Dynamic interactions between E-cadherin and Ankyrin-G mediate epithelial cell polarity maintenance.

E-cadherin is an essential cell‒cell adhesion protein that mediates canonical cadherin-catenin complex formation in epithelial lateral membranes. Ankyrin-G (AnkG), a scaffold protein linking membrane proteins to the spectrin-based cytoskeleton, coordinates with E-cadherin to maintain epithelial cell polarity. However, the molecular mechanisms governing this complex formation and its relationships with the cadherin-catenin complex remain elusive. Here, we report that AnkG employs a promiscuous manner to encapsulate three discrete sites of E-cadherin by the same region, a dynamic mechanism that is distinct from the canonical 1:1 molar ratio previously described for other AnkG or E-cadherin-mediated complexes. Moreover, we demonstrate that AnkG-binding-deficient E-cadherin exhibited defective accumulation at the lateral membranes and show that disruption of interactions resulted in cell polarity malfunction. Finally, we demonstrate that E-cadherin is capable of simultaneously anchoring to AnkG and ß-catenin, providing mechanistic insights into the functional orchestration of the ankyrin-spectrin complex with the cadherin-catenin complex. Collectively, our results show that complex formation between E-cadherin and AnkG is dynamic, which enables the maintenance of epithelial cell polarity by ensuring faithful targeting of the adhesion molecule-scaffold protein complex, thus providing molecular mechanisms for essential E-cadherin-mediated complex assembly at cell‒cell junctions.

The AAA-ATPase Yta4/ATAD1 interacts with the mitochondrial divisome to inhibit mitochondrial fission.

Mitochondria are in a constant balance of fusion and fission. Excessive fission or deficient fusion leads to mitochondrial fragmentation, causing mitochondrial dysfunction and physiological disorders. How the cell prevents excessive fission of mitochondria is not well understood. Here, we report that the fission yeast AAA-ATPase Yta4, which is the homolog of budding yeast Msp1 responsible for clearing mistargeted tail-anchored (TA) proteins on mitochondria, plays a critical role in preventing excessive mitochondrial fission. The absence of Yta4 leads to mild mitochondrial fragmentation in a Dnm1-dependent manner but severe mitochondrial fragmentation upon induction of mitochondrial depolarization. Overexpression of Yta4 delocalizes the receptor proteins of Dnm1, i.e., Fis1 (a TA protein) and Mdv1 (the bridging protein between Fis1 and Dnm1), from mitochondria and reduces the localization of Dnm1 to mitochondria. The effect of Yta4 overexpression on Fis1 and Mdv1, but not Dnm1, depends on the ATPase and translocase activities of Yta4. Moreover, Yta4 interacts with Dnm1, Mdv1, and Fis1. In addition, Yta4 competes with Dnm1 for binding Mdv1 and decreases the affinity of Dnm1 for GTP and inhibits Dnm1 assembly in vitro. These findings suggest a model, in which Yta4 inhibits mitochondrial fission by inhibiting the function of the mitochondrial divisome composed of Fis1, Mdv1, and Dnm1. Therefore, the present work reveals an uncharacterized molecular mechanism underlying the inhibition of mitochondrial fission.

A backbone-centred energy function of neural networks for protein design.

A protein backbone structure is designable if a substantial number of amino acid sequences exist that autonomously fold into it1,2. It has been suggested that the designability of backbones is governed mainly by side chain-independent or side chain type-insensitive molecular interactions3-5, indicating an approach for designing new backbones (ready for amino acid selection) based on continuous sampling and optimization of the backbone-centred energy surface. However, a sufficiently comprehensive and precise energy function has yet to be established for this purpose. Here we show that this goal is met by a statistical model named SCUBA (for Side Chain-Unknown Backbone Arrangement) that uses neural network-form energy terms. These terms are learned with a two-step approach that comprises kernel density estimation followed by neural network training and can analytically represent multidimensional, high-order correlations in known protein structures. We report the crystal structures of nine de novo proteins whose backbones were designed to high precision using SCUBA, four of which have novel, non-natural overall architectures. By eschewing use of fragments from existing protein structures, SCUBA-driven structure design facilitates far-reaching exploration of the designable backbone space, thus extending the novelty and diversity of the proteins amenable to de novo design.

