The outer membrane of Gram-negative bacteria is highly impermeable to hydrophilic molecules of larger than 600 Da, protecting these bacteria from toxins present in the environment. In order to transport nutrients across this impermeable membrane, Gram-negative bacteria utilize a diverse family of outer-membrane proteins called TonB-dependent transporters. The majority of the members of this family transport iron-containing substrates. However, it is becoming increasingly clear that TonB-dependent transporters target chemically diverse substrates. In this work, the structure and phylogenetic distribution of the TonB-dependent transporter YncD are investigated. It is shown that while YncD is present in some enteropathogens, including Escherichia coli and Salmonella spp., it is also widespread in Gammaproteobacteria and Betaproteobacteria of environmental origin. The structure of YncD was determined, showing that despite a distant evolutionary relationship, it shares structural features with the ferric citrate transporter FecA, including a compact positively charged substrate-binding site. Despite these shared features, it is shown that YncD does not contribute to the growth of E. coli in pure culture under iron-limiting conditions or with ferric citrate as an iron source. Previous studies of transcriptional regulation in E. coli show that YncD is not induced under iron-limiting conditions and is unresponsive to the ferric uptake regulator (Fur). These observations, combined with the data presented here, suggest that YncD is not responsible for the transport of an iron-containing substrate.

Aldo-keto reductases (AKRs) are NADPH/NADP+-dependent oxidoreductase enzymes that metabolize an aldehyde/ketone to the corresponding alcohol. AKR4C14 from rice exhibits a much higher efficiency in metabolizing malondialdehyde (MDA) than do the Arabidopsis enzymes AKR4C8 and AKR4C9, despite sharing greater than 60% amino-acid sequence identity. This study confirms the role of rice AKR4C14 in the detoxification of methylglyoxal and MDA, and demonstrates that the endogenous contents of both aldehydes in transgenic Arabidopsis ectopically expressing AKR4C14 are significantly lower than their levels in the wild type. The apo structure of indica rice AKR4C14 was also determined in the absence of the cofactor, revealing the stabilized open conformation. This is the first crystal structure in AKR subfamily 4C from rice to be observed in the apo form (without bound NADP+). The refined AKR4C14 structure reveals a stabilized open conformation of loop B, suggesting the initial phase prior to cofactor binding. Based on the X-ray crystal structure, the substrate- and cofactor-binding pockets of AKR4C14 are formed by loops A, B, C and β1α1. Moreover, the residues Ser211 and Asn220 on loop B are proposed as the hinge residues that are responsible for conformational alteration while the cofactor binds. The open conformation of loop B is proposed to involve Phe216 pointing out from the cofactor-binding site and the opening of the safety belt. Structural comparison with other AKRs in subfamily 4C emphasizes the role of the substrate-channel wall, consisting of Trp24, Trp115, Tyr206, Phe216, Leu291 and Phe295, in substrate discrimination. In particular, Leu291 could contribute greatly to substrate selectivity, explaining the preference of AKR4C14 for its straight-chain aldehyde substrate.

The biological processes related to protein homeostasis in Mycobacterium tuberculosis, the etiologic agent of tuberculosis, have recently been established as critical pathways for therapeutic intervention. Proteins of particular interest are ClpC1 and the ClpC1–ClpP1–ClpP2 proteasome complex. The structure of the potent antituberculosis macrocyclic depsipeptide ecumicin complexed with the N-terminal domain of ClpC1 (ClpC1-NTD) is presented here. Crystals of the ClpC1-NTD–ecumicin complex were monoclinic (unit-cell parameters a = 80.0, b = 130.0, c = 112.0 Å, β = 90.07°; space group P21; 12 complexes per asymmetric unit) and diffracted to 2.5 Å resolution. The structure was solved by molecular replacement using the self-rotation function to resolve space-group ambiguities. The new structure of the ecumicin complex showed a unique 1:2 (target:ligand) stoichiometry exploiting the intramolecular dyad in the α-helical fold of the target N-terminal domain. The structure of the ecumicin complex unveiled extensive interactions in the uniquely extended N-terminus, a critical binding site for the known cyclopeptide complexes. This structure, in comparison with the previously reported rufomycin I complex, revealed unique features that could be relevant for understanding the mechanism of action of these potential antituberculosis drug leads. Comparison of the ecumicin complex and the ClpC1-NTD-L92S/L96P double-mutant structure with the available structures of rufomycin I and cyclomarin A complexes revealed a range of conformational changes available to this small N-terminal helical domain and the minor helical alterations involved in the antibiotic-resistance mechanism. The different modes of binding and structural alterations could be related to distinct modes of action.

