Unique ATPase Site Architecture Triggers cis-Mediated Synchronized ATP Binding in Heptameric AAA+-ATPase Domain of Flagellar Regulatory Protein FlrC

Dey, Sanjay; Biswas, Maitree; Sen, U.; Dasgupta, J.

doi:10.1074/jbc.m114.611434

Cited by 17 publications

(29 citation statements)

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“…A similar argument as the VCP/p97 case can be applied here: It is likely that these Type II AAA+ also undergo large conformational changes upon ATP hydrolysis, which have yet been captured by the currently available structural studies. Besides NSF and ClpX, homomeric but asymmetric AAA+ ATPase ring has been observed for transcriptional activator NtrC1 [70,71], and more recently, the peroxisomal Pex1/Pex6 complex [72–74]. The relation between asymmetry and function has not been fully understood in these two cases.…”

Section: Comparison Of Nsf With Other Aaa+ Family Membersmentioning

confidence: 99%

Recent Advances in Deciphering the Structure and Molecular Mechanism of the AAA+ ATPase N-Ethylmaleimide-Sensitive Factor (NSF)

Zhao

Brunger

2016

Journal of Molecular Biology

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NSF (N-ethylmaleimide Sensitive Factor), first discovered in 1988, is a key factor for eukaryotic trafficking, including protein and hormone secretion and neurotransmitter release. It is a member of the AAA+ family (ATPases Associated with diverse cellular Activities). NSF disassembles SNARE (Soluble N-ethylmaleimide sensitive factor Attachment protein REceptors) complexes in conjunction with SNAP (Soluble N-ethylmaleimide sensitive factor Attachment Protein) adaptor proteins. Structural studies of NSF and its complex with SNARE and SNAP (known as 20S supercomplex) started about twenty years ago. Crystal structures of individual N and D2 domains of NSF and low-resolution electron microscopy structures of full-length NSF and 20S supercomplex have been reported over the years. Nevertheless, the molecular architecture of the 20S supercomplex and the molecular mechanism of NSF-mediated SNARE complex disassembly remained unclear until recently. Here we review recent atomic-resolution or near-atomic resolution structures of NSF and of the 20S supercomplex, and recent insights into the molecular mechanism and energy requirements of NSF. We also compare NSF with other known AAA+ family members.

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Section: Comparison Of Nsf With Other Aaa+ Family Membersmentioning

confidence: 99%

Recent Advances in Deciphering the Structure and Molecular Mechanism of the AAA+ ATPase N-Ethylmaleimide-Sensitive Factor (NSF)

Zhao

Brunger

2016

Journal of Molecular Biology

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“…1b) states (20). In both the cases FlrC C was found in heptameric state demonstrating that, unlike NtrC class of bEBPs, FlrC spontaneously forms heptamer without Ntdependent subunit remodeling (PDB codes: 4QHS, 4QHT) (20). The major presence of the heptameric species of FlrC C was also established in solution by size exclusion chromatography and dynamic light scattering (20).…”

Section: Introductionmentioning

confidence: 85%

“…1a) and AMP.PNP-bound ( Fig. 1b) states (20). In both the cases FlrC C was found in heptameric state demonstrating that, unlike NtrC class of bEBPs, FlrC spontaneously forms heptamer without Ntdependent subunit remodeling (PDB codes: 4QHS, 4QHT) (20).…”

Section: Introductionmentioning

confidence: 90%

“…The major presence of the heptameric species of FlrC C was also established in solution by size exclusion chromatography and dynamic light scattering (20). While asymmetric split-ring hexamer was observed for NtrC1 to contact RNAP- 54 complex at promoter DNA (18), heptameric FlrC C with much wider central pore was presumed to be adequate for the same purpose without any serious ring distortion (20).…”

Section: Introductionmentioning

confidence: 99%

“…Usually, oligomerization of bEBPs takes place through the AAA + domain. Despite that, bEBPs display remarkable diversity in terms of oligomerization, ATP binding and hydrolysis, mediated by different motifs within the AAA + domain, which possibly play roles in directing the proteins towards specific functions (16)(17)(18)(19)(20)(21)(22). Generally, in NtrC class of proteins, oligomerization is guided by Ntdependent subunit remodeling from inactive dimer to active hexa/heptamer (17,21,22).…”