The roles of Runx1 in skeletal development and osteoarthritis: A concise review.

Runt-related transcription factor-1 (Runx1) is well known for its functions in hematopoiesis and leukemia but recent research has focused on its role in skeletal development and osteoarthritis (OA). Deficiency of the Runx1 gene is fatal in early embryonic development, and specific knockout of Runx1 in cell lineages of cartilage and bone leads to delayed cartilage formation and impaired bone calcification. Runx1 can regulate genes including collagen type II (Col2a1) and X (Col10a1), SRY-box transcription factor 9 (Sox9), aggrecan (Acan) and matrix metalloproteinase 13 (MMP-13), and the up-regulation of Runx1 improves the homeostasis of the whole joint, even in the pathological state. Moreover, Runx1 is activated as a response to mechanical compression, but impaired in the joint with the pathological progress associated with osteoarthritis. Therefore, interpretation about the role of Runx1 could enlarge our understanding of key marker genes in the skeletal development and an increased understanding of Runx1 could be helpful to identify treatments for osteoarthritis. This review provides the most up-to-date advances in the roles and bio-mechanisms of Runx1 in healthy joints and osteoarthritis from all currently published articles and gives novel insights in therapeutic approaches to OA based on Runx1.

Assembly status transition offers an avenue for activity modulation of a supramolecular enzyme.

Nature has evolved many supramolecular proteins assembled in certain, sometimes even seemingly oversophisticated, morphological manners. The rationale behind such evolutionary efforts is often poorly understood. Here, we provide atomic-resolution insights into how the dynamic building of a structurally complex enzyme with higher order symmetry offers amenability to intricate regulation. We have established the functional coupling between enzymatic activity and protein morphological states of glutamine synthetase (GS), an old multi-subunit enzyme essential for cellular nitrogen metabolism. Cryo-EM structure determination of GS in both the catalytically active and inactive assembly states allows us to reveal an unanticipated self-assembly-induced disorder-order transition paradigm, in which the remote interactions between two subcomplex entities significantly rigidify the otherwise structurally fluctuating active sites, thereby regulating activity. We further show in vivo evidences that how the enzyme morphology transitions could be modulated by cellular factors on demand. Collectively, our data present an example of how assembly status transition offers an avenue for activity modulation, and sharpens our mechanistic understanding of the complex functional and regulatory properties of supramolecular enzymes.

Sandwiched-fusion strategy facilitates recombinant production of small labile proteins.

Efficient production of large quantities of soluble, properly folded proteins is of high demand in modern structural and functional genomics. Despite much advancement toward improving recombinant protein expression, many eukaryotic proteins especially small peptides often fail to be recovered due to rapid proteolytic degradation. Here we show that the sandwiched-fusion strategy, which is based on two protein tags incorporated both at the amino- and carboxyl-terminus of target protein, could be employed to overcome this obstacle. We have exploited this strategy on heterologous expression in Escherichia coli of eight small degradation-prone eukaryotic proteins, whose successful recombinant productions have yet to be achieved. These include seven mitochondria-derived peptides (MDPS), a class of unique metabolic regulators of human body, and a labile mosquito transcription factor, Guy1. We show here that the sandwiched-fusion strategy, which provides robust protection against proteolysis, affords an economical method to obtain large quantities of pure five MDPs and the transcription factor Guy1, in sharp contrast to otherwise unsuccessful recovery using the traditional amino-fusion method. Further biophysical characterization and interaction studies by NMR spectroscopy confirmed that the proteins produced by this novel approach are properly folded into their biologically active structures. We anticipate this strategy could be widely utilized in production of other labile protein systems.

Pursuing sustainable productivity with millions of smallholder farmers.

