Structure-function relationship of KaiC around dawn.

KaiC is a multifunctional enzyme functioning as the core of the circadian clock system in cyanobacteria: its N-terminal domain has adenosine triphosphatase (ATPase) activity, and its C-terminal domain has autokinase and autophosphatase activities targeting own S431 and T432. The coordination of these multiple biochemical activities is the molecular basis for robust circadian rhythmicity. Therefore, much effort has been devoted to elucidating the cooperative relationship between the two domains. However, structural and functional relationships between the two domains remain unclear especially with respect to the dawn phase, at which KaiC relieves its nocturnal history through autodephosphorylation. In this study, we attempted to design a double mutation of S431 and T432 that can capture KaiC as a fully dephosphorylated form with minimal impacts on its structure and function, and investigated the cooperative relationship between the two domains in the night to morning phases from many perspectives. The results revealed that both domains cooperate at the dawn phase through salt bridges formed between the domains, thereby non-locally co-activating two events, ATPase de-inhibition and S431 dephosphorylation. Our further analysis using existing crystal structures of KaiC suggests that the states of both domains are not always in one-to-one correspondence at every phase of the circadian cycle, and their coupling is affected by the interactions with KaiA or adjacent subunits within a KaiC hexamer.

Determination of the three-dimensional structure of bacteriophage Mu(-) tail fiber and its characterization.

Bacteriophage Mu is a temperate phage known to infect various species of Enterobacteria, playing a role in bacterial mutation induction and horizontal gene transfer. The phage possesses two types of tail fibers important for host recognition, which enable it to expand its range of hosts. The alternate tail fibers are formed through the action of genes 49-50 or 52-51, allowing the Mu phage to recognize different surfaces of host cells. In a previous study, we presented the X-ray crystal structure of the C-terminal lipopolysaccharide (LPS)-binding domain of gene product (gp) 49, one of the subunits comprising the Mu tail fiber. In this study, we have determined the structure of the alternative tail fiber subunit, gp52, and compared it with other tail fibers. The results revealed that Mu phage employs different structural motifs for two individual tail fibers for recognizing different hosts.

Crystallographic cyanide-probing for cytochrome c oxidase reveals structural bases suggesting that a putative proton transfer H-pathway pumps protons.

Cytochrome c oxidase (CcO) reduces O2 in the O2-reduction site by sequential four-electron donations through the low-potential metal sites (CuA and Fea). Redox-coupled X-ray crystal structural changes have been identified at five distinct sites including Asp51, Arg438, Glu198, the hydroxyfarnesyl ethyl group of heme a, and Ser382, respectively. These sites interact with the putative proton-pumping H-pathway. However, the metal sites responsible for each structural change have not been identified, since these changes were detected as structural differences between the fully reduced and fully oxidized CcOs. Thus, the roles of these structural changes in the CcO function are yet to be revealed. X-ray crystal structures of cyanide-bound CcOs under various oxidation states showed that the O2-reduction site controlled only the Ser382-including site, while the low-potential metal sites induced the other changes. This finding indicates that these low-potential site-inducible structural changes are triggered by sequential electron-extraction from the low-potential sites by the O2-reduction site and that each structural change is insensitive to the oxidation and ligand-binding states of the O2-reduction site. Because the proton/electron coupling efficiency is constant (1:1), regardless of the reaction progress in the O2-reduction site, the structural changes induced by the low-potential sites are assignable to those critically involved in the proton pumping, suggesting that the H-pathway, facilitating these low-potential site-inducible structural changes, pumps protons. Furthermore, a cyanide-bound CcO structure suggests that a hypoxia-inducible activator, Higd1a, activates the O2-reduction site without influencing the electron transfer mechanism through the low-potential sites, kinetically confirming that the low-potential sites facilitate proton pump.

Crystal structures of multidrug efflux transporters from Burkholderia pseudomallei suggest details of transport mechanism.

