Abstract

Neurotoxic misfolding of Cu, Zn-superoxide dismutase (SOD1) is implicated in causing amyotrophic lateral sclerosis, a devastating and incurable neurodegenerative disease. Disease-linked mutations in SOD1 have been proposed to promote misfolding and aggregation by decreasing protein stability and increasing the proportion of less folded forms of the protein. Here we report direct measurement of the thermodynamic effects of chemically and structurally diverse mutations on the stability of the dimer interface for metal free (apo) SOD1 using isothermal titration calorimetry and size exclusion chromatography. Remarkably, all mutations studied, even ones distant from the dimer interface, decrease interface stability, and increase the population of monomeric SOD1. We interpret the thermodynamic data to mean that substantial structural perturbations accompany dimer dissociation, resulting in the formation of poorly packed and malleable dissociated monomers. These findings provide key information for understanding the mechanisms and energetics underlying normal maturation of SOD1, as well as toxic SOD1 misfolding pathways associated with disease. Furthermore, accurate prediction of protein–protein association remains very difficult, especially when large structural changes are involved in the process, and our findings provide a quantitative set of data for such cases, to improve modelling of protein association.

ABSTRACT

To advance our understanding of protein tertiary structure, the development of the knob-socket model is completed in an analysis of the packing in irregular coil and turn secondary structure packing as well as between mixed secondary structure. The knob-socket model simplifies packing based on repeated patterns of two motifs: a three-residue socket for packing within secondary (2°) structure and a four-residue knob-socket for tertiary (3°) packing. For coil and turn secondary structure, knob-sockets allow identification of a correlation between amino acid composition and tertiary arrangements in space. Coil contributes almost as much as α-helices to tertiary packing. In irregular sockets, Gly, Pro, Asp, and Ser are favored, while in irregular knobs, the preference order is Arg, Asp, Pro, Asn, Thr, Leu, and Gly. Cys, His,Met, and Trp are not favored in either. In mixed packing, the knob amino acid preferences are a function of the socket that they are packing into, whereas the amino acid composition of the sockets does not depend on the secondary structure of the knob. A unique motif of a coil knob with an XYZ β-sheet socket may potentially function to inhibit β-sheet extension. In addition, analysis of the preferred crossing angles for strands within a β-sheet and mixed α-helice/β-sheet identifies canonical packing patterns useful in protein design. Lastly, the knob-socket model abstracts the complexity of protein tertiary structure into an intuitive packing surface topology map. Proteins 2015; 83:2147–2161. © 2015 Wiley Periodicals, Inc.

ABSTRACT

Here, we provide insights into the thermodynamic properties of A dissociation from an amyloid fibril using all-atom molecular dynamics simulations in explicit water. An umbrella sampling protocol is used to compute potentials of mean force (PMF) as a function of the distance ξ between centers-of-mass of the A peptide and the preformed fibril at nine temperatures. Changes in the enthalpy and the entropic energy are determined from the temperature dependence of these PMF(s) and the average volume of the simulation box is computed as a function of ξ. We find that the PMF at 310 K is dominated by enthalpy while the entropic energy does not change significantly during dissociation. The volume of the system decreases during dissociation. Moreover, the magnitude of this volume change also decreases with increasing temperature. By defining dock and lock states using the solvent accessible surface area (SASA), we find that the behavior of the electrostatic energy is different in these two states. It increases (unfavorable) and decreases (favorable) during dissociation in lock and dock states, respectively, while the energy due to Lennard-Jones interactions increases continuously in these states. Our simulations also highlight the importance of hydrophobic interactions in accounting for the stability of A . Proteins 2015; 83:1963–1972. © 2015 Wiley Periodicals, Inc.

