PNAS Plus Significance Statements

PNAS Plus Significance Statements

PNAS October 28, 2014 111 (43) 15293-15295; https://doi.org/10.1073/pnas.ss11143

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

Daniel S. Liu, Lucas G. Nivón, Florian Richter, Peter J. Goldman, Thomas J. Deerinck, Jennifer Z. Yao, Douglas Richardson, William S. Phipps, Anne Z. Ye, Mark H. Ellisman, Catherine L. Drennan, David Baker, and Alice Y. Ting

This work (pp. E4551–E4559) establishes a fluorescence labeling method that can be used in living cells to derivatize specific proteins of interest with a small red fluorophore, resorufin. The method has extremely high sequence specificity and is based on a computationally designed fluorophore ligase, derived from Escherichia coli lipoic acid ligase. These results demonstrate the power of computational design to majorly reengineer enzyme specificity. Extensive screening of rationally designed enzyme mutants failed to achieve the same result.

Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: Unravelling the role of Mg2+ in cell respiration

Elisabeth Gout, Fabrice Rébeillé, Roland Douce, and Richard Bligny

The respiration of heterotrophic cells, where most of the ATP demand is met by mitochondrial oxidative phosphorylation, is generally thought to be regulated either by the ATP/ADP ratio and/or energy charge or by nucleotide concentration. The way in which ADP and ATP may directly mediate respiration remains unclear, however. Furthermore, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP-synthase, Mg2+ compartmentation must be known. For this purpose, we performed (pp. E4560–E4567) simultaneous measurements of free and Mg-complexed nucleotides and Mg2+ in the cytosol and mitochondrial matrix using NMR-based techniques. Physiological alterations induced by Mg starvation helped unravel the key role of cytosolic and mitochondrial Mg2+ and free ADP in the regulation of cell respiration.

Aggregation propensities of superoxide dismutase G93 hotspot mutants mirror ALS clinical phenotypes

Ashley J. Pratt, David S. Shin, Gregory E. Merz, Robert P. Rambo, W. Andrew Lancaster, Kevin N. Dyer, Peter P. Borbat, Farris L. Poole II, Michael W. W. Adams, Jack H. Freed, Brian R. Crane, John A. Tainer, and Elizabeth D. Getzoff

Mutations in human Cu, Zn superoxide dismutase (SOD) cause the motor neuron disease ALS. To better understand why, we compared the aggregation, metal binding, and conformational dynamics of normal and mutant SOD proteins by using the biophysical techniques of X-ray scattering, inductively coupled plasma MS, and ESR spectroscopy. For SOD proteins with defects at a mutational hotspot, we found that copper deficiency, flexibility, and aggregation paralleled clinical severity in ALS patients. These data (pp. E4568–E4576) support a unifying protein framework destabilization mechanism for SOD-linked ALS and thereby point to potential therapies for this lethal condition with few treatment options.

eIF4AIII enhances translation of nuclear cap-binding complex–bound mRNAs by promoting disruption of secondary structures in 5′UTR

Junho Choe, Incheol Ryu, Ok Hyun Park, Joori Park, Hana Cho, Jin Seon Yoo, Sung Wook Chi, Min Kyung Kim, Hyun Kyu Song, and Yoon Ki Kim

Two major cap-binding components can drive mammalian translation initiation: the nuclear cap-binding complex (CBC) and eukaryotic translation initiation factor 4E (eIF4E). Although eIF4E-dependent translation has been well characterized, the mechanism of CBC-dependent translation remains unclear. Here (pp. E4577–E4586), we demonstrate the previously unappreciated role of eIF4AIII in CBC-dependent translation. eIF4AIII is traditionally considered a component of the exon junction complex loaded onto mRNAs after splicing. In addition, we found that eIF4AIII can be recruited to the 5′-end of CBC-associated mRNA and promotes the translation of CBC-associated mRNA by helping to unwind secondary structures in 5′UTR. Therefore, our data provide evidence that eIF4AIII is a translation initiation factor specifically required for translation of CBC-associated mRNAs.

