scholarly journals Ring-shaped multimeric structure enables the acceleration of KaiB-KaiC complex formation induced by the ADP/ATP exchange inhibition

2021 ◽  
Author(s):  
Shin-ichi Koda ◽  
Shinji Saito
Keyword(s):  
Complex Formation ◽  
Atp Hydrolysis ◽  
Reaction Model ◽  
Clock Period ◽  
Whole Process ◽  
Environmental Perturbations ◽  
Clock Proteins ◽  
C1 Domain ◽  
Forward Process ◽  
Hexameric Form

Circadian clocks tick a rhythm with a nearly 24-hour period in various organisms. The clock proteins of cyanobacteria, KaiA, KaiB, and KaiC, compose a minimum circadian clock. The slow KaiB-KaiC complex formation, which is essential in determining the clock period, occurs when the C1 domain of KaiC binds ADP produced by ATP hydrolysis. KaiC is considered to promote this complex formation by inhibiting the backward process, ADP/ATP exchange, rather than activating the forward process, ATP hydrolysis. Remarkably, although inhibition of backward process, in general, decelerates the whole process, KaiC oppositely accelerates the complex formation. In this article, by building a novel reaction model, we investigate the molecular mechanism of the simultaneous promotion and acceleration of the complex formation, which may play a significant role in keeping the period invariant under environmental perturbations. Based on several experimental results, we assume in this model that six KaiB monomers cooperatively and rapidly binds to C1 with the stabilization of the binding-competent conformation of C1 only when C1 binds six ADP. We find the cooperative KaiB binding effectively separates the pre-binding process of C1 into a fast transformation to binding-competent C1 requiring multiple ATP hydrolyses and its slow backward transformation. Since the ADP/ATP exchange retards the forward process, its inhibition results in the acceleration of the complex formation. We also find that, in a simplified monomeric model where KaiB binds to a KaiC monomer independently of the other monomers, the ADP/ATP exchange inhibition cannot accelerate the complex formation. In summary, we conclude that the ring-shaped hexameric form of KaiC enables the acceleration of the complex formation induced by the backward process inhibition because the cooperative KaiB binding arises from the structure of KaiC.

scholarly journals Synechocystis KaiC3 Displays Temperature- and KaiB-Dependent ATPase Activity and Is Important for Growth in Darkness

2019 ◽  
Vol 202 (4) ◽  
Author(s):  
Anika Wiegard ◽  
Christin Köbler ◽  
Katsuaki Oyama ◽  
Anja K. Dörrich ◽  
Chihiro Azai ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Circadian Clock ◽  
Atp Hydrolysis ◽  
Synechococcus Elongatus ◽  
Constant Darkness ◽  
Content Type ◽  
Clock Proteins ◽  
Pcc 7942 ◽  
Pcc 6803

ABSTRACT Cyanobacteria form a heterogeneous bacterial group with diverse lifestyles, acclimation strategies, and differences in the presence of circadian clock proteins. In Synechococcus elongatus PCC 7942, a unique posttranslational KaiABC oscillator drives circadian rhythms. ATPase activity of KaiC correlates with the period of the clock and mediates temperature compensation. Synechocystis sp. strain PCC 6803 expresses additional Kai proteins, of which KaiB3 and KaiC3 proteins were suggested to fine-tune the standard KaiAB1C1 oscillator. In the present study, we therefore characterized the enzymatic activity of KaiC3 as a representative of nonstandard KaiC homologs in vitro. KaiC3 displayed ATPase activity lower than that of the Synechococcus elongatus PCC 7942 KaiC protein. ATP hydrolysis was temperature dependent. Hence, KaiC3 is missing a defining feature of the model cyanobacterial circadian oscillator. Yeast two-hybrid analysis showed that KaiC3 interacts with KaiB3, KaiC1, and KaiB1. Further, KaiB3 and KaiB1 reduced in vitro ATP hydrolysis by KaiC3. Spot assays showed that chemoheterotrophic growth in constant darkness is completely abolished after deletion of ΔkaiAB1C1 and reduced in the absence of kaiC3. We therefore suggest a role for adaptation to darkness for KaiC3 as well as a cross talk between the KaiC1- and KaiC3-based systems. IMPORTANCE The circadian clock influences the cyanobacterial metabolism, and deeper understanding of its regulation will be important for metabolic optimizations in the context of industrial applications. Due to the heterogeneity of cyanobacteria, characterization of clock systems in organisms apart from the circadian model Synechococcus elongatus PCC 7942 is required. Synechocystis sp. strain PCC 6803 represents a major cyanobacterial model organism and harbors phylogenetically diverged homologs of the clock proteins, which are present in various other noncyanobacterial prokaryotes. By our in vitro studies we unravel the interplay of the multiple Synechocystis Kai proteins and characterize enzymatic activities of the nonstandard clock homolog KaiC3. We show that the deletion of kaiC3 affects growth in constant darkness, suggesting its involvement in the regulation of nonphotosynthetic metabolic pathways.