Abstract

Messenger RNA is recruited to the eukaryotic ribosome by a complex including the eukaryotic initiation factor (eIF) 4E (the cap-binding protein), the scaffold protein eIF4G and the RNA helicase eIF4A. To shut-off host–cell protein synthesis, eIF4G is cleaved during picornaviral infection by a virally encoded proteinase; the structural basis of this reaction and its stimulation by eIF4E is unclear. We have structurally and biochemically investigated the interaction of purified foot-and-mouth disease virus (FMDV) leader proteinase (Lbpro), human rhinovirus 2 (HRV2) 2A proteinase (2Apro) and coxsackievirus B4 (CVB4) 2Apro with purified eIF4GII, eIF4E and the eIF4GII/eIF4E complex. Using nuclear magnetic resonance (NMR), we completed 13C/15N sequential backbone assignment of human eIF4GII residues 551–745 and examined their binding to murine eIF4E. eIF4GII551–745 is intrinsically unstructured and remains so when bound to eIF4E. NMR and biophysical techniques for determining stoichiometry and binding constants revealed that the papain-like Lbpro only forms a stable complex with eIF4GII551–745 in the presence of eIF4E, with KD values in the low nanomolar range; Lbpro contacts both eIF4GII and eIF4E. Furthermore, the unrelated chymotrypsin-like 2Apro from HRV2 and CVB4 also build a stable complex with eIF4GII/eIF4E, but with KD values in the low micromolar range. The HRV2 enzyme also forms a stable complex with eIF4E; however, none of the proteinases tested complex stably with eIF4GII alone. Thus, these three picornaviral proteinases have independently evolved to establish distinct triangular heterotrimeric protein complexes that may actively target ribosomes involved in mRNA recruitment to ensure efficient host cell shut-off.

Projections from neocortex mediate top-down control of memory retrieval

Nature 526, 7575 (2015). doi:10.1038/nature15389

Authors: Priyamvada Rajasethupathy, Sethuraman Sankaran, James H. Marshel, Christina K. Kim, Emily Ferenczi, Soo Yeun Lee, Andre Berndt, Charu Ramakrishnan, Anna Jaffe, Maisie Lo, Conor Liston & Karl Deisseroth

Top-down prefrontal cortex inputs to the hippocampus have been hypothesized to be important in memory consolidation, retrieval, and the pathophysiology of major psychiatric diseases; however, no such direct projections have been identified and functionally described. Here we report the discovery of a monosynaptic prefrontal cortex

Hedgehog actively maintains adult lung quiescence and regulates repair and regeneration

Nature 526, 7574 (2015). doi:10.1038/nature14984

Authors: Tien Peng, David B. Frank, Rachel S. Kadzik, Michael P. Morley, Komal S. Rathi, Tao Wang, Su Zhou, Lan Cheng, Min Min Lu & Edward E. Morrisey

Postnatal tissue quiescence is thought to be a default state in the absence of a proliferative stimulus such as injury. Although previous studies have demonstrated that certain embryonic developmental programs are reactivated aberrantly in adult organs to drive repair and regeneration, it is not well understood how quiescence is maintained in organs such as the lung, which displays a remarkably low level of cellular turnover. Here we demonstrate that quiescence in the adult lung is an actively maintained state and is regulated by hedgehog signalling. Epithelial-specific deletion of sonic hedgehog (Shh) during postnatal homeostasis in the murine lung results in a proliferative expansion of the adjacent lung mesenchyme. Hedgehog signalling is initially downregulated during the acute phase of epithelial injury as the mesenchyme proliferates in response, but returns to baseline during injury resolution as quiescence is restored. Activation of hedgehog during acute epithelial injury attenuates the proliferative expansion of the lung mesenchyme, whereas inactivation of hedgehog signalling prevents the restoration of quiescence during injury resolution. Finally, we show that hedgehog also regulates epithelial quiescence and regeneration in response to injury via a mesenchymal feedback mechanism. These results demonstrate that epithelial–mesenchymal interactions coordinated by hedgehog actively maintain postnatal tissue homeostasis, and deregulation of hedgehog during injury leads to aberrant repair and regeneration in the lung.

