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Ge, X., Oliveira, A., Hjort, K., Bergfors, T., Gutiérrez-de-Terán, H., Andersson, D. I., . . . Åqvist, J. (2019). Inhibition of translation termination by small molecules targeting ribosomal release factors. Scientific Reports, 9, Article ID 15424.
Open this publication in new window or tab >>Inhibition of translation termination by small molecules targeting ribosomal release factors
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2019 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 15424Article in journal (Refereed) Published
Abstract [en]

The bacterial ribosome is an important drug target for antibiotics that can inhibit different stages of protein synthesis. Among the various classes of compounds that impair translation there are, however, no known small-molecule inhibitors that specifically target ribosomal release factors (RFs). The class I RFs are essential for correct termination of translation and they differ considerably between bacteria and eukaryotes, making them potential targets for inhibiting bacterial protein synthesis. We carried out virtual screening of a large compound library against 3D structures of free and ribosome-bound RFs in order to search for small molecules that could potentially inhibit termination by binding to the RFs. Here, we report identification of two such compounds which are found both to bind free RFs in solution and to inhibit peptide release on the ribosome, without affecting peptide bond formation.

Place, publisher, year, edition, pages
Nature Publishing Group, 2019
National Category
Medicinal Chemistry Structural Biology
Identifiers
urn:nbn:se:uu:diva-396310 (URN)10.1038/s41598-019-51977-1 (DOI)000492832300009 ()31659219 (PubMedID)
Funder
Swedish Research CouncilKnut and Alice Wallenberg FoundationSwedish National Infrastructure for Computing (SNIC)
Available from: 2019-11-01 Created: 2019-11-01 Last updated: 2019-11-18Bibliographically approved
Ge, X., Mandava, C. S., Lind, C., Åqvist, J. & Sanyal, S. (2018). Complementary charge-based interaction between the ribosomal-stalk protein L7/12 and IF2 is the key to rapid subunit association. Proceedings of the National Academy of Sciences of the United States of America, 115(18), 4649-4654
Open this publication in new window or tab >>Complementary charge-based interaction between the ribosomal-stalk protein L7/12 and IF2 is the key to rapid subunit association
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2018 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 18, p. 4649-4654Article in journal (Refereed) Published
Abstract [en]

The interaction between the ribosomal-stalk protein L7/12 (L12) and initiation factor 2 (IF2) is essential for rapid subunit association, but the underlying mechanism is unknown. Here, we have characterized the L12–IF2 interaction on Escherichia coli ribosomes using site-directed mutagenesis, fast kinetics, and molecular dynamics (MD) simulations. Fifteen individual point mutations were introduced into the C-terminal domain of L12 (L12-CTD) at helices 4 and 5, which constitute the common interaction site for translational GTPases. In parallel, 15 point mutations were also introduced into IF2 between the G4 and G5 motifs, which we hypothesized as the potential L12 interaction sites. The L12 and IF2 mutants were tested in ribosomal subunit association assay in a stopped-flow instrument. Those amino acids that caused defective subunit association upon substitution were identified as the molecular determinants of L12–IF2 interaction. Further, MD simulations of IF2 docked onto the L12-CTD pinpointed the exact interacting partners—all of which were positively charged on L12 and negatively charged on IF2, connected by salt bridges. Lastly, we tested two pairs of charge-reversed mutants of L12 and IF2, which significantly restored the yield and the rate of formation of the 70S initiation complex. We conclude that complementary charge-based interaction between L12-CTD and IF2 is the key for fast subunit association. Considering the homology of the G domain, similar mechanisms may apply for L12 interactions with other translational GTPases.