BpeB and BpeF are multidrug efflux transporters from Burkholderia pseudomallei that enable multidrug resistance. Here, we report the crystal structures of BpeB and BpeF at 2.94 Å and 3.0 Å resolution, respectively. BpeB was found as an asymmetric trimer, consistent with the widely-accepted functional rotation mechanism for this type of transporter. One of the monomers has a distinct structure that we interpret as an intermediate along this functional cycle. Additionally, a detergent molecule bound in a previously undescribed binding site provides insights into substrate translocation through the pathway. BpeF shares structural similarities with the crystal structure of OqxB from Klebsiella pneumoniae, where both are symmetric trimers composed of three "binding"-state monomers. The structures of BpeB and BpeF further our understanding of the functional mechanisms of transporters belonging to the HAE1-RND superfamily.

Multifunctional chemical inhibitors of the florigen activation complex discovered by structure-based high-throughput screening.

Structure-based high-throughput screening of chemical compounds that target protein-protein interactions (PPIs) is a promising technology for gaining insight into how plant development is regulated, leading to many potential agricultural applications. At present, there are no examples of using high-throughput screening to identify chemicals that target plant transcriptional complexes, some of which are responsible for regulating multiple physiological functions. Florigen, a protein encoded by FLOWERING LOCUS T (FT), was initially identified as a molecule that promotes flowering and has since been shown to regulate flowering and other developmental phenomena such as tuber formation in potato (Solanum tuberosum). FT functions as a component of the florigen activation complex (FAC) with a 14-3-3 scaffold protein and FD, a bZIP transcription factor that activates downstream gene expression. Although 14-3-3 is an important component of FAC, little is known about the function of the 14-3-3 protein itself. Here, we report the results of a high-throughput in vitro fluorescence resonance energy transfer (FRET) screening of chemical libraries that enabled us to identify small molecules capable of inhibiting FAC formation. These molecules abrogate the in vitro interaction between the 14-3-3 protein and the OsFD1 peptide, a rice (Oryza sativa) FD, by directly binding to the 14-3-3 protein. Treatment with S4, a specific hit molecule, strongly inhibited FAC activity and flowering in duckweed, tuber formation in potato, and branching in rice in a dose-dependent manner. Our results demonstrate that the high-throughput screening approach based on the three-dimensional structure of PPIs is suitable in plants. In this study, we have proposed good candidate compounds for future modification to obtain inhibitors of florigen-dependent processes through inhibition of FAC formation.

Crystal structure of potato 14-3-3 protein St14f revealed the importance of helix I in StFDL1 recognition.

In potato (Solanum tuberosum L.), 14-3-3 protein forms a protein complex with the FLOWERING LOCUS T (FT)-like protein StSP6A and the FD-like protein StFDL1 to activate potato tuber formation. Eleven 14-3-3 isoforms were reported in potato, designated as St14a-k. In this study, the crystal structure of the free form of St14f was determined at 2.5 Å resolution. Three chains were included in the asymmetric unit of the St14f free form crystal, and the structural deviation among the three chain structures was found on the C-terminal helix H and I. The St14f free form structure in solution was also investigated by nuclear magnetic resonance (NMR) residual dipolar coupling analysis, and the chain B in the crystal structure was consistent with NMR data. Compared to other crystal structures, St14f helix I exhibited a different conformation with larger B-factor values. Larger B-factor values on helix I were also found in the 14-3-3 free form structure with higher solvent contents. The mutation in St14f Helix I stabilized the complex with StFDL1. These data clearly showed that the flexibility of helix I of 14-3-3 protein plays an important role in the recognition of target protein.

Highly sensitive tryptophan fluorescence probe for detecting rhythmic conformational changes of KaiC in the cyanobacterial circadian clock system.

