New insights into the human 26S proteasome function and regulation

SummaryThe 26S proteasome is a protease complex essential for proteostasis and strict regulation of diverse critical physiological processes, the mechanisms of which are still not fully described. The human 26S proteasome purification was optimized without exogenous nucleotides, to preserve the endogenous nucleotide occupancy and conformation of its AAA-ATPase subunits. This unveiled important effects on the proteasome function and structure resulting from exposure to Ca2+ or Mg2+, with important physiological implications. This sample, with an added model degron designed to mimic the minimum canonical ubiquitin signal for proteasomal recognition, was analysed by high-resolution cryo-EM. Two proteasome conformations were resolved, with only one capable of degron binding. The structural data show that this occurs without major conformation rearrangements and allows to infer into the allosteric communication between ubiquitin degron binding and the peptidase activities. These results revise existing concepts on the 26S proteasome function and regulation, opening important opportunities for further research.

Determining the gait of Miocene, Pliocene, and Pleistocene horses from fossilized trackways

Much work has been done on the study of vertebrate gaits over the past several decades and efforts undertaken to apply this to fossil tracks, especially dinosaurs and mammals such as cats, dogs, camels, and horses. This work seeks to expand upon such studies and in particular to study footprints laid down in sand by modern horses and apply such studies to determine the gaits of fossil horse trackways. It thus builds upon the work of Renders (1984a, b) and Kienapfel et al. (2014) and suggests additional measurements that can be taken on horse footprints. In this study the footprints left in the sand by 15 horses of various breeds with various gaits were videotaped, photographed, described, and measured in order to determine characteristics useful in distinguishing gaits. These results were then applied to two new sets of fossil footprints, those of the middle Miocene merychippine horse Scaphohippus intermontanus that I personally examined and measured and those from the late Pleistocene horse Equus conversidens, previously illustrated and described in the literature (McNeil et al., 2007). The latter horse exhibits a fast gallop of around 9.4 m/s, but it is the former whose footprints are quite unique. The quantitative and visual features of these prints are suggestive of a medium-fast gait involving apparent “understepping” of diagonal couplets and hind feet that overlap the centerline. The gait that most closely matches the footprints of Scaphohippus is the “artificial” gait of a slow rack or tölt, or pace, around 1.9 m/s, though an atypical trot of a horse with major conformation issues or which is weaving (swaying) from side to side is a less likely possibility. This intimates, along with the earlier study of Renders (1984a, b), who found the artificial gait of the running walk displayed by Pliocene hipparionine horses, that ancient horses possessed a much greater variety of gaits than modern horses and that over time they lost these abilities with the exception of certain gaited breeds.

GapR binds DNA through dynamic opening of its tetrameric interface

GapR is a nucleoid-associated protein that is an essential regulator of chromosome replication in the cell cycle model Caulobacter crescentus. Here, we demonstrate that free GapR is a homotetramer, but not a dimer as previously reported (Guo et al., Cell 175: 583–597, 2018). We have determined the crystal structure of GapR in complex with a 10-bp A-tract DNA, which has an open tetrameric conformation, different from the closed clamp conformation in the previously reported crystal structure of GapR/DNA complex. The free GapR adopts multiple conformations in dynamic exchange equilibrium, with the major conformation resembling the closed tetrameric conformation, while the open tetrameric conformation is a representative of minor conformers. As it is impossible for the circular genomic DNA to get into the central DNA binding tunnel of the major conformation, we propose that GapR initially binds DNA through the open conformation, and then undergoes structural rearrangement to form the closed conformation which fully encircles the DNA. GapR prefers to bind DNA with 10-bp consecutive A/T base pairs nonselectively (Kd ∼12 nM), while it can also bind GC-rich DNA sequence with a reasonable affinity of about 120 nM. Besides, our results suggest that GapR binding results in widening the minor groove of AT-rich DNA, instead of overtwisting DNA.

Crystal structure of methyl α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranoside monohydrate

The title compound, C13H24O9·H2O, a structural model for part of bacterial O-antigen polysaccharides from Shigella flexneri and Escherichia coli, crystallizes with four independent disaccharide molecules and four water molecules in the asymmetric unit. The conformation at the glycosidic linkage joining the two rhamnosyl residues is described by the torsion angles φH of 39, 30, 37 and 37°, and ψH of −32, −35, −31 and −32°, which are the major conformation region known to be populated in an aqueous solution. The hexopyranose rings have the 1 C 4 chair conformation. In the crystal, the disaccharide and water molecules are associated through O—H...O hydrogen bonds, forming a layer parallel to the bc plane. The layers stack along the a axis via hydrophobic interactions between the methyl groups.