Sustainably feeding a growing population is a grand challenge, and one that is particularly difficult in regions that are dominated by smallholder farming. Despite local successes, mobilizing vast smallholder communities with science- and evidence-based management practices to simultaneously address production and pollution problems has been infeasible. Here we report the outcome of concerted efforts in engaging millions of Chinese smallholder farmers to adopt enhanced management practices for greater yield and environmental performance. First, we conducted field trials across China's major agroecological zones to develop locally applicable recommendations using a comprehensive decision-support program. Engaging farmers to adopt those recommendations involved the collaboration of a core network of 1,152 researchers with numerous extension agents and agribusiness personnel. From 2005 to 2015, about 20.9 million farmers in 452 counties adopted enhanced management practices in fields with a total of 37.7 million cumulative hectares over the years. Average yields (maize, rice and wheat) increased by 10.8-11.5%, generating a net grain output of 33 million tonnes (Mt). At the same time, application of nitrogen decreased by 14.7-18.1%, saving 1.2 Mt of nitrogen fertilizers. The increased grain output and decreased nitrogen fertilizer use were equivalent to US$12.2 billion. Estimated reactive nitrogen losses averaged 4.5-4.7 kg nitrogen per Megagram (Mg) with the intervention compared to 6.0-6.4 kg nitrogen per Mg without. Greenhouse gas emissions were 328 kg, 812 kg and 434 kg CO2 equivalent per Mg of maize, rice and wheat produced, respectively, compared to 422 kg, 941 kg and 549 kg CO2 equivalent per Mg without the intervention. On the basis of a large-scale survey (8.6 million farmer participants) and scenario analyses, we further demonstrate the potential impacts of implementing the enhanced management practices on China's food security and sustainability outlook.

Automatic methyl assignment in large proteins by the MAGIC algorithm.

Selective methyl labeling is an extremely powerful approach to study the structure, dynamics and function of biomolecules by NMR. Despite spectacular progress in the field, such studies remain rather limited in number. One of the main obstacles remains the assignment of the methyl resonances, which is labor intensive and error prone. Typically, NOESY crosspeak patterns are manually correlated to the available crystal structure or an in silico template model of the protein. Here, we propose methyl assignment by graphing inference construct, an exhaustive search algorithm with no peak network definition requirement. In order to overcome the combinatorial problem, the exhaustive search is performed locally, i.e. for a small number of methyls connected through-space according to experimental 3D methyl NOESY data. The local network approach drastically reduces the search space. Only the best local assignments are combined to provide the final output. Assignments that match the data with comparable scores are made available to the user for cross-validation by additional experiments such as methyl-amide NOEs. Several NMR datasets for proteins in the 25-50 kDa range were used during development and for performance evaluation against the manually assigned data. We show that the algorithm is robust, reliable and greatly speeds up the methyl assignment task.

Enhancing the sensitivity of multidimensional NMR experiments by using triply-compensated π pulses.

In multidimensional solution NMR experiments, π pulses are used extensively for inversion and refocusing operations on 1H, 13C and 15N nuclei. Pulse miscalibration, off-resonance effects, and J-coupling evolution during π pulse execution result in severe signal losses that are exacerbated at high magnetic fields. Here, we report the implementation of a triply-compensated π pulse (G5) optimized for both inversion and refocusing in widely used 2- and 3-dimensional experiments. By replacing most of the hard π pulses, adiabatic or composite pulses on the 1H, 13C and 15N channels with G5 pulses, we obtained signal enhancements ranging from 80 to 240%. We anticipate that triply-compensated pulses will be crucial for improving the performance of multidimensional and multinuclear pulse sequences at ultra-high fields.

Optimization of 1H decoupling eliminates sideband artifacts in 3D TROSY-based triple resonance experiments.

TROSY-based triple resonance experiments are essential for protein backbone assignment of large biomolecular systems by solution NMR spectroscopy. In a survey of the current Bruker pulse sequence library for TROSY-based experiments we found that several sequences were plagued by artifacts that affect spectral quality and hamper data analysis. Specifically, these experiments produce sidebands in the 13C(t 1) dimension with inverted phase corresponding to 1HN resonance frequencies, with approximately 5% intensity of the parent 13C crosspeaks. These artifacts originate from the modulation of the 1HN frequency onto the resonance frequency of 13Cα and/or 13Cß and are due to 180° pulses imperfections used for 1H decoupling during the 13C(t 1) evolution period. These sidebands can become severe for CAi, CAi-1 and/or CBi, CBi-1 correlation experiments such as TROSY-HNCACB. Here, we implement three alternative decoupling strategies that suppress these artifacts and, depending on the scheme employed, boost the sensitivity up to 14% on Bruker spectrometers. A class of comparable Agilent/Varian pulse sequences that use WALTZ16 1H decoupling can also be improved by this method resulting in up to 60-80% increase in sensitivity.