KaiC, a core protein of the cyanobacterial circadian clock, consists of an N-terminal CI domain and a C-terminal CII domain, and assembles into a double-ring hexamer upon binding with ATP. KaiC rhythmically phosphorylates and dephosphorylates its own two adjacent residues Ser431 and Thr432 at the CII domain with a period of ∼24 h through assembly and disassembly with the other clock proteins, KaiA and/or KaiB. In this study, to understand how KaiC alters its conformation as the source of circadian rhythm, we investigated structural changes of an inner-radius side of the CII ring using time-resolved Trp fluorescence spectroscopy. A KaiC mutant harboring a Trp fluorescence probe at a position of 419 exhibited a robust circadian rhythm with little temperature sensitivity in the presence of KaiA and KaiB. Our fluorescence observations show a remarkable environmental change at the inner-radius side of the CII ring during circadian oscillation. Crystallographic analysis revealed that a side chain of Trp at the position of 419 was oriented toward a region undergoing a helix-coil transition, which is considered to be a key event to allosterically regulate the CI ring that plays a crucial role in determining the cycle period. The present study provides a dynamical insight into how KaiC generates circadian oscillation.

Cooperative regulation of PBI1 and MAPKs controls WRKY45 transcription factor in rice immunity.

The U-box type ubiquitin ligase PUB44 positively regulates pattern-triggered immunity in rice. Here, we identify PBI1, a protein that interacts with PUB44. Crystal structure analysis indicates that PBI1 forms a four-helix bundle structure. PBI1 also interacts with WRKY45, a master transcriptional activator of rice immunity, and negatively regulates its activity. PBI1 is degraded upon perception of chitin, and this is suppressed by silencing of PUB44 or expression of XopP, indicating that PBI1 degradation depends on PUB44. These data suggest that PBI1 suppresses WRKY45 activity when cells are in an unelicited state, and during chitin signaling, PUB44-mediated degradation of PBI1 leads to activation of WRKY45. In addition, chitin-induced MAP kinase activation is required for WRKY45 activation and PBI1 degradation. These results demonstrate that chitin-induced activation of WRKY45 is regulated by the cooperation between MAP kinase-mediated phosphorylation and PUB44-mediated PBI1 degradation.

Regulation mechanisms of the dual ATPase in KaiC.

KaiC is a dual adenosine triphosphatase (ATPase), with one active site in its N-terminal domain and another in its C-terminal domain, that drives the circadian clock system of cyanobacteria through sophisticated coordination of the two sites. To elucidate the coordination mechanism, we studied the contribution of the dual-ATPase activities in the ring-shaped KaiC hexamer and these structural bases for activation and inactivation. At the N-terminal active site, a lytic water molecule is sequestered between the N-terminal domains, and its reactivity to adenosine triphosphate (ATP) is controlled by the quaternary structure of the N-terminal ring. The C-terminal ATPase activity is regulated mostly by water-incorporating voids between the C-terminal domains, and the size of these voids is sensitive to phosphoryl modification of S431. The up-regulatory effect on the N-terminal ATPase activity inversely correlates with the affinity of KaiC for KaiB, a clock protein constitutes the circadian oscillator together with KaiC and KaiA, and the complete dissociation of KaiB from KaiC requires KaiA-assisted activation of the dual ATPase. Delicate interactions between the N-terminal and C-terminal rings make it possible for the components of the dual ATPase to work together, thereby driving the assembly and disassembly cycle of KaiA and KaiB.

Theoretical Model of the Far-Red-Light-Adapted Photosystem I Reaction Center of Cyanobacterium Acaryochloris marina Using Chlorophyll d and the Effect of Chlorophyll Exchange.