Keywords
protein synthesis, ribosomal protein L7/12, protein-protein interaction, ribosome, translation initiation
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-350185 (URN)10.1073/pnas.1802001115 (DOI)000431119600050 ()29686090 (PubMedID)
Funder
Swedish Research Council, 2014-4423; 2016-06264Knut and Alice Wallenberg Foundation, 2011.0081VINNOVA, 2013-8778
Available from: 2018-05-07 Created: 2018-05-07 Last updated: 2018-07-13Bibliographically approved
Li, Z., Ge, X., Zhang, Y., Zheng, L., Sanyal, S. & Gao, N. (2018). Cryo-EM structure of Mycobacterium smegmatis ribosome reveals two unidentified ribosomal proteins close to the functional centers [Letter to the editor]. Protein & cell, 9(4), 384-388
Open this publication in new window or tab >>Cryo-EM structure of Mycobacterium smegmatis ribosome reveals two unidentified ribosomal proteins close to the functional centers
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2018 (English)In: Protein & cell, ISSN 1674-8018, Vol. 9, no 4, p. 384-388Article in journal, Letter (Other academic) Published
National Category
Microbiology Cell Biology
Identifiers
urn:nbn:se:uu:diva-344575 (URN)10.1007/s13238-017-0456-9 (DOI)000428849800007 ()28875450 (PubMedID)
Funder
Swedish Research Council, 2013-8778; 2014-4423; 2016-06264; 2008-6593Knut and Alice Wallenberg Foundation, KAW 2011.0081
Available from: 2018-03-06 Created: 2018-03-06 Last updated: 2018-08-10Bibliographically approved
Kacar, B., Ge, X., Sanyal, S. & Gaucher, E. A. (2017). Experimental Evolution of Escherichia coli Harboring an Ancient Translation Protein. Journal of Molecular Evolution, 84(2-3), 69-84
Open this publication in new window or tab >>Experimental Evolution of Escherichia coli Harboring an Ancient Translation Protein
2017 (English)In: Journal of Molecular Evolution, ISSN 0022-2844, E-ISSN 1432-1432, Vol. 84, no 2-3, p. 69-84Article in journal (Refereed) Published
Abstract [en]

The ability to design synthetic genes and engineer biological systems at the genome scale opens new means by which to characterize phenotypic states and the responses of biological systems to perturbations. One emerging method involves inserting artificial genes into bacterial genomes and examining how the genome and its new genes adapt to each other. Here we report the development and implementation of a modified approach to this method, in which phylogenetically inferred genes are inserted into a microbial genome, and laboratory evolution is then used to examine the adaptive potential of the resulting hybrid genome. Specifically, we engineered an approximately 700-million-year-old inferred ancestral variant of tufB, an essential gene encoding elongation factor Tu, and inserted it in a modern Escherichia coli genome in place of the native tufB gene. While the ancient homolog was not lethal to the cell, it did cause a twofold decrease in organismal fitness, mainly due to reduced protein dosage. We subsequently evolved replicate hybrid bacterial populations for 2000 generations in the laboratory and examined the adaptive response via fitness assays, whole genome sequencing, proteomics, and biochemical assays. Hybrid lineages exhibit a general adaptive strategy in which the fitness cost of the ancient gene was ameliorated in part by upregulation of protein production. Our results suggest that an ancient-modern recombinant method may pave the way for the synthesis of organisms that exhibit ancient phenotypes, and that laboratory evolution of these organisms may prove useful in elucidating insights into historical adaptive processes.

Place, publisher, year, edition, pages
SPRINGER, 2017
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-320859 (URN)10.1007/s00239-017-9781-0 (DOI)000398294900001 ()28233029 (PubMedID)
Funder
Swedish Research Council, 2013-8778 2014-4423 2008-6593Knut and Alice Wallenberg Foundation, KAW2011.0081
Available from: 2017-04-26 Created: 2017-04-26 Last updated: 2017-04-26Bibliographically approved
Qiang, X., Liotta, A. S., Shiloach, J., Gutierrez, J. C., Wang, H., Ochani, M., . . . Roth, J. (2017). New melanocortin-like peptide of E. coli can suppress inflammation via the mammalian melanocortin-1 receptor (MC1R): possible endocrine-like function for microbes of the gut.. NPJ biofilms and microbiomes, 3, Article ID 31.
Open this publication in new window or tab >>New melanocortin-like peptide of E. coli can suppress inflammation via the mammalian melanocortin-1 receptor (MC1R): possible endocrine-like function for microbes of the gut.
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2017 (English)In: NPJ biofilms and microbiomes, ISSN 2055-5008, Vol. 3, article id 31Article in journal (Refereed) Published
Abstract [en]