Pullulanase (EC 3.2.1.41) is a well known starch-debranching enzyme that catalyzes the cleavage of α-1,6-glycosidic linkages in α-glucans such as starch and pullulan. Crystal structures of a type I pullulanase from Paenibacillus barengoltzii (PbPulA) and of PbPulA in complex with maltopentaose (G5), maltohexaose (G6)/α-cyclodextrin (α-CD) and β-cyclodextrin (β-CD) were determined in order to better understand substrate binding to this enzyme. PbPulA belongs to glycoside hydrolase (GH) family 13 subfamily 14 and is composed of three domains (CBM48, A and C). Three carbohydrate-binding sites identified in PbPulA were located in CBM48, near the active site and in domain C, respectively. The binding site in CBM48 was specific for β-CD, while that in domain C has not been reported for other pullulanases. The domain C binding site had higher affinity for α-CD than for G6; a small motif (FGGEH) seemed to be one of the major determinants for carbohydrate binding in this domain. Structure-based mutations of several surface-exposed aromatic residues in CBM48 and domain C had a debilitating effect on the activity of the enzyme. These results suggest that both CBM48 and domain C play a role in binding substrates. The crystal forms described contribute to the understanding of pullulanase domain–carbohydrate interactions.

The new book Subsistence Economies of Indigenous North American Societies provides a comprehensive and in-depth documentation of how Native American societies met the challenges of […]

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Yinlong Song, Yikan Zhang, Ying Pan, Jianfeng He, Yan Wang, Wei Chen, Jing Guo, Haiteng Deng, Yi Xue, Xianyang Fang, and Xin Liang

Microtubules dynamics is regulated by the plus end-tracking proteins (+TIPs) in cells. End binding protein 1 (EB1) acts as a master regulator in +TIPs networks by targeting microtubule growing ends and recruiting other factors. However, the molecular mechanism of how EB1 binds to microtubule ends with a high affinity remains to be an open question. Using single-molecule imaging, we show that the end-binding kinetics of EB1 changes along with the polymerizing and hydrolysis rate of tubulin dimers, confirming the binding of EB1 to GTP/GDP-Pi tubulin at microtubule growing ends. The affinity of wild-type EB1 to these sites is higher than monomeric EB1 mutants, suggesting that two CH domains in the dimer contribute to the end-binding. Introducing phosphomimicking mutations into the linker domain of EB1 weakens the end-binding affinity and confers a more curved conformation to EB1 dimer without compromising dimerization, suggesting that the overall architecture of EB1 is important for the end-binding affinity. Taken together, our results provide insights into understanding how the high-affinity end-binding of EB1 can be achieved and how this activity may be regulated in cells.

Archan Chakraborty, Wei-Cheng Lin, Yu-Tsun Lin, Kuang-Jing Huang, Pei-Yu Wang, Yi-Feng Chang, Hsiang-Iu Wang, Kung-Ting Ma, Chun-Yen Wang, Xuan-Rong Huang, Yen-Hsien Lee, Bi-Chang Chen, Ya-Ju Hsieh, Kun-Yi Chien, Tzu-Yang Lin, Ji-Long Liu, Li-Ying Sung, Jau-Song Yu, Yu-Sun Chang, and Li-Mei Pai

Under metabolic stress, cellular components can assemble into distinct membraneless organelles for adaptation. One such example is cytidine 5'-triphosphate synthase (CTPS), which forms filamentous structures under glutamine deprivation. We have previously demonstrated that histidine (His)-mediated methylation regulates the formation of CTPS filaments to suppress enzymatic activity and preserve the CTPS protein under Gln deprivation, which promotes cancer cell growth after stress alleviation. However, it remains unclear where and how these enigmatic structures are assembled. Using CTPS-APEX2-mediated in vivo proximity labeling, we found that SNAP29 regulates the spatiotemporal filament assembly of CTPS along the cytokeratin network in a keratin 8 (KRT8)-dependent manner. Knockdown of synaptosome-associated protein 29 (SNAP29) interfered with assembly and relaxed the filament-induced suppression of CTPS enzymatic activity. Furthermore, APEX2 proximity labeling of keratin 18 (KRT18) revealed a spatiotemporal association of SNAP29 with cytokeratin in response to stress. Super-resolution imaging suggests that during CTPS filament formation, SNAP29 interacts with CTPS along the cytokeratin network. This study links the cytokeratin network to the regulation of metabolism by compartmentalization of metabolic enzymes during nutrient deprivation.

