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Deficiencies in mitochondrial protein import are associated with a number of diseases. However, although nonimported mitochondrial proteins are at great risk of aggregation, it remains largely unclear how their accumulation causes cell dysfunction. Here, we show that nonimported citrate synthase is targeted for proteasomal degradation by the ubiquitin ligase SCF Ucc1 . Unexpectedly, our structural and genetic analyses revealed that nonimported citrate synthase appears to form an enzymatically active conformation in the cytosol. Its excess accumulation caused ectopic citrate synthesis, which, in turn, led to an imbalance in carbon flux of sugar, a reduction of the pool of amino acids and nucleotides, and a growth defect. Under these conditions, translation repression is induced and acts as a protective mechanism that mitigates the growth defect. We propose that the consequence of mitochondrial import failure is not limited to proteotoxic insults, but that the accumulation of a nonimported metabolic enzyme elicits ectopic metabolic stress.

Citrate synthase (CS) catalyzes the conversion of oxaloacetate and acetyl coenzyme A into citrate and coenzyme A in the mitochondrial tricarboxylic acid (TCA) cycle. In plants, mitochondrial metabolism, including the TCA cycle, occurs in interaction with photosynthetic metabolism. The controlled regulation of several enzymes in the TCA cycle, such as CS, is important in plants. Here, the first crystal structure of a plant mitochondrial CS, CSY4 from Arabidopsis thaliana (AtCSY4), has been determined. Structural comparison of AtCSY4 with mitochondrial CSs revealed a high level of similarity. Inhibition analysis showed a similar manner of inhibition as in mitochondrial CSs. The effect of oxidation on one of a pair of cysteine residues in AtCSY4 was speculated upon based on the folded structure.

Real-time traffic signal information has recently become available thanks to further developments in Intelligent Transportation System (ITS) technology. Drivers may change their driving behavior by receiving information on the status of upcoming traffic signals. The behavior of a driver who reacts to such information with the purpose of smoothly passing through an upcoming signalized intersection is described as reinforcement learning in this study. The influence of reactive drivers on traffic flows is analyzed by a multi-agent traffic flow simulation on an arterial road composed of four signalized intersections. Furthermore, the relationship between the ratio of reactive drivers and traffic flows is clarified, under the assumption that there is a mixture of reactive and nonreactive drivers on the road. The results of the multi-agent traffic flow simulation showed that the average stop time decreased as the ratio of reactive drivers increased. Moreover, the threshold of the ratio of reactive drivers to begin to influence largely vehicle stop situations was around 50%. The average travel time was almost constant regardless of the ratio of reactive drivers. It was also demonstrated that reacting to the traffic signal information and decelerating accordingly did not cause time delays. This suggests that the provision of the traffic signal information and appropriate reaction to the provided information may help reduce the amount of CO2 emissions from a vehicle approaching a signalized intersection and probably contribute to alleviating progress of global warming.

The SCF ubiquitin ligase comprises four components: Skp1, Cul1, Rbx1 and a variable-subunit F-box protein. The F-box protein Fbs1, which recognizes the N-linked glycoproteins, is involved in the endoplasmic reticulum-associated degradation pathway. Although FBG3, another F-box protein, shares 51% sequence identity with Fbs1, FBG3 does not bind glycoproteins. To investigate the sequence–structure relationship of the substrate-binding pocket, the crystal structure of a mutant substrate-binding domain of Fbs1 in which the six nonconserved regions (β1, β2–β3, β3–β4, β5–β6, β7–β8 and β9–β10) of Fbs1 were substituted with those of FBG3 was determined. The substrate-binding pocket of this model exhibits structural features that differ from those of Fsb1.

Malate dehydrogenase (MDH), a carbohydrate and energy metabolism enzyme in eukaryotes, catalyzes the interconversion of malate to oxaloacetate (OAA) in conjunction with that of nicotinamide adenine dinucleotide (NAD+) to NADH. Three isozymes of MDH have been reported inSaccharomyces cerevisiae: MDH1, MDH2 and MDH3. MDH1 is a mitochondrial enzyme and a member of the tricarboxylic acid cycle, whereas MDH2 is a cytosolic enzyme that functions in the glyoxylate cycle. MDH3 is a glyoxysomal enzyme that is involved in the reoxidation of NADH, which is produced during fatty-acid β-oxidation. The affinity of MDH3 for OAA is lower than those of MDH1 and MDH2. Here, the crystal structures of yeast apo MDH3, the MDH3–NAD+complex and the MDH3–NAD+–OAA ternary complex were determined. The structure of the ternary complex suggests that the active-site loop is in the open conformation, differing from the closed conformations in mitochondrial and cytosolic malate dehydrogenases.

Local anesthetics (LAs) inhibit endoplasmic reticulum-associated protein degradation, however the mechanisms remain elusive. Here, we show that the clinically used LAs pilsicainide and lidocaine bind directly to the 20S proteasome and inhibit its activity. Molecular dynamic calculation indicated that these LAs were bound to the β5 subunit of the 20S proteasome, and not to the other active subunits, β1 and β2. Consistently, pilsicainide inhibited only chymotrypsin-like activity, whereas it did not inhibit the caspase-like and trypsin-like activities. In addition, we confirmed that the aromatic ring of these LAs was critical for inhibiting the proteasome. These LAs stabilized p53 and suppressed proliferation of p53-positive but not of p53-negative cancer cells.