E. coli releases a 33 amino acid peptide melanocortin-like peptide of E. coli (MECO-1) that is identical to the C-terminus of the E. coli elongation factor-G (EF-G) and has interesting similarities to two prominent mammalian melanocortin hormones, alpha-melanocyte-stimulating hormone (alpha-MSH) and adrenocorticotropin (ACTH). Note that MECO-1 lacks HFRW, the common pharmacophore of the known mammalian melanocortin peptides. MECO-1 and the two hormones were equally effective in severely blunting release of cytokines (HMGB1 and TNF) from macrophage-like cells in response to (i) endotoxin (lipopolysaccharide) or (ii) pro-inflammatory cytokine HMGB-1. The in vitro anti-inflammatoty effects of MECO-1 and of alpha-MSH were abrogated by (i) antibody against melanocortin-1 receptor (MC1R) and by (ii) agouti, an endogenous inverse agonist of MC1R. In vivo MECO-1 was even more potent than alpha-MSH in rescuing mice from death due to (i) lethal doses of LPS endotoxin or (ii) cecal ligation and puncture, models of sterile and infectious sepsis, respectively.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:uu:diva-344574 (URN)10.1038/s41522-017-0039-9 (DOI)000416775100001 ()29152323 (PubMedID)
Available from: 2018-03-06 Created: 2018-03-06 Last updated: 2018-03-06Bibliographically approved
Mohapatra, S., Choi, H., Ge, X., Sanyal, S. & Weisshaar, J. C. (2017). Spatial Distribution and Ribosome-Binding Dynamics of EF-P in Live Escherichia coli. mBio, 8(3), Article ID e00300-17.
Open this publication in new window or tab >>Spatial Distribution and Ribosome-Binding Dynamics of EF-P in Live Escherichia coli
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2017 (English)In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 8, no 3, article id e00300-17Article in journal (Refereed) Published
Abstract [en]

In vitro assays find that ribosomes form peptide bonds to proline (Pro) residues more slowly than to other residues. Ribosome profiling shows that stalling at Pro-Pro-X triplets is especially severe but is largely alleviated in Escherichia coli by the action of elongation factor EF-P. EF-P and its eukaryotic/archaeal homolog IF5A enhance the peptidyl transfer step of elongation. Here, a superresolution fluorescence localization and tracking study of EF-P-mEos2 in live E. coli provides the first in vivo information about the spatial distribution and on-off binding kinetics of EF-P. Fast imaging at 2 ms/frame helps to distinguish ribosome-bound (slowly diffusing) EF-P from free (rapidly diffusing) EF-P. Wild-type EF-P exhibits a three-peaked axial spatial distribution similar to that of ribosomes, indicating substantial binding. The mutant EF-P-K34A exhibits a homogeneous distribution, indicating little or no binding. Some 30% of EF-P copies are bound to ribosomes at a given time. Two-state modeling and copy number estimates indicate that EF-P binds to 70S ribosomes during 25 to 100% of translation cycles. The timescale of the typical diffusive search by free EF-P for a ribosome-binding site is tau(free) approximate to 16 ms. The typical residence time of an EF-P on the ribosome is very short, tau(bound) approximate to 7 ms. Evidently, EF-P binds to ribosomes during many or most elongation cycles, much more often than the frequency of Pro-Pro motifs. Emptying of the E site during part of the cycle is consistent with recent in vitro experiments indicating dissociation of the deacylated tRNA upon translocation. IMPORTANCE Ribosomes translate the codon sequence within mRNA into the corresponding sequence of amino acids within the nascent polypeptide chain, which in turn ultimately folds into functional protein. At each codon, bacterial ribosomes are assisted by two well-known elongation factors: EF-Tu, which aids binding of the correct aminoacyl-tRNA to the ribosome, and EF-G, which promotes tRNA translocation after formation of the new peptide bond. A third factor, EF-P, has been shown to alleviate ribosomal pausing at rare Pro-Pro motifs, which are translated very slowly without EF-P. Here, we use superresolution fluorescence imaging to study the spatial distribution and ribosome-binding dynamics of EF-P in live E. coli cells. We were surprised to learn that EF-P binds to and unbinds from translating ribosomes during at least 25% of all elongation events; it may bind during every elongation cycle.

Place, publisher, year, edition, pages
AMER SOC MICROBIOLOGY, 2017
Keywords
EF-P, binding dynamics, live E. coli, superresolution fluorescence
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-330056 (URN)10.1128/mBio.00300-17 (DOI)000404733300004 ()
Available from: 2017-11-16 Created: 2017-11-16 Last updated: 2017-11-16Bibliographically approved
Degiacomi, G., Personne, Y., Mondesert, G., Ge, X., Mandava, C. S., Hartkoorn, R. C., . . . Manganelli, R. (2016). Micrococcin P1-A bactericidal thiopeptide active against Mycobacterium tuberculosis. Tuberculosis, 100, 95-101
Open this publication in new window or tab >>Micrococcin P1-A bactericidal thiopeptide active against Mycobacterium tuberculosis
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2016 (English)In: Tuberculosis, ISSN 1472-9792, E-ISSN 1873-281X, Vol. 100, p. 95-101Article in journal (Refereed) Published
Abstract [en]