Abstract

We introduce a new structure for a set of points in the plane and an angle \(\alpha \) , which is similar in flavor to a bounded-degree MST. We name this structure \(\alpha \) -MST. Let P be a set of points in the plane and let \(0 < \alpha \le 2\pi \) be an angle. An \(\alpha \) -ST of P is a spanning tree of the complete Euclidean graph induced by P, with the additional property that for each point \(p \in P\) , the smallest angle around p containing all the edges adjacent to p is at most \(\alpha \) . An \(\alpha \) -MST of P is then an \(\alpha \) -ST of P of minimum weight, where the weight of an \(\alpha \) -ST is the sum of the lengths of its edges. For \(\alpha < \pi /3\) , an \(\alpha \) -ST does not always exist, and, for \(\alpha \ge \pi /3\) , it always exists (Ackerman et al. in Comput Geom Theory Appl 46(3):213–218, 2013; Aichholzer et al. in Comput Geom Theory Appl 46(1):17–28, 2013; Carmi et al. in Comput Geom Theory Appl 44(9):477–485, 2011). In this paper, we study the problem of computing an \(\alpha \) -MST for several common values of \(\alpha \) . Motivated by wireless networks, we formulate the problem in terms of directional antennas. With each point \(p \in P\) , we associate a wedge \({\textsc {w}_{p}}\) of angle \(\alpha \) and apex p. The goal is to assign an orientation and a radius \(r_p\) to each wedge \({\textsc {w}_{p}}\) , such that the resulting graph is connected and its MST is an \(\alpha \) -MST (we draw an edge between p and q if \(p \in {\textsc {w}_{q}}\) , \(q \in {\textsc {w}_{p}}\) , and \(|pq| \le r_p, r_q\) ). We prove that the problem of computing an \(\alpha \) -MST is NP-hard, at least for \(\alpha =\pi \) and \(\alpha =2\pi /3\) , and present constant-factor approximation algorithms for \(\alpha = \pi /2, 2\pi /3, \pi \) . One of our major results is a surprising theorem for \(\alpha = 2\pi /3\) , which, besides being interesting from a geometric point of view, has important applications. For example, the theorem guarantees that given any set P of 3n points in the plane and any partitioning of the points into n triplets, one can orient the wedges of each triplet independently, such that the graph induced by P is connected. We apply the theorem to the antenna conversion problem and to the orientation and power assignment problem.

ABSTRACT

The backbone NH groups of proteins can form N1N3-bridges to δ-ve or anionic acceptor atoms when the tripeptide in which they occur orients them appropriately, as in the RL and LR nest motifs, which have dihedral angles 1,2-αRαL and 1,2-αLαR, respectively. We searched a protein database for structures with backbone N1N3-bridging to anionic atoms of the polypeptide chain and found that RL and LR nests together accounted for 92% of examples found (88% RL nests, 4% LR nests). Almost all the remaining 8% of N1N3-bridges were found within a third tripeptide motif which has not been described previously. We term this a “crown,” because of the disposition of the tripeptide CO groups relative to the three NH groups and the acceptor oxygen anion, and the crown together with its bridged anion we term a “crown bridge.” At position 2 of these structures the dihedral angles have a tight αR distribution, but at position 1 they have a wider distribution, with ϕ and ψ values generally being lower than those at position 1. Over half of crown bridges involve the backbone CO group three residues N-terminal to the tripeptide, the remainder being to other main-chain or side-chain carbonyl groups. As with nests, bridging of crowns to oxygen atoms within ligands was observed, as was bridging to the sulfur atom of an iron-sulfur cluster. This latter property may be of significance for protein evolution. Proteins 2015; 83:2067–2076. © 2015 Wiley Periodicals, Inc.

Abstract

In this paper, we study the problem of augmenting a planar graph such that it becomes \(k\) -regular, \(c\) -connected and remains planar, either in the sense that the augmented graph is planar, or in the sense that the input graph has a fixed (topological) planar embedding that can be extended to a planar embedding of the augmented graph. We fully classify the complexity of this problem for all values of  \(k\) and  \(c\) in both, the variable embedding and the fixed embedding case. For  \(k \le 2\) all problems are simple and for  \(k \ge 4\) all problems are NP-complete. Our main results are efficient algorithms (with running time \(O(n^{1.5}))\) for deciding the existence of a \(c\) -connected, 3-regular augmentation of a graph with a fixed planar embedding for  \(c=0,1,2\) and a corresponding hardness result for  \(c=3\) . The algorithms are such that for yes-instances a corresponding augmentation can be constructed in the same running time.