To understand the basis for the lower activity of the tryptophan synthase β2 subunit in comparison to the α2β2 complex, we determined the crystal structures of apo-β2 and holo-β2 from Escherichia coli at 3.0 and 2.9 A resolutions, respectively. To our knowledge, this is the first report of both β2 subunit structures with and without pyridoxal-5′-phosphate. The apo-type molecule retained a dimeric form in solution, as in the case of the holo-β2 subunit. The subunit structures of both the apo-β2 and the holo-β2 forms consisted of two domains, namely the N domain and the C domain. Although there were significant structural differences between the apo- and holo-structures, they could be easily superimposed with a 22° rigid body rotation of the C domain. The pyridoxal-5′-phosphate-bound holo-form had multiple interactions between the two domains and a long loop (residues 260–310), which were missing in the apo-form. Comparison of the structures of holo-Ecβ2 and Stβ2 in the α2β2 complex from Salmonella typhimurium (Stα2β2) identified the cause of the lower enzymatic activity of holo-Ecβ2 in comparison with Stα2β2. The substrate (indole) gate residues, Tyr279 and Phe280, block entry of the substrate into the β2 subunit, although the indole can directly access the active site as a result of a wider cleft between the N and C domains in the holo-Ecβ2 subunit. In addition, the structure around βAsp305 of the holo-Ecβ2 subunit was similar to the open state of Stα2β2 with low activity, resulting in lower activity of holo-Ecβ2. Structured digital abstract • MINT-7712009: Ecβ2 (uniprotkb:P0A879) and Ecβ2 (uniprotkb:P0A879) bind (MI:0407) by x-ray crystallography (MI:0114) • MINT-7712032: Ecβ2 (uniprotkb:P0A879) and Ecβ2 (uniprotkb:P0A879) bind (MI:0407) by biophysical (MI:0013)

Adhesion pili (fimbriae) play a critical role in initiating the events that lead to intestinal colonization and diarrheal disease by enterotoxigenic Escherichia coli (ETEC), an E. coli pathotype that inflicts an enormous global disease burden. We elucidate atomic structures of an ETEC major pilin subunit, CfaB, from colonization factor antigen I (CFA/I) fimbriae. These data are used to construct models for 2 morphological forms of CFA/I fimbriae that are both observed in vivo: the helical filament into which it is typically assembled, and an extended, unwound conformation. Modeling and corroborative mutational data indicate that proline isomerization is involved in the conversion between these helical and extended forms. Our findings affirm the strong structural similarities seen between class 5 fimbriae (from bacteria primarily causing gastrointestinal disease) and class 1 pili (from bacteria that cause urinary, respiratory, and other infections) in the absence of significant primary sequence similarity. They also suggest that morphological and biochemical differences between fimbrial types, regardless of class, provide structural specialization that facilitates survival of each bacterial pathotype in its preferred host microenvironment. Last, we present structural evidence for bacterial use of antigenic variation to evade host immune responses, in that residues occupying the predicted surface-exposed face of CfaB and related class 5 pilins show much higher genetic sequence variability than the remainder of the pilin protein.

The Skp1-Cul1-F-box protein (SCF) complex catalyzes protein ubiquitination in diverse cellular processes and is one of the best-characterized ubiquitin ligases. F-box proteins determine the substrate specificities of SCF ubiquitin ligases. Among these, Fbs1/FBG1/FBXO2, Fbs2/FBG2/FBXO6, and Fbs3/FBG5/FBXO27 recognize the N-glycans of glycoproteins, whereas FBG3/FBXO44 has no sugar-binding activity, despite the high sequence homology and conservation of the residues necessary for oligosaccharide binding between Fbs1.3 and FBG3. Here we determined the crystal structure of the Skp1.FBG3 complex at a resolution of 2.6 A. The substrate-binding domain of FBG3 is composed of a 10-stranded antiparallel β-sandwich with three helices. Although the overall structure of FBG3 is similar to that of Fbs1, the residues that form the Fbs1 carbohydrate-binding pocket failed to be superposed with the corresponding residues of FBG3. Structure-based mutational analysis shows that distinct hydrogen bond networks of four FBG3 loops, i.e., β2-β3, β5-β6, β7-β8, and β9-β10, prevent the formation of the carbohydrate-binding pocket shown in Fbs1.

To understand the basis for the lower activity of the tryptophan synthase beta(2) subunit in comparison to the alpha(2)beta(2) complex, we determined the crystal structures of apo-beta(2) and holo-beta(2) from Escherichia coli at 3.0 and 2.9 A resolutions, respectively. To our knowledge, this is the first report of both beta(2) subunit structures with and without pyridoxal-5'-phosphate. The apo-type molecule retained a dimeric form in solution, as in the case of the holo-beta(2) subunit. The subunit structures of both the apo-beta(2) and the holo-beta(2) forms consisted of two domains, namely the N domain and the C domain. Although there were significant structural differences between the apo- and holo-structures, they could be easily superimposed with a 22 degrees rigid body rotation of the C domain. The pyridoxal-5'-phosphate-bound holo-form had multiple interactions between the two domains and a long loop (residues 260-310), which were missing in the apo-form. Comparison of the structures of holo-Ecbeta(2) and Stbeta(2) in the alpha(2)beta(2) complex from Salmonella typhimurium (Stalpha(2)beta(2)) identified the cause of the lower enzymatic activity of holo-Ecbeta(2) in comparison with Stalpha(2)beta(2). The substrate (indole) gate residues, Tyr279 and Phe280, block entry of the substrate into the beta(2) subunit, although the indole can directly access the active site as a result of a wider cleft between the N and C domains in the holo-Ecbeta(2) subunit. In addition, the structure around betaAsp305 of the holo-Ecbeta(2) subunit was similar to the open state of Stalpha(2)beta(2) with low activity, resulting in lower activity of holo-Ecbeta(2).