scholarly journals The ORC/Cdc6/MCM2-7 complex facilitates MCM2-7 dimerization during prereplicative complex formation

2013 ◽  
Vol 42 (4) ◽  
pp. 2257-2269 ◽  
Author(s):  
Cecile Evrin ◽  
Alejandra Fernández-Cid ◽  
Alberto Riera ◽  
Juergen Zech ◽  
Pippa Clarke ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Complex Formation ◽  
Cell Cycle Arrest ◽  
Atp Hydrolysis ◽  
Cycle Arrest ◽  
Helicase Loading ◽  
Limiting Step ◽  
Prereplicative Complex ◽  
Dominant Lethality

Abstract The replicative mini-chromosome-maintenance 2–7 (MCM2-7) helicase is loaded in Saccharomyces cerevisiae and other eukaryotes as a head-to-head double-hexamer around origin DNA. At first, ORC/Cdc6 recruits with the help of Cdt1 a single MCM2-7 hexamer to form an ‘initial’ ORC/Cdc6/Cdt1/MCM2-7 complex. Then, on ATP hydrolysis and Cdt1 release, the ‘initial’ complex is transformed into an ORC/Cdc6/MCM2-7 (OCM) complex. However, it remains unclear how the OCM is subsequently converted into a MCM2-7 double-hexamer. Through analysis of MCM2-7 hexamer-interface mutants we discovered a complex competent for MCM2-7 dimerization. We demonstrate that these MCM2-7 mutants arrest during prereplicative complex (pre-RC) assembly after OCM formation, but before MCM2-7 double-hexamer assembly. Remarkably, only the OCM complex, but not the ‘initial’ ORC/Cdc6/Cdt1/MCM2-7 complex, is competent for MCM2-7 dimerization. The MCM2-7 dimer, in contrast to the MCM2-7 double-hexamer, interacts with ORC/Cdc6 and is salt-sensitive, classifying the arrested complex as a helicase-loading intermediate. Accordingly, we found that overexpression of the mutants cause cell-cycle arrest and dominant lethality. Our work identifies the OCM complex as competent for MCM2-7 dimerization, reveals MCM2-7 dimerization as a limiting step during pre-RC formation and defines critical mechanisms that explain how origins are licensed.

scholarly journals An alternative interpretation of the slow KaiB-KaiC binding of the cyanobacterial clock proteins

2020 ◽  
Author(s):  
Shin-ichi Koda ◽  
Shinji Saito
Keyword(s):  
Circadian Rhythm ◽  
Conformational Change ◽  
Conformational Transition ◽  
Biological Clock ◽  
Alternative Interpretation ◽  
Experimental Results ◽  
Present Scheme ◽  
Long Period ◽  
Clock Proteins ◽  
Hexameric Form

ABSTRACTThe biological clock of cyanobacteria is composed of three proteins, KaiA, KaiB, and KaiC. The KaiB-KaiC binding brings the slowness into the system, which is essential for the long period of the circadian rhythm. However, there is no consensus as to the origin of the slowness due to the pre-binding conformational transition of either KaiB or KaiC. In this study, we propose a simple KaiB-KaiC binding scheme in a hexameric form with an attractive interaction between adjacent bound KaiB monomers, which is independent of KaiB’s conformational change. We then show that the present scheme can explain several important experimental results on the binding, including that used as evidence for the slow conformational transition of KaiB. The present result thus indicates that the slowness arises from KaiC rather than KaiB.

scholarly journals Mechanism of Antiactivation at the Pseudomonas sp. Strain ADP σN-Dependent PatzTPromoter

2016 ◽  
Vol 82 (14) ◽  
pp. 4350-4362 ◽  
Author(s):  
Ana Isabel Platero ◽  
Aroa López-Sánchez ◽  
Laura Tomás-Gallardo ◽  
Eduardo Santero ◽  
Fernando Govantes
Keyword(s):  
Complex Formation ◽  
Promoter Region ◽  
Atp Hydrolysis ◽  
Cyanuric Acid ◽  
Regulatory Protein ◽  
Dna Interaction ◽  
Integration Host Factor ◽  
Open Complex Formation