Abstract

We design a sublinear Fourier sampling algorithm for a case of sparse off-grid frequency recovery. These are signals with the form \(f(t) = \sum _{j=1}^k a_j \mathrm{e}^{i\omega _j t} + \int \nu (\omega )\mathrm{e}^{i\omega t}d\mu (\omega )\) ; i.e., exponential polynomials with a noise term. The frequencies \(\{\omega _j\}\) satisfy \(\omega _j\in [\eta ,2\pi -\eta ]\) and \(\min _{i\ne j} |\omega _i-\omega _j|\ge \eta \) for some \(\eta > 0\) . We design a sublinear time randomized algorithm which, for any \(\epsilon \in (0,\eta /k]\) , which takes \(O(k\log k\log (1/\epsilon )(\log k+\log (\Vert a\Vert _1/\Vert \nu \Vert _1))\) samples of \(f(t)\) and runs in time proportional to number of samples, recovering \(\omega _j'\approx \omega _j\) and \(a_j'\approx a_j\) such that, with probability \(\varOmega (1)\) , the approximation error satisfies \(|\omega _j'-\omega _j|\le \epsilon \) and \(|a_j-a_j'|\le \Vert \nu \Vert _1/k\) for all \(j\) with \(|a_j|\ge \Vert \nu \Vert _1/k\) . We apply our model and algorithm to bearing estimation or source localization and discuss their implications for receiver array processing.

Abstract

We consider the facility location problem with submodular penalties (FLPSP) and the facility location problem with linear penalties (FLPLP), two extensions of the classical facility location problem (FLP). First, we introduce a general algorithmic framework for a class of covering problems with submodular penalties, extending the recent result of Geunes et al. (Math Program 130:85–106, 2011) with linear penalties. This framework leverages existing approximation results for the original covering problems to obtain corresponding results for their counterparts with submodular penalties. Specifically, any LP-based \(\alpha \) -approximation for the original covering problem can be leveraged to obtain an \(\left( 1-e^{-1/\alpha }\right) ^{-1}\) -approximation algorithm for the counterpart with submodular penalties. Consequently, any LP-based approximation algorithm for the classical FLP (as a covering problem) can yield, via this framework, an approximation algorithm for the counterpart with submodular penalties. Second, by exploiting some special properties of submodular/linear penalty function, we present an LP rounding algorithm which has the currently best \(2\) -approximation ratio over the previously best \(2.375\) by Li et al. (Theoret Comput Sci 476:109–117, 2013) for the FLPSP and another LP-rounding algorithm which has the currently best \(1.5148\) -approximation ratio over the previously best \(1.853\) by Xu and Xu (J Comb Optim 17:424–436, 2008) for the FLPLP, respectively.

Publication date: Available online 28 September 2015
Source:Journal of Structural Biology

Author(s): Rui Zhang, Eva Nogales

Microtubules (MTs) are cylindrical polymers of αβ-tubulin that display pseudo-helical symmetry due to the presence of a lattice seam of heterologous lateral contacts. The structural similarity between α- and β-tubulin makes it difficult to computationally distinguish them in the noisy cryo-EM images, unless a marker protein for the tubulin dimer, such as kinesin motor domain, is present. We have developed a new data processing protocol that can accurately determine αβ-tubulin register and seam location for MT segments. Our strategy can deal with difficult situations, where the marker protein is relatively small or the decoration of marker protein is sparse. Using this new seam-search protocol, combined with movie processing for data from a direct electron detection camera, we were able to determine the cryo-EM structures of MT at 3.5 Å resolution in different functional states. The successful distinction of α- and β-tubulin allowed us to visualize the nucleotide state at the E-site and the configuration of lateral contacts at the seam.