A theoretical model of the far-red-light-adapted photosystem I (PSI) reaction center (RC) complex of a cyanobacterium, Acaryochloris marina (AmPSI), was constructed based on the exciton theory and the recently identified molecular structure of AmPSI by Hamaguchi et al. (Nat. Commun., 2021, 12, 2333). A. marina performs photosynthesis under the visible to far-red light (400-750 nm), which is absorbed by chlorophyll d (Chl-d). It is in contrast to the situation of all the other oxygenic photosynthetic processes of cyanobacteria and plants, which contains chlorophyll a (Chl-a) that absorbs only 400-700 nm visible light. AmPSI contains 70 Chl-d, 1 Chl-d', 2 pheophytin a (Pheo-a), and 12 carotenoids in the currently available structure. A special pair of Chl-d/Chl-d' acts as the electron donor (P740) and two Pheo-a act as the primary electron acceptor A0 as the counterparts of P700 and Chl-a, respectively, of Chl-a-type PSIs. The exciton Hamiltonian of AmPSI was constructed considering the excitonic coupling strength and site energy shift of individual pigments using the Poisson-TrESP (P-TrESP) and charge density coupling (CDC) methods. The model was constructed to fit the experimentally measured spectra of absorption and circular dichroism (CD) spectra during downhill/uphill excitation energy transfer processes. The constructed theoretical model of AmPSI was further compared with the Chl-a-type PSI of Thermosynechococcus elongatus (TePSI), which contains only Chl-a and Chl-a'. The functional properties of AmPSI and TePSI were further examined by the in silico exchange of Chl-d by Chl-a in the models.

Elucidation of master allostery essential for circadian clock oscillation in cyanobacteria.

Spatiotemporal allostery is the source of complex but ordered biological phenomena. To identify the structural basis for allostery that drives the cyanobacterial circadian clock, we crystallized the clock protein KaiC in four distinct states, which cover a whole cycle of phosphor-transfer events at Ser431 and Thr432. The minimal set of allosteric events required for oscillatory nature is a bidirectional coupling between the coil-to-helix transition of the Ser431-dependent phospho-switch in the C-terminal domain of KaiC and adenosine 5'-diphosphate release from its N-terminal domain during adenosine triphosphatase cycle. An engineered KaiC protein oscillator consisting of a minimal set of the identified master allosteric events exhibited a monophosphorylation cycle of Ser431 with a temperature-compensated circadian period, providing design principles for simple posttranslational biochemical circadian oscillators.

Structure and function relationship of OqxB efflux pump from Klebsiella pneumoniae.

OqxB is an RND (Resistance-Nodulation-Division) efflux pump that has emerged as a factor contributing to the antibiotic resistance in Klebsiella pneumoniae. OqxB underwent horizontal gene transfer and is now seen in other Gram-negative bacterial pathogens including Escherichia coli, Enterobacter cloacae and Salmonella spp., further disseminating multi-drug resistance. In this study, we describe crystal structure of OqxB with n-dodecyl-ß-D-maltoside (DDM) molecules bound in its substrate-binding pocket, at 1.85 Å resolution. We utilize this structure in computational studies to predict the key amino acids contributing to the efflux of fluoroquinolones by OqxB, distinct from analogous residues in related transporters AcrB and MexB. Finally, our complementation assays with mutated OqxB and minimum inhibitory concentration (MIC) experiments with clinical isolates of E. coli provide further evidence that the predicted structural features are indeed involved in ciprofloxacin efflux.

Critical roles of the CuB site in efficient proton pumping as revealed by crystal structures of mammalian cytochrome c oxidase catalytic intermediates.