Structures of Large Protein Complexes Determined by Nuclear Magnetic Resonance Spectroscopy.

Nuclear magnetic resonance (NMR) spectroscopy has been instrumental during the past two decades in providing high-resolution structures of protein complexes. It has been the method of choice for determining the structure of dynamic protein complexes, which are typically recalcitrant to other structural techniques. Until recently, NMR spectroscopy has yielded structures of small or medium-sized protein complexes, up to approximately 30-40 kDa. Major breakthroughs during the past decade, especially in isotope-labeling techniques, have enabled NMR characterization of large protein systems with molecular weights of hundreds of kDa. This has provided unique insights into the binding, dynamic, and allosteric properties of large systems. Notably, there is now a slowly but steadily growing list of large, dynamic protein complexes whose atomic structure has been determined by NMR. Many of these complexes are characterized by a high degree of flexibility and, thus, their structures could not have been obtained using other structural methods. Especially in the field of molecular chaperones, NMR has recently provided the first-ever high-resolution structures of their complexes with unfolded proteins. Further technological advances will establish NMR as the primary tool for obtaining atomic structures of challenging systems with even higher complexity.

Structural basis for the antifolding activity of a molecular chaperone.

Molecular chaperones act on non-native proteins in the cell to prevent their aggregation, premature folding or misfolding. Different chaperones often exert distinct effects, such as acceleration or delay of folding, on client proteins via mechanisms that are poorly understood. Here we report the solution structure of SecB, a chaperone that exhibits strong antifolding activity, in complex with alkaline phosphatase and maltose-binding protein captured in their unfolded states. SecB uses long hydrophobic grooves that run around its disk-like shape to recognize and bind to multiple hydrophobic segments across the length of non-native proteins. The multivalent binding mode results in proteins wrapping around SecB. This unique complex architecture alters the kinetics of protein binding to SecB and confers strong antifolding activity on the chaperone. The data show how the different architectures of chaperones result in distinct binding modes with non-native proteins that ultimately define the activity of the chaperone.

Correlation between weighted spectral distribution and average path length in evolving networks.

The weighted spectral distribution (WSD) is a metric defined on the normalized Laplacian spectrum. In this study, synchronic random graphs are first used to rigorously analyze the metric's scaling feature, which indicates that the metric grows sublinearly as the network size increases, and the metric's scaling feature is demonstrated to be common in networks with Gaussian, exponential, and power-law degree distributions. Furthermore, a deterministic model of diachronic graphs is developed to illustrate the correlation between the slope coefficient of the metric's asymptotic line and the average path length, and the similarities and differences between synchronic and diachronic random graphs are investigated to better understand the correlation. Finally, numerical analysis is presented based on simulated and real-world data of evolving networks, which shows that the ratio of the WSD to the network size is a good indicator of the average path length.

Economic Performance and Sustainability of a Novel Intercropping System on the North China Plain.

Double cropping of wheat and maize is common on the North China Plain, but it provides limited income to rural households due to the small farm sizes in the region. Local farmers in Quzhou County have therefore innovated their production system by integration of watermelon as a companion cash crop into the system. We examine the economic performance and sustainability of this novel intercropping system using crop yield data from 2010 to 2012 and farm household survey data collected in 2012. Our results show that the gross margin of the intercropping system exceeded that of the double cropping system by more than 50% in 2012. Labor use in the intercropping system was more than three times that in double cropping. The lower returns per labor hour in intercropping, however, exceeded the average off-farm wage in the region by a significant margin. Nutrient surpluses and irrigation water use are significant larger under the intercropping system. We conclude that the novel wheat-maize/watermelon intercropping system contributes to rural poverty alleviation and household-level food security, by raising farm incomes and generating more employment, but needs further improvement to enhance its sustainability.

Genetic regulation of vesiculogenesis and immunomodulation in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) restrains immune responses well enough to escape eradication but elicits enough immunopathology to ensure its transmission. Here we provide evidence that this host-pathogen relationship is regulated in part by a cytosolic, membrane-associated protein with a unique structural fold, encoded by the Mtb gene rv0431. The protein acts by regulating the quantity of Mtb-derived membrane vesicles bearing Toll-like receptor 2 ligands, including the lipoproteins LpqH and SodC. We propose that rv0431 be named "vesiculogenesis and immune response regulator."