Publication date: Available online 28 September 2015
Source:Journal of Structural Biology

Author(s): Elena P. Sablin, Raymond D. Blind, Rubatharshini Uthayaruban, H.J. Chiu, Ashley M. Deacon, Debanu Das, Holly A. Ingraham, Robert J. Fletterick

The nuclear receptor LRH-1 (Liver Receptor Homolog-1, NR5A2) is a transcription factor that regulates gene expression programs critical for many aspects of metabolism and reproduction. Although LRH-1 is able to bind phospholipids, it is still considered an orphan nuclear receptor (NR) with an unknown regulatory hormone. Our prior cellular and structural studies demonstrated that the signaling phosphatidylinositols PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3) bind and regulate SF-1 (Steroidogenic Factor-1, NR5A1), a close homolog of LRH-1. Here, we describe the crystal structure of human LRH-1 ligand binding domain (LBD) bound by PIP3 - the first phospholipid with a head group endogenous to mammals. We show that the phospholipid hormone binds LRH-1 with high affinity, stabilizing the receptor LBD. While the hydrophobic PIP3 tails (C16/C16) are buried inside the LRH-1 ligand binding pocket, the negatively charged PIP3 head group is presented on the receptor surface, similar to the phosphatidylinositol binding mode observed in the PIP3-SF-1 structure. Thus, data presented in this work reinforce our earlier findings demonstrating that signaling phosphatidylinositols regulate the NR5A receptors LRH-1 and SF-1.

Publication date: Available online 28 September 2015
Source:Journal of Structural Biology

Author(s): Allan H. Pang, Sylvie Garneau-Tsodikova, Oleg V. Tsodikov

Pyoluteorin is an antifungal agent composed of a 4,5-dichlorinated pyrrole group linked to a resorcinol moiety. The pyoluteorin biosynthetic gene cluster in Pseudomonas fluorescens Pf-5 encodes the halogenase PltA, which has been previously demonstrated to perform both chlorinations in vitro. PltA selectively accepts as a substrate a pyrrole moiety covalently tethered to a nonribosomal peptide thiolation domain PltL (pyrrolyl-S-PltL) for FAD-dependent di-chlorination, yielding 4,5-dichloropyrrolyl-S-PltL. We report a 2.75 Å-resolution crystal structure of PltA in complex with FAD and chloride. PltA is a dimeric enzyme, containing a flavin-binding fold conserved in flavin-dependent halogenases and monooxygenases, and an additional unique helical region at the C-terminus. This C-terminal region blocks a putative substrate-binding cleft, suggesting that a conformational change involving repositioning of this region is necessary to allow binding of the pyrrolyl-S-PltL substrate for its dichlorination by PltA.

Publication date: Available online 28 September 2015
Source:Journal of Structural Biology

Author(s): Willy Wriggers, Jing He

We are describing best practices and assessment strategies for the atomic interpretation of cryo-Electron Microscopy (cryo-EM) maps. Multiscale numerical geometry strategies in the Situs package and in secondary structure detection software are currently evolving due to the recent increases in cryo-EM resolution. Criteria that aim to predict the accuracy of fitted atomic models at low (worse than 8 Å) and medium (4-8 Å) resolution remain challenging. However, a high level of confidence in atomic models can be achieved by combining such criteria. The observed errors are due to map-model discrepancies and due to the effect of imperfect global docking strategies. Extending the earlier motion capture approach developed for flexible fitting, we use simulated fiducials (pseudoatoms) at varying levels of coarse graining to track the local drift of structural features. We compare three tracking approaches: naïve vector quantization, a smoothly deformable model, and a tessellation of the structure into rigid Voronoi cells which are fitted using a multi-fragment refinement approach. The lowest error is an upper bound for the (small) discrepancy between crystal structure and EM map due to different conditions in their structure determination. When internal features such as secondary structures are visible in medium-resolution EM maps, it is possible to extend the idea of point-based fiducials to more complex geometric representations such as helical axes, strands, and skeletons. We propose a quantitative strategies to assess map-model pairs when such secondary structure patterns are prominent.