Assembly and comparative analyses of the mitochondrial genome of Castanospermum australe (Papilionoideae, Leguminosae)

Plant mitochondrial genomes are often difficult to assemble because of frequent recombination mediated by repeats. Only a few mitochondrial genomes have been characterised in subfamily Papilionoideae of Leguminosae. Here, we report the complete mitochondrial genome of Castanospermum australe A.Cunn. & C.Fraser, an important medicinal and ornamental species in the Aldinoid clade of Papilionoideae. By mapping paired-end reads, seven hypothetical subgenomic conformations were rejected and two hypothetical complete isometric mitochondrial genome conformations that differed by a 64-kb inversion were strongly supported. Quantitative assessment of repeat-spanning read pairs showed a major conformation (MC1) and a minor conformation (MC2). The complete mitochondrial genome of C. australe was, thus, generated as 542079bp in length, with a high depth of coverage (~389.7×). Annotation of this mitochondrial genome yielded 58 genes encoding 37 proteins, 18 tRNAs and three rRNAs, as well as 17 introns and three medium-sized repeats (133, 119 and 114bp). Comparison of 10 mitochondrial genomes from Papilionoideae demonstrated significant variation in genome size, structure, gene content and RNA editing sites. In addition, mitochondrial genes were shown to be potentially useful in resolving the deep relationships of Papilionoideae.

Redetermined crystal structure of β-DL-methionine at 320 K

The structure of β-DL-methionine, C5H11NO2S, in the space groupC2/c, is here confirmed to be fully ordered all the way up to the phase transition at approximately 326 K, where displacive sliding of molecular bilayers gives the disorderedP21/cα form [data at 340 K; Görbitz (2014).Acta Cryst.E70, 341–343]. The geometry of hydrogen bonds in LD–LD hydrogen-bonding patterns [Görbitzet al.(2009).Acta Cryst.B65, 393–400] at the hydrophilic core of each molecular bilayer are virtually unperturbed by the phase shift, but the C—C—S—C torsion angle of the side chain changes fromtransat 320 K togauche+ for the major conformation at 340 K.

Cooperative Conformational Regulation in N-Heterocyclic Fluorohydrins

Seven-membered N-heterocycles are flexible ring structures, and their conformational control is important to their bioactivity. Our prior work shows that stereoselective monofluorination, if installed diastereoselectively, can bias a seven-membered, substituted azepane ring to one major conformation. However, multiple fluorination may not provide as much conformational bias due to conflicting effects. Here we show in our model azepane system that fluorohydrins can confer strong conformational bias if the relative configuration of the fluorine and hydroxy substitutent is appropriate to enable cooperative conformational control.

Diethyl (E)-2,3-bis[(E)-(2-methyl-2-phenylhydrazin-1-ylidene)methyl]but-2-enedioate

The complete molecule of the title compound, C24H28N4O4, is generated by crystallographic inversion symmetry. The ethyl side chain is disordered over two sets of sites in a 0.57 (4):0.43 (4) ratio. The dihedral angles between the methylidene group and the phenyl ring and ester side chain (major conformation) are 7.61 (8) and 86.95 (8)°, respectively. In the crystal, molecules are linkedviaC—H...O hydrogen bonds, forming corrugated sheets lying parallel to (010).

Altered chromatographic behaviour of mitochondrial ADP/ATP translocase induced by stabilization of the protein by binding of 6′-O-fluorescein-atractyloside

Atractyloside (ATR) is a high-affinity specific inhibitor of the mitochondrial ADP/ATP translocase (AAT). The binding of a fluorescent derivative, 6´-O-fluorescein-ATR (FATR), to mitochondria has been characterized. The binding constants obtained are in agreement with previously published values for ATR, demonstrating that FATR is a suitable probe of the AAT. AAT inhibited by FATR (FATR-AAT) was solubilized in dodecyl maltoside and purified by two separate ion-exchange chromatography steps at different pHs, which allowed FATR-AAT to be purified to homogeneity. The presence of the bound fluorescent probe enabled the inhibited AAT to be distinguished from the unliganded protein during chromatography, as they were markedly different in their chromatographic behaviour. The purified FATR-AAT was dimeric and in a single major conformation containing 1 mole FATR per mole of AAT dimer. In contrast, uninhibited AAT was monomeric and conformationally unstable. Use of the fluorescent ATR derivative in the development of the protocol enabled the stable dimeric AAT to be monitored directly and purified more effectively. The purification protocol was repeated using non-derivatized ATR, and highly pure AAT was obtained that was devoid of other members of the mitochondrial carrier family.