Crystal structure of Mycobacterium tuberculosis ClpP1P2 suggests a model for peptidase activation by AAA+ partner binding and substrate delivery

Karl R. Schmitz, Daniel W. Carney, Jason K. Sello, and Robert T. Sauer

Caseinolytic peptidase P (ClpP) normally collaborates with ATPases associated with diverse activities (AAA+) partner proteins, such as ClpX and ClpC, to carry out energy-dependent degradation of proteins within cells. The ClpP enzyme from Mycobacterium tuberculosis is required for survival of this human pathogen, is a validated drug target, and is unusual in consisting of discrete ClpP1 and ClpP2 rings. We solved the crystal structure of ClpP1P2 bound to peptides that mimic binding of protein substrates and small molecules that mimic binding of a AAA+ partner and cause unregulated rogue proteolysis. These studies (pp. E4587–E4595) explain why two different ClpP rings are required for peptidase activity and provide a foundation for the rational development of drugs that target ClpP1P2 and kill M. tuberculosis.

Structural basis of thymosin-β4/profilin exchange leading to actin filament polymerization

Bo Xue, Cedric Leyrat, Jonathan M. Grimes, and Robert C. Robinson

Thymosin-β4 (Tβ4) sequesters actin monomers to help maintain the high concentrations of unpolymerized actin in higher eukaryotic cells. Despite more than two decades of research investigating the Tβ4–actin interaction, the X-ray structure of the full-length Tβ4:actin complex remained unresolved. Here (pp. E4596–E4605), we report two X-ray structures of Tβ4:actin complexes. The first structure reveals that Tβ4 has two helices that bind at the barbed and pointed faces of actin, whereas the second structure displays a more open actin nucleotide binding cleft and a disruption of the Tβ4 C-terminal helix interaction. These structures, combined with biochemical assays and molecular dynamics simulations, reveal how Tβ4 prevents monomeric actin from joining actin filaments but participates in the exchange of actin with profilin to ensure controlled actin polymerization.

Capsid expansion mechanism of bacteriophage T7 revealed by multistate atomic models derived from cryo-EM reconstructions

Fei Guo, Zheng Liu, Ping-An Fang, Qinfen Zhang, Elena T. Wright, Weimin Wu, Ci Zhang, Frank Vago, Yue Ren, Joanita Jakana, Wah Chiu, Philip Serwer, and Wen Jiang

T7 phage has been used as a model system to study dsDNA virus capsid assembly and maturation. Yet, atomic capsid models and details of capsid transformations are not elucidated. From our cryo-EM study we have derived near-atomic resolution reconstructions of the DNA-free procapsid, a DNA packaging intermediate, and the DNA-packaged, mature phage capsid. From these structures, we have derived (pp. E4606–E4614) the first near-atomic-level model of T7 capsid maturation. The structural knowledge obtained from this study can serve as a platform for analysis of other dsDNA viruses as well as a platform for the development of molecular tools such as improved phage display systems.

Locating folds of the in-register parallel β-sheet of the Sup35p prion domain infectious amyloid

Anton Gorkovskiy, Kent R. Thurber, Robert Tycko, and Reed B. Wickner

Infectious proteins (prions) are capable of encoding genetic information by templating their conformation, just as DNA templates its sequence. The mechanism of this templating has not been clear. We provide (pp. E4615–E4622) definitive proof that the architecture of amyloid of the prion domain of yeast prion protein Sup35p is a folded in-register parallel β-sheet, and our data identify some of the sites of folds in the sheet. This architecture naturally suggests a templating mechanism based on favorable interactions among aligned side chains of identical amino acids. This is the only mechanism suggested to date for such a conformation templating.