ABSTRACTPatzTis an internal promoter of theatzRSTUVWoperon that directs the synthesis of AtzT, AtzU, AtzV, and AtzW, components of an ABC-type cyanuric acid transport system. PatzTis σNdependent, activated by the general nitrogen control regulator NtrC with the assistance of protein integration host factor (IHF), and repressed by the LysR-type transcriptional regulator (LTTR) AtzR. We have used a variety ofin vivoandin vitrogene expression and protein-DNA interaction assays to assess the mechanisms underlying AtzR-dependent repression of PatzT. Here, we show that repression only occurs when AtzR and NtrC interact simultaneously with the PatzTpromoter region, indicating that AtzR acts as an antiactivator to antagonize activation by NtrC. Furthermore, repression requires precise rotational orientation of the AtzR and NtrC binding sites, strongly suggesting protein-protein interaction between the two proteins on the promoter region. Further exploration of the antiactivation mechanism showed that although AtzR-dependent repression occurs prior to open complex formation, AtzR does not alter the oligomerization state of NtrC or inhibit NtrC ATPase activity when bound to the PatzTpromoter region. Taken together, these results strongly suggest that PatzT-bound AtzR interacts with NtrC to prevent the coupling of NtrC-mediated ATP hydrolysis with the remodeling of the interactions between E-σNand PatzTthat lead to open complex formation.IMPORTANCEHere, we describe a unique mechanism by which the regulatory protein AtzR prevents the activation of the σN-dependent promoter PatzT. Promoters of this family are always positively regulated, but there are a few examples of overlapping negative regulation. The mechanism described here is highly unconventional and involves an interaction between the repressor and activator proteins to prevent the action of the repressor protein on the RNA polymerase-promoter complex.

scholarly journals The role of ATP hydrolysis in the function of the chaperonin GroEL: dynamic complex formation with GroES

FEBS Letters ◽  
1995 ◽  
Vol 369 (2-3) ◽  
pp. 283-286 ◽  
Author(s):  
Yasushi Kawata ◽  
Kunihiro Hongo ◽  
Koji Nosaka ◽  
Yoshinobu Furutsu ◽  
Tomohiro Mizobata ◽  
...  
Keyword(s):  
Complex Formation ◽  
Atp Hydrolysis ◽  
Chaperonin Groel

scholarly journals Mutant Forms of Salmonella typhimuriumς54 Defective in Transcription Initiation but Not Promoter Binding Activity

1999 ◽  
Vol 181 (11) ◽  
pp. 3351-3357 ◽  
Author(s):  
Mary T. Kelly ◽  
Timothy R. Hoover
Keyword(s):  
Complex Formation ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Atp Hydrolysis ◽  
Random Mutagenesis ◽  
Binding Activity ◽  
Mutant Proteins ◽  
Promoter Dna ◽  
Mutant Forms ◽  
Rna Polymerase Holoenzyme

ABSTRACT Transcription initiation with ς54-RNA polymerase holoenzyme (ς54-holoenzyme) has absolute requirements for an activator protein and ATP hydrolysis. ς54’s binding to core RNA polymerase and promoter DNA has been well studied, but little is known about its role in the subsequent steps of transcription initiation. Following random mutagenesis, we isolated eight mutant forms of Salmonella typhimurium ς54 that were deficient in transcription initiation but still directed ς54-holoenzyme to the promoter to form a closed complex. Four of these mutant proteins had amino acid substitutions in region I, which had been shown previously to be required for ς54-holoenzyme to respond to the activator. From the remaining mutants, we identified four residues in region III which when altered affect the function of ς54 at some point after closed-complex formation. These results suggest that in addition to its role in core and DNA binding, region III participates in one or more steps of transcription initiation that follow closed-complex formation.

Repressor Forms of the Enhancer-binding Protein NtrC: Some Fail in Coupling ATP Hydrolysis to Open Complex Formation by σ54-Holoenzyme

1996 ◽  
Vol 260 (3) ◽  
pp. 317-331 ◽  
Author(s):  
Anne K. North ◽  
David S. Weiss ◽  
Hideyuki Suzuki ◽  
Yehuda Flashner ◽  
Sydney Kustu
Keyword(s):  
Complex Formation ◽  
Atp Hydrolysis ◽  
Binding Protein ◽  
Enhancer Binding Protein ◽  
Open Complex Formation

scholarly journals Attempts to characterize the NBD heterodimer of MRP1: transient complex formation involves Gly771 of the ABC signature sequence but does not enhance the intrinsic ATPase activity

2005 ◽  
Vol 391 (3) ◽  
pp. 481-490 ◽  
Author(s):  
Odile Ramaen ◽  
Christina Sizun ◽  
Olivier Pamlard ◽  
Eric Jacquet ◽  
Jean-Yves Lallemand
Keyword(s):  
Complex Formation ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Chemotherapeutic Agents ◽  
Activation Mechanism ◽  
Hydrolysis Rate ◽  
Sequential Assignment ◽  
Atp Binding ◽  
Native Page ◽  
Transient Complex