Mammalian cytochrome c oxidase (CcO) reduces O2 to water in a bimetallic site including Fea3 and CuB giving intermediate molecules, termed A-, P-, F-, O-, E-, and R-forms. From the P-form on, each reaction step is driven by single-electron donations from cytochrome c coupled with the pumping of a single proton through the H-pathway, a proton-conducting pathway composed of a hydrogen-bond network and a water channel. The proton-gradient formed is utilized for ATP production by F-ATPase. For elucidation of the proton pumping mechanism, crystal structural determination of these intermediate forms is necessary. Here we report X-ray crystallographic analysis at ∼1.8 Å resolution of fully reduced CcO crystals treated with O2 for three different time periods. Our disentanglement of intermediate forms from crystals that were composed of multiple forms determined that these three crystallographic data sets contained ∼45% of the O-form structure, ∼45% of the E-form structure, and ∼20% of an oxymyoglobin-type structure consistent with the A-form, respectively. The O- and E-forms exhibit an unusually long CuB2+-OH- distance and CuB1+-H2O structure keeping Fea33+-OH- state, respectively, suggesting that the O- and E-forms have high electron affinities that cause the O→E and E→R transitions to be essentially irreversible and thus enable tightly coupled proton pumping. The water channel of the H-pathway is closed in the O- and E-forms and partially open in the R-form. These structures, together with those of the recently reported P- and F-forms, indicate that closure of the H-pathway water channel avoids back-leaking of protons for facilitating the effective proton pumping.

Structure of the far-red light utilizing photosystem I of Acaryochloris marina.

Acaryochloris marina is one of the cyanobacterial species that can use far-red light to drive photochemical reactions for oxygenic photosynthesis. Here, we report the structure of A. marina photosystem I (PSI) reaction center, determined by cryo-electron microscopy at 2.58 Å resolution. The structure reveals an arrangement of electron carriers and light-harvesting pigments distinct from other type I reaction centers. The paired chlorophyll, or special pair (also referred to as P740 in this case), is a dimer of chlorophyll d and its epimer chlorophyll d'. The primary electron acceptor is pheophytin a, a metal-less chlorin. We show the architecture of this PSI reaction center is composed of 11 subunits and we identify key components that help explain how the low energy yield from far-red light is efficiently utilized for driving oxygenic photosynthesis.

The 1.3-Å resolution structure of bovine cytochrome c oxidase suggests a dimerization mechanism.

Cytochrome c oxidase (CcO) in the respiratory chain catalyzes oxygen reduction by coupling electron and proton transfer through the enzyme and proton pumping across the membrane. Although the functional unit of CcO is monomeric, mitochondrial CcO forms a monomer and a dimer, as well as a supercomplex with respiratory complexes I and III. A recent study showed that dimeric CcO has lower activity than monomeric CcO and proposed that dimeric CcO is a standby form for enzymatic activation in the mitochondrial membrane. Other studies have suggested that the dimerization is dependent on specifically arranged lipid molecules, peptide segments, and post-translationally modified amino acid residues. To re-examine the structural basis of dimerization, we improved the resolution of the crystallographic structure to 1.3 Å by optimizing the method for cryoprotectant soaking. The observed electron density map revealed many weakly bound detergent and lipid molecules at the interface of the dimer. The dimer interface also contained hydrogen bonds with tightly bound cholate molecules, hydrophobic interactions between the transmembrane helices, and a Met-Met interaction between the extramembrane regions. These results imply that binding of physiological ligands structurally similar to cholate could trigger dimerization in the mitochondrial membrane and that non-specifically bound lipid molecules at the transmembrane surface between monomers support the stabilization of the dimer. The weak interactions involving the transmembrane helices and extramembrane regions may play a role in positioning each monomer at the correct orientation in the dimer.

X-ray structures of catalytic intermediates of cytochrome c oxidase provide insights into its O2 activation and unidirectional proton-pump mechanisms.