Eukaryotic N-glycosylation occurs via the membrane-anchored C-terminal domain of the Stt3p subunit of oligosaccharyltransferase.

N-glycosylation is an essential and highly conserved protein modification. In eukaryotes, it is catalyzed by a multisubunit membrane-associated enzyme, oligosaccharyltransferase (OT). We report the high resolution structure of the C-terminal domain of eukaryotic Stt3p. Unlike its soluble ß-sheet-rich prokaryotic counterparts, our model reveals that the C-terminal domain of yeast Stt3p is highly helical and has an overall oblate spheroid-shaped structure containing a membrane-embedded region. Anchoring of this protein segment to the endoplasmic reticulum membrane is likely to bring the membrane-embedded donor substrate closer, thus facilitating glycosylation efficiency. Structural comparison of the region near the WWDYG signature motif revealed that the acceptor substrate-binding site of yeast OT strikingly resembles its prokaryotic counterparts, suggesting a conserved mechanism of N-glycosylation from prokaryotes to eukaryotes. Furthermore, comparison of the NMR and cryo-EM structures of yeast OT revealed that the molecular architecture of this acceptor substrate-recognizing domain has interesting spatial specificity for interactions with other essential OT subunits.

Dynamic flexibility of the ATPase p97 is important for its interprotomer motion transmission.

The hexameric protein p97, a very abundant type II AAA ATPase (ATPase associated with various cellular activities), is involved in a diverse range of cellular functions. During its ATPase cycle p97 functions as an ATP motor, converting the chemical energy released upon hydrolysis of ATP to ADP into mechanical work, which is then directed toward the proteins that serve as substrates. A key question in this process is: How is the nucleotide-induced motion transmitted from the C-terminal ATPase domain (the D2 domain) of p97 to the distant N-terminal substrate-processing domain? We have previously reported the surprising finding that motion transmission between the two ATPase domains (the D2 and D1 domains) is mediated by the D1-D2 linker region of its neighboring protomer. In this study we report efforts to better understand this process. Our findings suggest that the amino acid sequence containing Gly-Gly that is located at the C terminus of the D1-D2 linker functions as a pivoting point that allows the dynamic movement of the D1-D2 linker. Furthermore, we found that locking the D1-D2 linker to the D2 domain by introducing disulfide bonds significantly impaired the motion-transmission process. These results support our previous model for interprotomer motion transmission, and provide more detailed information on how the motion transmission between the two ATPase domains of p97 is relayed by the flexible movement of the D1-D2 linker from its neighboring protomer.

Interprotomer motion-transmission mechanism for the hexameric AAA ATPase p97.

Multimeric AAA ATPases represent a structurally homologous yet functionally diverse family of proteins. The essential and highly abundant hexameric AAA ATPase p97 is perhaps the best studied AAA protein, playing an essential role in various important cellular activities. During ATP-hydrolysis process, p97 undergoes dramatic conformational changes, and these changes are initiated in the C-terminal ATPase domain and transmitted across the entire length of the molecule to the N-terminal effector domain. However, the detailed mechanism of the motion transmission remains unclear. Here, we report an interprotomer motion-transmission mechanism to explain this process: The nucleotide-dependent motion transmission between the two ATPase domains of one protomer is mediated by its neighboring protomer. This finding reveals a strict requirement for interprotomer coordination of p97 during the motion-transmission process and may shed light on studies of other AAA ATPases.

Challenging the limit: NMR assignment of a 31 kDa helical membrane protein.

Structural determination of membrane proteins by NMR spectroscopy remains a challenge, especially for helical membrane proteins. Here we report the NMR assignment and secondary structure of a 31 kDa helical membrane protein, the C-terminal domain of Stt3p. The C-terminal domain of Stt3p has been proposed to be the catalytic domain of yeast oligosaccharyl transferase (OT), a multisubunit membrane-associated enzyme complex catalyzing N-glycosylation, which is an essential and highly conserved protein modification. NMR assignment is the first critical step in the determination of the high-resolution solution structure and further structure-function studies.