Ana Loncar, Sergio A. Rincon, Manuel Lera Ramirez, Anne Paoletti, and Phong T. Tran

To segregate the chromosomes faithfully during cell division, cells assemble a spindle that captures the kinetochores and pulls them towards opposite poles. Proper spindle function requires correct interplay between microtubule motors and non-motor proteins. Defects in spindle assembly or changes in spindle dynamics are associated with diseases like cancer or developmental disorders. Here we compared mitotic and meiotic spindles in fission yeast. We show that even though mitotic and meiotic spindles undergo the typical three phases of spindle elongation, they have distinct features. We found that the relative concentration of kinesin-14 Pkl1 is decreased in meiosis I compared to mitosis, while the concentration of kinesin-5 Cut7 remains constant. We identified the second kinesin-14 Klp2 and microtubule dynamics as factors necessary for proper meiotic spindle assembly. This work defines differences between mitotic and meiotic spindles in fission yeast, and provides prospect for future comparative studies.

Samantha-Su Z. Taylor, Nicole L. Jacobsen, Tasha K. Pontifex, Paul Langlais, and Janis M. Burt

Connexin 37 (Cx37) expression profoundly suppresses proliferation of rat insulinoma (Rin) cells in a manner dependent on gap junction channel (GJCh) functionality and the presence and phosphorylation status of its carboxyl-terminus (CT). In Rin cells growth arrested by induced Cx37 expression, serine 319 (S319) is frequently phosphorylated. Preventing phosphorylation at this site (alanine substitution; S319A) relieved Cx37 of its growth suppressive effect whereas mimicking phosphorylation at this site (aspartate substitution; S319D) enhanced Cx37's growth suppressive properties. Like Cx37-WT, -S319D GJChs and hemichannels (HChs) preferred the closed state, rarely opening fully, and gated slowly. In contrast, Cx37-S319A channels preferred open states, opened fully, and gated rapidly. These data indicate that phosphorylation-dependent conformational differences in Cx37 protein and channel function underlie Cx37-induced growth arrest vs. growth permissive phenotypes. That the closed state of -WT and Cx37-S319D GJChs and HChs favors growth arrest suggests that rather than specific permeants mediating cell cycle arrest, the closed conformation instead supports interaction of Cx37 with growth regulatory proteins that result in growth arrest.

Ganlu Deng, Yihong Chen, Cao Guo, Ling Yin, Ying Han, Yiyi Li, Yaojie Fu, Changjing Cai, Hong Shen, and Shan Zeng

Epithelial-mesenchymal transition (EMT) is a crucial process for cancer cells to acquire metastatic potential, which primarily causes death in gastric cancer (GC) patients. Bone morphogenetic protein 4 (BMP4) is a member of the TGF-β family that plays an indispensable role in human cancers. However, little is known about its roles in GC metastasis. In this study, BMP4 was found to be frequently overexpressed in GC tissues and was correlated with patient's poor prognosis. BMP4 was upregulated in GC cell lines and promoted EMT and metastasis of GC cells both in vitro and in vivo, while knockdown of BMP4 significantly inhibited EMT and metastasis of GC cells. Meanwhile, the inhibitor of DNA binding 1 (Id1) was identified as a downstream target of BMP4 by PCR arrays and upregulated via Smad1/5/8 phosphorylation. Id1 knockdown attenuated BMP4-induced EMT and invasion in GC cells. Moreover, Id1 overexpression in BMP4 knockdown cells restored the promotion of EMT and cell invasion. In summary, BMP4 induced EMT to promote GC metastasis by upregulating Id1 expression. Antagonizing BMP4 may be a potential therapeutic strategy in GC metastasis.

Mei Wang, Luping Yu, Shu Wang, Fan Yang, Min Wang, Lufan Li, and Xin Wu

RNA-binding protein LIN28A is required for maintaining tissue homeostasis, including the reproductive system, but the underlying mechanisms on how LIN28A regulates germline progenitors remain unclear. Here, we dissected LIN28A-binding targets using high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) in the mouse testes. LIN28A preferentially binds to CDS or 3'UTR regions through these sites with GGAG(A) sequences enriched within mRNAs. Further investigation of Lin28a null mouse testes indicated that meiosis-associated mRNAs mediated by LIN28A were differentially expressed. Next, ribosome profiling revealed that the mRNA levels of these targets were significantly reduced in polysome fractions, and their protein expression levels decreased in the Lin28a null mouse testes, even when meiotic arrest in null mouse testes was not apparent. Collectively, these findings provide a set of binding targets that are regulated by LIN28A, which may potentially be the mechanism for the prominent role of LIN28A in regulating mammalian undifferentiated spermatogonia fates and male fertility.