Cytochrome c oxidase (CcO) reduces O2 to water, coupled with a proton-pumping process. The structure of the O2-reduction site of CcO contains two reducing equivalents, Fe a32+ and CuB1+, and suggests that a peroxide-bound state (Fe a33+-O--O--CuB2+) rather than an O2-bound state (Fe a32+-O2) is the initial catalytic intermediate. Unexpectedly, however, resonance Raman spectroscopy results have shown that the initial intermediate is Fe a32+-O2, whereas Fe a33+-O--O--CuB2+ is undetectable. Based on X-ray structures of static noncatalytic CcO forms and mutation analyses for bovine CcO, a proton-pumping mechanism has been proposed. It involves a proton-conducting pathway (the H-pathway) comprising a tandem hydrogen-bond network and a water channel located between the N- and P-side surfaces. However, a system for unidirectional proton-transport has not been experimentally identified. Here, an essentially identical X-ray structure for the two catalytic intermediates (P and F) of bovine CcO was determined at 1.8 Šresolution. A 1.70 ŠFe-O distance of the ferryl center could best be described as Fe a34+ = O2-, not as Fe a34+-OH- The distance suggests an ∼800-cm-1 Raman stretching band. We found an interstitial water molecule that could trigger a rapid proton-coupled electron transfer from tyrosine-OH to the slowly forming Fe a33+-O--O--CuB2+ state, preventing its detection, consistent with the unexpected Raman results. The H-pathway structures of both intermediates indicated that during proton-pumping from the hydrogen-bond network to the P-side, a transmembrane helix closes the water channel connecting the N-side with the hydrogen-bond network, facilitating unidirectional proton-pumping during the P-to-F transition.

Author Correction: Conformational rearrangements of the C1 ring in KaiC measure the timing of assembly with KaiB.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Monomeric structure of an active form of bovine cytochrome c oxidase.

Cytochrome c oxidase (CcO), a membrane enzyme in the respiratory chain, catalyzes oxygen reduction by coupling electron and proton transfer through the enzyme with a proton pump across the membrane. In all crystals reported to date, bovine CcO exists as a dimer with the same intermonomer contacts, whereas CcOs and related enzymes from prokaryotes exist as monomers. Recent structural analyses of the mitochondrial respiratory supercomplex revealed that CcO monomer associates with complex I and complex III, indicating that the monomeric state is functionally important. In this study, we prepared monomeric and dimeric bovine CcO, stabilized using amphipol, and showed that the monomer had high activity. In addition, using a newly synthesized detergent, we determined the oxidized and reduced structures of monomer with resolutions of 1.85 and 1.95 Å, respectively. Structural comparison of the monomer and dimer revealed that a hydrogen bond network of water molecules is formed at the entry surface of the proton transfer pathway, termed the K-pathway, in monomeric CcO, whereas this network is altered in dimeric CcO. Based on these results, we propose that the monomer is the activated form, whereas the dimer can be regarded as a physiological standby form in the mitochondrial membrane. We also determined phospholipid structures based on electron density together with the anomalous scattering effect of phosphorus atoms. Two cardiolipins are found at the interface region of the supercomplex. We discuss formation of the monomeric CcO, dimeric CcO, and supercomplex, as well as their role in regulation of CcO activity.

Observation of unexpected molecular binding activity for Mu phage tail fibre chaperones.

In the history of viral research, one of the important biological features of bacteriophage Mu is the ability to expand its host range. For extending the host range, the Mu phage encodes two alternate tail fibre genes. Classical amber mutation experiments and genome sequence analysis of Mu phage suggested that gene products (gp) of geneS (gpS = gp49) and gene S' (gpS' = gp52) are tail fibres and that gene products of geneU (gpU = gp50) and geneU' (gpU' = gp51) work for tail fibre assembly or tail fibre chaperones. Depending on the gene orientation, a pair of genes 49-50 or 52-51 is expressed for producing different tail fibres that enable Mu phage to recognize different host cell surface. Since several fibrous proteins including some phage tail fibres employ their specific chaperone to facilitate folding and prevent aggregation, we expected that gp50 or gp51 would be a specific chaperone for gp49 and gp52, respectively. However, heterologous overexpression results for gp49 or gp52 (tail fibre subunit) together with gp51 and gp50, respectively, were also effective in producing soluble Mu tail fibres. Moreover, we successfully purified non-native gp49-gp51 and gp52-gp50 complexes. These facts showed that gp50 and gp51 were fungible and functional for both gp49 and gp52 each other.