The lack of proper treatment for serious infectious diseases due to the emergence of multidrug resistance reinforces the need for the discovery of novel antibiotics. This is particularly true for tuberculosis (TB) for which 3.7% of new cases and 20% of previously treated cases are estimated to be caused by multi-drug resistant strains. In addition, in the case of TB, which claimed 1.5 million lives in 2014, the treatment of the least complicated, drug sensitive cases is lengthy and disagreeable. Therefore, new drugs with novel targets are urgently needed to control resistant Mycobacterium tuberculosis strains. In this manuscript we report the characterization of the thiopeptide micrococcin P1 as an anti-tubercular agent. Our biochemical experiments show that this antibiotic inhibits the elongation step of protein synthesis in mycobacteria. We have further identified micrococcin resistant mutations in the ribosomal protein L11 (RplK); the mutations were located in the proline loop at the N-terminus. Reintroduction of the mutations into a clean genetic background, confirmed that they conferred resistance, while introduction of the wild type RplK allele into resistant strains re-established sensitivity. We also identified a mutation in the 23S rRNA gene. These data, in good agreement with previous structural studies suggest that also in M. tuberculosis micrococcin P1 functions by binding to the cleft between the 23S rRNA and the L11 protein loop, thus interfering with the binding of elongation factors Tu and G (EF-Tu and EF-G) and inhibiting protein translocation.

Keywords
Tuberculosis, Drug development, Translation, Thiopeptides
National Category
Cell and Molecular Biology Respiratory Medicine and Allergy
Identifiers
urn:nbn:se:uu:diva-307870 (URN)10.1016/j.tube.2016.07.011 (DOI)000382201400013 ()27553416 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 260872Swedish Research Council, 2014-4423
Available from: 2016-11-22 Created: 2016-11-22 Last updated: 2019-11-08Bibliographically approved
Chai, Q., Singh, B., Peisker, K., Metzendorf, N., Ge, X., Dasgupta, S. & Sanyal, S. (2014). Organization of Ribosomes and Nucleoids in Escherichia coli Cells during Growth and in Quiescence. Journal of Biological Chemistry, 289(16), 11342-11352
Open this publication in new window or tab >>Organization of Ribosomes and Nucleoids in Escherichia coli Cells during Growth and in Quiescence
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2014 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 289, no 16, p. 11342-11352Article in journal (Refereed) Published
Abstract [en]

Background: We studied ribosome and nucleoid distribution in Escherichia coli under growth and quiescence. Results: Spatially segregated ribosomes and nucleoids show drastically altered distribution in stationary phase or when treated with drugs affecting translation, transcription, nucleoid-topology, or cytoskeleton. Ribosome inheritance in daughter cells is frequently unequal. Conclusion: Cellular growth processes modulate ribosome and nucleoid distribution. Significance: This provides insight into subcellular organization of molecular machines. We have examined the distribution of ribosomes and nucleoids in live Escherichia coli cells under conditions of growth, division, and in quiescence. In exponentially growing cells translating ribosomes are interspersed among and around the nucleoid lobes, appearing as alternative bands under a fluorescence microscope. In contrast, inactive ribosomes either in stationary phase or after treatment with translation inhibitors such as chloramphenicol, tetracycline, and streptomycin gather predominantly at the cell poles and boundaries with concomitant compaction of the nucleoid. However, under all conditions, spatial segregation of the ribosomes and the nucleoids is well maintained. In dividing cells, ribosomes accumulate on both sides of the FtsZ ring at the mid cell. However, the distribution of the ribosomes among the new daughter cells is often unequal. Both the shape of the nucleoid and the pattern of ribosome distribution are also modified when the cells are exposed to rifampicin (transcription inhibitor), nalidixic acid (gyrase inhibitor), or A22 (MreB-cytoskeleton disruptor). Thus we conclude that the intracellular organization of the ribosomes and the nucleoids in bacteria are dynamic and critically dependent on cellular growth processes (replication, transcription, and translation) as well as on the integrity of the MreB cytoskeleton.