MRP1 (multidrug-resistance-associated protein 1; also known as ABCC1) is a member of the human ABC (ATP-binding cassette) transporter superfamily that confers cell resistance to chemotherapeutic agents. Considering the structural and functional similarities to the other ABC proteins, the interaction between its two NBDs (nucleotide-binding domains), NBD1 (N-terminal NBD) and NBD2 (C-terminal NBD), is proposed to be essential for the regulation of the ATP-binding/ATP-hydrolysis cycle of MRP1. We were interested in the ability of recombinant NBD1 and NBD2 to interact with each other and to influence ATPase activity. We purified NBD1 (Asn642–Ser871) and NBD2 (Ser1286–Val1531) as soluble monomers under native conditions. We measured extremely low intrinsic ATPase activity of NBD1 (10−5 s−1) and NBD2 (6×10−6 s−1) and no increase in the ATP-hydrolysis rate could be detected in an NBD1+NBD2 mixture, with concentrations up to 200 μM. Despite the fact that both monomers bind ATP, no stable NBD1·NBD2 heterodimer could be isolated by gel-filtration chromatography or native-PAGE, but we observed some significant modifications of the heteronuclear single-quantum correlation NMR spectrum of 15N-NBD1 in the presence of NBD2. This apparent NBD1·NBD2 interaction only occurred in the presence of Mg2+ and ATP. Partial sequential assignment of the NBD1 backbone resonances shows that residue Gly771 of the LSGGQ sequence is involved in NBD1·NBD2 complex formation. This is the first NMR observation of a direct interaction between the ABC signature and the opposite NBD. Our study also reveals that the NBD1·NBD2 heterodimer of MRP1 is a transient complex. This labile interaction is not sufficient to induce an ATPase co-operativity of the NBDs and suggests that other structures are required for the ATPase activation mechanism.

scholarly journals ATP-Dependent Simian Virus 40 T-Antigen–Hsc70 Complex Formation

2001 ◽  
Vol 75 (4) ◽  
pp. 1601-1610 ◽  
Author(s):  
Christopher S. Sullivan ◽  
Susan P. Gilbert ◽  
James M. Pipas
Keyword(s):  
Complex Formation ◽  
Dissociation Constant ◽  
Atp Hydrolysis ◽  
Biological Activities ◽  
Simian Virus 40 ◽  
Cellular Transformation ◽  
Simian Virus ◽  
T Antigen ◽  
Amino Terminal ◽  
J Domain

ABSTRACT Simian virus 40 large T antigen is a multifunctional oncoprotein that is required for numerous viral functions and the induction of cellular transformation. T antigen contains a J domain that is required for many of its activities including viral DNA replication, transformation, and virion assembly. J-domain-containing proteins interact with Hsc70 (a cellular chaperone) to perform multiple biological activities, usually involving a change in the conformation of target substrates. It is thought that Hsc70 associates with T antigen to assist in performing its numerous activities. However, it is not clear if T antigen binds to Hsc70 directly or induces the binding of Hsc70 to other T-antigen binding proteins such as pRb or p53. In this report, we show that T antigen binds Hsc70 directly with a stoichiometry of 1:1 (dissociation constant = 310 nM Hsc70). Furthermore, the T-antigen–Hsc70 complex formation is dependent upon ATP hydrolysis at the active site of Hsc70 (ATP dissociation constant = 0.16 μM), but T-antigen–Hsc70 complex formation does not require nucleotide hydrolysis at the T-antigen ATP binding site. N136, a J domain-containing fragment of T antigen, does not stably associate with Hsc70 but can form a transient complex as assayed by centrifugation analysis. Finally, T antigen does not associate stably with either of two yeast Hsc70 homologues or an amino-terminal fragment of Hsc70 containing the ATPase domain. These results provide direct evidence that the T-antigen–Hsc70 interaction is specific and that this association requires multiple domains of both T antigen and Hsc70. This is the first demonstration of a nucleotide requirement for the association of T antigen and Hsc70 and lays the foundation for future reconstitution studies of chaperone-dependent tumorigenesis induced by T antigen.

scholarly journals Protein folding in mitochondria requires complex formation with hsp60 and ATP hydrolysis

Nature ◽  
1989 ◽  
Vol 341 (6238) ◽  
pp. 125-130 ◽  
Author(s):  
Joachim Ostermann ◽  
Arthur L. Horwich ◽  
Walter Neupert ◽  
F.-Ulrich Hartl
Keyword(s):  
Protein Folding ◽  
Complex Formation ◽  
Atp Hydrolysis
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