Miniature camels and horses, a rhinoceros and a giant bear-dog are among fossils unearthed in the recent excavations of the Panama Canal expansion project. These […]

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As average annual temperatures increase, mosquitoes have also been on the move—up the mountains of the Hawaiian islands. Once a refuge for native birds susceptible […]

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Serial crystallography records still diffraction patterns from single, randomly oriented crystals, then merges data from hundreds or thousands of them to form a complete data set. To process the data, the diffraction patterns must first be indexed, equivalent to determining the orientation of each crystal. A novel automatic indexing algorithm is presented, which in tests usually gives significantly higher indexing rates than alternative programs currently available for this task. The algorithm does not require prior knowledge of the lattice parameters but can make use of that information if provided, and also allows indexing of diffraction patterns generated by several crystals in the beam. Cases with a small number of Bragg spots per pattern appear to particularly benefit from the new approach. The algorithm has been implemented and optimized for fast execution, making it suitable for real-time feedback during serial crystallography experiments. It is implemented in an open-source C++ library and distributed under the LGPLv3 licence. An interface to it has been added to the CrystFEL software suite.

A crystallographic indexing algorithm, pinkIndexer, is presented for the analysis of snapshot diffraction patterns. It can be used in a variety of contexts including measurements made with a monochromatic radiation source, a polychromatic source or with radiation of very short wavelength. As such, the algorithm is particularly suited to automated data processing for two emerging measurement techniques for macromolecular structure determination: serial pink-beam X-ray crystallography and serial electron crystallography, which until now lacked reliable programs for analyzing many individual diffraction patterns from crystals of uncorrelated orientation. The algorithm requires approximate knowledge of the unit-cell parameters of the crystal, but not the wavelengths associated with each Bragg spot. The use of pinkIndexer is demonstrated by obtaining 1005 lattices from a published pink-beam serial crystallography data set that had previously yielded 140 indexed lattices. Additionally, in tests on experimental serial crystallography diffraction data recorded with quasi-monochromatic X-rays and with electrons the algorithm indexed more patterns than other programs tested.

Crystal structure identification of thin organic films entails a number of technical and methodological challenges. In particular, if molecular crystals are epitaxially grown on single-crystalline substrates a complex scenario of multiple preferred orientations of the adsorbate, several symmetry-related in-plane alignments and the occurrence of unknown polymorphs is frequently observed. In theory, the parameters of the reduced unit cell and its orientation can simply be obtained from the matrix of three linearly independent reciprocal-space vectors. However, if the sample exhibits unit cells in various orientations and/or with different lattice parameters, it is necessary to assign all experimentally obtained reflections to their associated individual origin. In the present work, an effective algorithm is described to accomplish this task in order to determine the unit-cell parameters of complex systems comprising different orientations and polymorphs. This method is applied to a polycrystalline thin film of the conjugated organic material 6,13-pentacene­quinone (PQ) epitaxially grown on an Ag(111) surface. All reciprocal vectors can be allocated to unit cells of the same lattice constants but grown in various orientations [sixfold rotational symmetry for the contact planes (102) and (102)]. The as-determined unit cell is identical to that reported in a previous study determined for a fibre-textured PQ film. Preliminary results further indicate that the algorithm is especially effective in analysing epitaxially grown crystallites not only for various orientations, but also if different polymorphs are present in the film.

Recent developments of two-color operation modes at X-ray free-electron laser facilities provide new research opportunities, such as X-ray pump/X-ray probe experiments and multiple-wavelength anomalous dispersion phasing methods. However, most existing indexing methods were developed for indexing diffraction data from monochromatic X-ray beams. Here, a new algorithm is presented for indexing two-color diffraction data, as an extension of the sparse-pattern indexing algorithm SPIND, which has been demonstrated to be capable of indexing diffraction patterns with as few as five peaks. The principle and implementation of the two-color indexing method, SPIND-TC, are reported in this paper. The algorithm was tested on both simulated and experimental data of protein crystals. The results show that the diffraction data can be accurately indexed in both cases. Source codes are publicly available at https://github.com/lixx11/SPIND-TC.

Smithsonian Scientist Helen James dives into dormant Hawaiian volcanoes to find bird fossils. Her work to identify these species (some new species, some extinct) helps us learn about the past--specifically, how humans might have caused the extinction of certain bird species.

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What's possibly the most calming yet nerve-racking job in the world? Come behind the scenes of the Smithsonian's Freer Gallery of Art to find out!

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The post Smithsonian conservators open 2001 Jim Thorpe Wheaties Box from the Smithsonian’s National Museum of the American Indian appeared first on Smithsonian Insider.

The post What will invasive round goby fish do to Great Lakes streams? Join SERC post doc Matt Kornis and find out. appeared first on Smithsonian Insider.

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Inupiaq artist and teacher Holly Nordlum visited the National Museum of the American Indian to study Arctic objects and archival photographs in the museum’s collections. […]

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As American power and population grew in the 19th century, the United States gradually rejected the main principle of treaty-making—that tribes were self-governing nations—and initiated […]

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