Publication date: Available online 26 September 2015
Source:Journal of Structural Biology

Author(s): Plínio Salmazo Vieira, Priscila Oliveira de Giuseppe, Arthur Henrique Cavalcante de Oliveira, Mario Tyago Murakami

Nucleoside diphosphate kinase (NDK) is a housekeeping enzyme that plays key roles in nucleotide recycling and homeostasis in trypanosomatids. Moreover, it is secreted by the intracellular parasite Leishmania to modulate the host response. These functions make NDK an attractive target for drug design and for studies aiming at a better understanding of the mechanisms mediating host-pathogen interactions. Here, we report the crystal structures of three mutants of the NDK from Leishmania major (LmNDK) that affects the stability of the hexameric biological assembly including P95S, Δ5Ct (lacking the last five residues) and the double mutant P100S/Δ5Ct. Although P95S and Δ5Ct variants conserve the hexameric structure of the wild-type protein, the double mutant becomes a dimer as shown by in solution studies. Free energy calculation of dimer-dimer interfaces and enzymatic assays indicate that P95S, Δ5Ct and P100S/Δ5Ct mutations progressively decrease the hexamer stability and enzyme activity. These results demonstrate that the mutated regions play a role in protein function through stabilizing the quaternary arrangement.

Publication date: Available online 25 September 2015
Source:Journal of Structural Biology

Author(s): Kalvis Brangulis, Kristaps Jaudzems, Ivars Petrovskis, Inara Akopjana, Andris Kazaks, Kaspars Tars

Spirochete Borrelia burgdorferi is the causative agent of Lyme disease and is transmitted from infected Ixodes ticks to a mammalian host after a tick bite. The outer surface protein BB0689 from B. burgdorferi is up-regulated when the tick feeds, which indicates a potential role for BB0689 in Lyme disease pathogenesis. We have determined the crystal structure of BB0689, which revealed that the protein belongs to the CAP superfamily. Though the CAP domain is widespread in all three cellular domains of life, thus far the CAP domain has been studied only in eukaryotes, in which it is usually linked to certain other domains to form a multi-domain protein and is associated with the mammalian reproductive tract, the plant response to pathogens, venom allergens from insects and reptiles, and the growth of human brain tumors. Though the exact function of the isolated CAP domain remains ambiguous, several functions, including the binding of cholesterol, lipids and heparan sulfate, have been recently attributed to different CAP domain proteins. In this study, the bacterial CAP domain structure was analyzed and compared with the previously solved crystal structures of representative CAPs, and the function of BB0689 was examined. To determine the potential function of BB0689 and ascertain whether the functions that have been attributed to the CAP domain proteins are conserved, the binding of previously reported CAP domain interaction partners was analyzed, and the results suggested that BB0689 has a unique function that is yet to be discovered.

Publication date: Available online 25 September 2015
Source:Journal of Structural Biology

Author(s): Julien Ochala, Gianina Ravenscroft, Elyshia McNamara, Kristen J. Nowak, Hiroyuki Iwamoto

In humans, mutant skeletal muscle α-actin proteins are associated with contractile dysfunction, skeletal muscle weakness and a wide range of primarily skeletal muscle diseases. Despite this knowledge, the exact molecular mechanisms triggering the contractile dysfunction remain unknown. Here, we aimed to unravel these. Hence, we used a transgenic mouse model expressing a well-described D286G mutant skeletal muscle α-actin protein and recapitulating the human condition of contractile deregulation and severe skeletal muscle weakness. We then recorded and analysed the small-angle x-ray diffraction patterns of isolated membrane-permeabilized myofibres. Results showed that upon addition of Ca2+, the intensity changes of the second (1/19 nm−1) and sixth (1/5.9 nm−1) actin layer lines and of the first myosin meridional reflection (1/14.3 nm-1) were disrupted when the thin-thick filament overlap was optimal (sarcomere length of 2.5-2.6 μm). However these reflections were normal when the thin and thick filaments were not interacting (sarcomere length &gt;3.6 μm). These findings demonstrate, for the first time, that the replacement of just one amino acid in the skeletal muscle α-actin protein partly prevents actin conformational changes during activation, disrupting the strong binding of myosin molecules. This leads to a limited myosin-related tropomyosin movement over the thin filaments, further affecting the amount of cross-bridges, explaining the contractile dysfunction.
Graphical abstract