'Glu-switch' E286, facilitates ATP binding and hydrolysis. Fluorescence quenching and ATPase assays on FlrCC and mutants revealed that while R349 of sensor II, positioned by trans-acting E286 and Y290, acts as a key residue to bind and hydrolyse ATP, R319 of α7 anchors ribose and controls the rate of ATP hydrolysis by retarding the expulsion of ADP. Heptameric state of FlrCC is restored in solution even with the transition state mimicking ADP+AlF3. Structural results and pull-down assays indicated that L3 renders an in-built geometry to L1 and L2 causing σ54-FlrCC interaction independent of Nt-binding. Collectively, our results underscore a novel mechanism of ATP binding and σ54 interaction that drives to understand the transcription mechanism of the bEBPs, which probably interact directly with RNAP-σ54 complex without DNA looping.
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