Section: Introductionmentioning

confidence: 99%

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The heptameric structure of the flagellar regulatory protein FlrC is indispensable for ATPase activity and disassembled by cyclic-di-GMP

Chakraborty

Biswas

Dey

et al. 2020

Journal of Biological Chemistry

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The bacterial enhancer binding protein (bEBP) FlrC, controls motility and colonization of Vibrio cholerae by regulating the transcription of class-III flagellar genes in σ54-dependent manner. However, the mechanism by which FlrC regulates transcription is not fully elucidated. While, most bEBPs require nucleotides to stimulate the oligomerization necessary for function, our previous study showed that the central domain of FlrC (FlrCC) forms heptamer in a nucleotide-independent manner. Furthermore, heptameric FlrCC binds ATP in ‘cis-mediated’ style without any contribution from sensor I motif 285REDXXYR291 of the trans protomer. This atypical ATP binding raises the question of whether heptamerization of FlrC is solely required for transcription regulation, or if it is also critical for ATPase activity. ATPase assays and size exclusion chromatography of the trans-variants FlrCC-Y290A and FlrCC-R291A showed destabilization of heptameric assembly with concomitant abrogation of ATPase activity. Crystal structures showed that in the cis-variant FlrCC-R349A drastic shift of Walker A encroached ATP binding site whereas the site remained occupied by ADP in FlrCC-Y290A. We postulated that FlrCC heptamerizes through concentration dependent cooperativity for maximal ATPase activity and upon heptamerization, packing of trans-acting Tyr290 against cis-acting Arg349 compels Arg349 to maintain proper conformation of Walker A. Finally, a Trp quenching study revealed binding of cyclic-di-GMP with FlrCC. Excess cyclic-di-GMP repressed ATPase activity of FlrCC through destabilization of heptameric assembly, especially at low concentration of protein. Systematic phylogenetic analysis allowed us to propose similar regulatory mechanisms for FlrCs of several Vibrio species and a set of monotrichous Gram negative bacteria.

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The N‐terminal FleQ domain of the Vibrio cholerae flagellar master regulator FlrA plays pivotal structural roles in stabilizing its active state

et al. 2023

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In Vibrio cholerae, the master regulator FlrA controls transcription of downstream flagellar genes in a σ54‐dependent manner. However, the molecular basis of regulation by VcFlrA, which contains a phosphorylation‐deficient N‐terminal FleQ domain, has remained elusive. Our studies on VcFlrA, four of its constructs, and a mutant showed that the AAA+ domain of VcFlrA, with or without the linker ‘L’, remains in ATPase‐deficient monomeric states. By contrast, the FleQ domain plays a pivotal role in promoting higher‐order functional oligomers, providing the required conformation to ‘L’ for ATP/cyclic di‐GMP (c‐di‐GMP) binding. The crystal structure of VcFlrA‐FleQ at 2.0 Å suggests that distinct structural features of VcFlrA‐FleQ presumably assist in inter‐domain packing. VcFlrA at a high concentration forms ATPase‐efficient oligomers when the intracellular c‐di‐GMP level is low. Conversely, excess c‐di‐GMP locks VcFlrA in a non‐functional lower oligomeric state, causing repression of flagellar biosynthesis.

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Unique ATPase Site Architecture Triggers cis-Mediated Synchronized ATP Binding in Heptameric AAA+-ATPase Domain of Flagellar Regulatory Protein FlrC

Abstract: Background: ATP binding, hydrolysis, and 54 interactions by oligomeric bEBPs are poorly understood. Results: First structure of the flagellar regulatory protein FlrC revealed a heptameric AAA ϩ domain with unique cis-mediated

Cited by 17 publications

References 50 publications

Recent Advances in Deciphering the Structure and Molecular Mechanism of the AAA+ ATPase N-Ethylmaleimide-Sensitive Factor (NSF)

Recent Advances in Deciphering the Structure and Molecular Mechanism of the AAA+ ATPase N-Ethylmaleimide-Sensitive Factor (NSF)

The heptameric structure of the flagellar regulatory protein FlrC is indispensable for ATPase activity and disassembled by cyclic-di-GMP

The N‐terminal FleQ domain of the Vibrio cholerae flagellar master regulator FlrA plays pivotal structural roles in stabilizing its active state

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