Dynamic architecture of a protein kinase

Christopher L. McClendon, Alexandr P. Kornev, Michael K. Gilson, and Susan S. Taylor

Protein kinases represent a critically important family of regulatory enzymes. Their activity can be altered by mutations and binding events distant from the active site. To understand the nature of these long-distance effects, we used microsecond-timescale molecular dynamic simulation to subdivide a prototypical kinase, protein kinase A, into contiguous communities that exhibit internally correlated motions. Surprisingly, most of these unconventional structural entities were centered around known protein kinase functions. We thus propose a new framework for analysis of protein kinase structure and function that differs from traditional representations based simply on sequence motifs and secondary structure elements. These results (pp. E4623–E4631) extend our view on the dynamic nature of protein kinases and open a door to understanding of allosteric signaling in these enzymes.

Facilitated sequence counting and assembly by template mutagenesis

Dan Levy and Michael Wigler

Despite vast improvements in DNA sequencing, many problems of interpretation arise when trying to count or assemble molecules (templates) that are largely identical. We show (pp. E4632–E4637) through simulations that by randomly mutagenizing DNA templates before amplification, many of these problems are resolved. We can obtain more accurate counting of templates by observing the number of unique patterns. By introducing distinctive patterns onto otherwise identical spans, we enhance our ability to correctly assemble sequences. This idea can be implemented with currently available mutagenesis protocols and such techniques can have applications in RNA expression analysis, haplotype phasing, copy number determination, and genome assembly. Template mutagenesis solves counting problems and effectively generates long reads from short-read sequence output.

Spleen supports a pool of innate-like B cells in white adipose tissue that protects against obesity-associated insulin resistance

Lan Wu, Vrajesh V. Parekh, Joseph Hsiao, Daisuke Kitamura, and Luc Van Kaer

The rise in obesity-associated diseases has led to increased efforts to identify new therapeutic targets. Lipid accumulation in obesity triggers a low-grade inflammation that links obesity to its associated diseases, notably insulin resistance and type 2 diabetes. In this report (pp. E4638–E4647), we have studied a group of innate-like B lymphocytes in visceral white adipose tissue (VAT) of mice that are (i) capable of producing the anti-inflammatory mediator IL-10; (ii) replenished from precursor cells in spleen during diet-induced obesity (DIO); and (iii) impaired in VAT of DIO mice, resulting in diminished protection against obesity-associated insulin resistance that can be ameliorated by supplementation of these cells. These findings identify spleen-supplied innate-like B cells in VAT as previously unidentified therapeutic targets for obesity-associated diseases.

Molecular basis of mycobacterial lipid antigen presentation by CD1c and its recognition by αβ T cells

Sobhan Roy, Dalam Ly, Nan-Sheng Li, John D. Altman, Joseph A. Piccirilli, D. Branch Moody, and Erin J. Adams

Mycobacterium tuberculosis infects more than one-third of humans yet no effective vaccine exists. This study shows how a subset of αβ T cells targets M. tuberculosis lipid antigens that are presented by the MHC molecule CD1c. In contrast to many T cells that recognize CD1d, these αβ T cells express diverse T-cell receptors and have differing footprints on CD1c during lipid recognition. This study (pp. E4648–E4657) also shows that some CD1c-specific αβ T cells are exquisitely specific for the lipid presented, whereas others have a more promiscuous reactivity, demonstrating that the αβ T-cell response to CD1c lipid presentation is diverse and adaptable. These data may provide additional resources for development of MHC-independent vaccines against M. tuberculosis.

Intestinal myofibroblast-specific Tpl2-Cox-2-PGE2 pathway links innate sensing to epithelial homeostasis

Manolis Roulis, Christoforos Nikolaou, Elena Kotsaki, Eleanna Kaffe, Niki Karagianni, Vasiliki Koliaraki, Klelia Salpea, Jiannis Ragoussis, Vassilis Aidinis, Eva Martini, Christoph Becker, Harvey R. Herschman, Stefania Vetrano, Silvio Danese, and George Kollias

