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

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.

Antagonistic control of DDK binding to licensed replication origins by Mcm2 and Rad53

Eukaryotic replication origins are licensed by the loading of the replicative DNA helicase, Mcm2-7, in inactive double hexameric form around DNA. Subsequent origin activation is under control of multiple protein kinases that either promote or inhibit origin activation, which is important for genome maintenance. Using the reconstituted budding yeast DNA replication system, we find that the flexible N-terminal extension (NTE) of Mcm2 promotes the stable recruitment of Dbf4-dependent kinase (DDK) to Mcm2-7 double hexamers, which in turn promotes DDK phosphorylation of Mcm4 and −6 and subsequent origin activation. Conversely, we demonstrate that the checkpoint kinase, Rad53, inhibits DDK binding to Mcm2-7 double hexamers. Unexpectedly, this function is not dependent on Rad53 kinase activity, suggesting steric inhibition of DDK by activated Rad53. These findings identify critical determinants of the origin activation reaction and uncover a novel mechanism for checkpoint-dependent origin inhibition.

Antagonistic control of DDK binding to licensed replication origins by Mcm2 and Rad53

ABSTRACTEukaryotic replication origins are licensed by the loading of the replicative DNA helicase, Mcm2-7, in inactive double hexameric form around DNA. Subsequent origin activation is under control of multiple protein kinases that either promote or inhibit origin activation, which is important for genome maintenance. Using the reconstituted budding yeast DNA replication system, we find that the flexible N-terminal tail of Mcm2 promotes the stable recruitment of Dbf4-dependent kinase (DDK) to Mcm2-7 double hexamers, which in turn promotes DDK phosphorylation of Mcm4 and -6 and subsequent origin activation. Conversely, we demonstrate that the checkpoint kinase, Rad53, inhibits DDK binding to Mcm2-7 double hexamers. Unexpectedly, this function is not dependent on Rad53 kinase activity, but requires Rad53 activation by trans-autophosphorylation, suggesting steric inhibition of DDK by activated Rad53. These findings identify critical determinants of the origin activation reaction and uncover a novel mechanism for checkpoint-dependent origin inhibition.

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

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.

The Subcellular Localization and Oligomerization Preferences of NME1/NME2 upon Radiation-Induced DNA Damage

Nucleoside diphosphate kinases (NDPK/NME/Nm23) are enzymes composed of subunits NME1/NDPK A and NME2/NDPK B, responsible for the maintenance of the cellular (d)NTP pool and involved in other cellular processes, such as metastasis suppression and DNA damage repair. Although eukaryotic NDPKs are active only as hexamers, it is unclear whether other NME functions require the hexameric form, and how the isoenzyme composition varies in different cellular compartments. To examine the effect of DNA damage on intracellular localization of NME1 and NME2 and the composition of NME oligomers in the nucleus and the cytoplasm, we used live-cell imaging and the FRET/FLIM technique. We showed that exogenous NME1 and NME2 proteins co-localize in the cytoplasm of non-irradiated cells, and move simultaneously to the nucleus after gamma irradiation. The FRET/FLIM experiments imply that, after DNA damage, there is a slight shift in the homomer/heteromer balance between the nucleus and the cytoplasm. Collectively, our results indicate that, after irradiation, NME1 and NME2 engage in mutual functions in the nucleus, possibly performing specific functions in their homomeric states. Finally, we demonstrated that fluorophores fused to the N-termini of NME polypeptides produce the largest FRET effect and thus recommend this orientation for use in similar studies.

Phosphatidic acid inhibits SNARE priming by inducing conformational changes in Sec18 protomers

SUMMARYEukaryotic homeostasis relies on membrane fusion catalyzed by SNARE proteins. Inactive SNARE bundles are re-activated by Sec18/NSF driven disassembly to enable a new round of fusion. We previously found that phosphatidic acid (PA) binds Sec18 to sequester it from SNAREs. Dephosphorylation of PA dissociates Sec18 from the membrane allowing it to engage SNARE complexes. We now report that PA induces conformational changes in Sec18 protomers, while hexameric Sec18 cannot bind PA membranes. The association of Sec18 with PA was shown to be sensitive to membrane curvature, suggesting that regulation could vary on different organelles in a curvature dependent manner. Molecular dynamics showed that PA binding sites exist on the D1 and D2 domains of Sec18 and that residues needed for binding were masked in the hexameric form of the protein. Together these data indicate that PA regulates Sec18 function through altering protein architecture and stabilizing membrane-bound protomers.

The structure of the hexameric atrazine chlorohydrolase AtzA

Atrazine chlorohydrolase (AtzA) was discovered and purified in the early 1990s from soil that had been exposed to the widely used herbicide atrazine. It was subsequently found that this enzyme catalyzes the first and necessary step in the breakdown of atrazine by the soil organismPseudomonassp. strain ADP. Although it has taken 20 years, a crystal structure of the full hexameric form of AtzA has now been obtained. AtzA is less well adapted to its physiological role (i.e.atrazine dechlorination) than the alternative metal-dependent atrazine chlorohydrolase (TrzN), with a substrate-binding pocket that is under considerable strain and for which the substrate is a poor fit.

HIV-1 capsid binds host cofactors to mediate nuclear import

The capsid (CA) protein of HIV-1, which forms the core of the virus, has been shown to have an increasingly important role in the early stages of the virus lifecycle, in particular during reverse transcription and nuclear import. We recently solved the structure of a fragment of the human cofactor CPSF6 in complex with the N-terminal domain of HIV-1 CA, revealing a previously unknown interface used by the virus to recruit CPSF6, which is required for the virus to successfully complete the early stages of its lifecycle. Using a recently developed hexameric unit of CA, we have solved the structure of the CPSF6 peptide with CA in a context that more closely resembles an intact CA lattice. This has revealed that CPSF6 contacts HIV-1 CA using an additional second site only present in the hexameric form of CA. Furthermore, we have now solved the structure of a fragment of NUP153 (an HIV-1 cofactor that is integral to the nuclear pore) in complex with hexameric CA and discovered that this also forms contacts specific to hexameric CA. Moreover, the binding sites for CPSF6 and NUP153 on CA overlap at one crucial residue, which is remarkably mimicked by two drugs independently discovered to bind at this same site. Together, these data provide evidence for an essential role for CA in HIV-1 infection, and highlights CA as an important target for antiretroviral drugs.