Tumor progression locus-2 (Tpl2) is a proinflammatory gene genetically associated with inflammatory bowel diseases. This study (pp. E4658–E4667) provides a mechanistic interpretation for this association showing a dominant Tpl2-mediated homeostatic mechanism protecting mice from epithelial injury-induced colitis. This function of Tpl2 is mediated specifically by subepithelial intestinal myofibroblasts, a cell type supporting crypt stem cells. Tpl2 in myofibroblasts is essential for the compensatory proliferative response of the epithelium by promoting arachidonic acid metabolism and cyclooxygenase-2 (Cox-2)/prostaglandin E2 activation. Notably, in Crohn’s Disease patients, Tpl2 is downregulated in myofibroblasts isolated from the inflamed ileum. These results challenge current concepts on a solely proinflammatory function of Tpl2 and highlight the dominant role of subepithelial myofibroblasts in sensing inflammation and tissue damage and promoting intestinal homeostasis through Tpl2-Cox-2-prostaglandin E2.

A nonpyrrolysine member of the widely distributed trimethylamine methyltransferase family is a glycine betaine methyltransferase

Tomislav Ticak, Duncan J. Kountz, Kimberly E. Girosky, Joseph A. Krzycki, and Donald J. Ferguson Jr.

Pyrrolysine, the 22nd amino acid, is found in few proteins. One, the trimethylamine methyltransferase MttB, forms a small portion of a large family of proteins. Most in this family lack pyrrolysine and have no known activity. We show that one such protein, MtgB, is a glycine betaine methyltransferase, providing functional context that may explain the relationship between family members with and without pyrrolysine. Close relatives of MtgB are encoded in many of the abundant bacteria in the oceans, as well as different microbes undertaking symbioses ranging from plants to humans. This finding (pp. E4668–E4676) implies that MtgB might partake in a widespread and underappreciated pathway of GB metabolism contributing significantly to global carbon and nitrogen cycling as well as human health.

Low-frequency calcium oscillations accompany deoxyhemoglobin oscillations in rat somatosensory cortex

Congwu Du, Nora D. Volkow, Alan P. Koretsky, and Yingtian Pan

Spontaneous low-frequency oscillations (LFOs) of blood-oxygen-level-dependent (BOLD) signals in brain constitute the basis for mapping resting functional connectivity with functional MRI (fMRI). However the origin of these LFOs is not well understood. Using optical imaging we provide evidence that (i) LFOs in calcium (marker of cellular oscillations) show frequencies similar to those of deoxyhemoglobin (main contributor to the BOLD signal) and precede them by 5–6 s; (ii) hemodynamic slow oscillations (including LFOs in deoxyhemoglobin) also correlate with spontaneous neuronal firing activity (as assessed with slow local field potentials); and (iii) LFOs of deoxyhemoglobin (HbR) are observed in arteries, veins, and capillaries. These findings (pp. E4677–E4686) therefore corroborate the cellular basis underlying resting-state fMRI and indicate that oscillating HbR signals are detectable across the vascular tree.

Coupled neural systems underlie the production and comprehension of naturalistic narrative speech

Lauren J. Silbert, Christopher J. Honey, Erez Simony, David Poeppel, and Uri Hasson

Successful verbal communication requires the finely orchestrated interaction between production-based processes in the speaker’s brain and comprehension-based processes in the listener’s brain. Here we first develop a time-warping tool that enables us to map all brain areas reliably activated during the production of real-world speech. The results indicate that speech production is not localized to the left hemisphere but recruits an extensive bilateral network of linguistic and extralinguistic brain areas. We then directly compare the neural responses during speech production and comprehension and find that the two systems respond in similar ways. Our results (pp. E4687–E4696) argue that a shared neural mechanism supporting both production and comprehension facilitates communication and underline the importance of studying comprehension and production within unified frameworks.

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PNAS Plus Significance Statements
Proceedings of the National Academy of Sciences Oct 2014, 111 (43) 15293-15295; DOI: 10.1073/pnas.ss11143
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Proceedings of the National Academy of Sciences: 111 (43)
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