Keywords
Antibiotics, Cell Division, Cytoskeleton, DNA Replication, Ribosomes, Subcellular Organelles, Transcription, Translation, FtsZ Ring, Nucleoid
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-227282 (URN)10.1074/jbc.M114.557348 (DOI)000334638500036 ()
Funder
Swedish Research Council, 2010-2619, 2011-6088, 2008-6593Carl Tryggers foundation , 09:341, 10:330Knut and Alice Wallenberg Foundation, 2011.0081
Available from: 2014-06-30 Created: 2014-06-24 Last updated: 2017-12-05Bibliographically approved
Mandava, C. S., Peisker, K., Ederth, J., Kumar, R., Ge, X., Szaflarski, W. & Sanyal, S. (2012). Bacterial ribosome requires multiple L12 dimers for efficient initiation and elongation of protein synthesis involving IF2 and EF-G. Nucleic Acids Research, 40(5), 2054-2064
Open this publication in new window or tab >>Bacterial ribosome requires multiple L12 dimers for efficient initiation and elongation of protein synthesis involving IF2 and EF-G
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2012 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 5, p. 2054-2064Article in journal (Refereed) Published
Abstract [en]

The ribosomal stalk in bacteria is composed of four or six copies of L12 proteins arranged in dimers that bind to the adjacent sites on protein L10, spanning 10 amino acids each from the L10 C-terminus. To study why multiple L12 dimers are required on the ribosome, we created a chromosomally engineered Escherichia coli strain, JE105, in which the peripheral L12 dimer binding site was deleted. Thus JE105 harbors ribosomes with only a single L12 dimer. Compared to MG1655, the parental strain with two L12 dimers, JE105 showed significant growth defect suggesting suboptimal function of the ribosomes with one L12 dimer. When tested in a cell-free reconstituted transcription-translation assay the synthesis of a full-length protein, firefly luciferase, was notably slower with JE105 70S ribosomes and 50S subunits. Further, in vitro analysis by fast kinetics revealed that single L12 dimer ribosomes from JE105 are defective in two major steps of translation, namely initiation and elongation involving translational GTPases IF2 and EF-G. Varying number of L12 dimers on the ribosome can be a mechanism in bacteria for modulating the rate of translation in response to growth condition.

Keywords
ribosome, translation initiation, L12, IF2, subunit association, protein synthesis
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Biology
Identifiers
urn:nbn:se:uu:diva-157694 (URN)10.1093/nar/gkr1031 (DOI)000302019900022 ()22102582 (PubMedID)
Available from: 2012-03-13 Created: 2011-08-22 Last updated: 2017-12-08Bibliographically approved
Gallwitz, M., Enoksson, M., Thorpe, M., Ge, X. & Hellman, L. (2010). The extended substrate recognition profile of the dog mast cell chymase reveals similarities and differences to the human chymase. International Immunology, 22(6), 421-431
Open this publication in new window or tab >>The extended substrate recognition profile of the dog mast cell chymase reveals similarities and differences to the human chymase
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2010 (English)In: International Immunology, ISSN 0953-8178, E-ISSN 1460-2377, Vol. 22, no 6, p. 421-431Article in journal (Refereed) Published
Abstract [en]

Human chymase (HC) constitutes a major granule protease in one of the two human mast cell (MC) types. The main biological role of this haematopoietic serine protease is probably not yet known, although it has been implicated in a large number of functions. Dogs, like humans, have only one chymase. This enzyme is closely related to its human homologue, and the MC subtypes of human and dog appear to be similar as well. Therefore, the functions of the dog chymase (DC) may closely reflect the functions of the HC. Moreover, dogs may serve as good models for studies of human MC functions and MC-related diseases. To reveal functional similarities and differences between the DC and HC, we have determined the extended cleavage specificity of the DC by substrate phage display. This method allows the simultaneous permutation of primed and unprimed substrate positions. The DC was found to have very similar preferences to its human counterpart for substrate positions P1, P3, P4 and P3', whereas their preferences differ at positions P2, P1' and P2'. Therefore, the HC and DC may have co-evolved with a substrate where positions P1, P3, P4 and P3' are conserved between dogs and humans, whereas positions P2 and P1' are not and P2' differs to a minor extent. The differences observed between these two enzymes suggest that results obtained from dog models cannot be directly extrapolated to human clinical settings but need to be evaluated carefully concerning potential differences in substrate preferences.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-95246 (URN)10.1093/intimm/dxq021 (DOI)000279109000002 ()
Available from: 2006-11-29 Created: 2006-11-29 Last updated: 2017-12-14
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ORCID iD: ORCID iD iconorcid.org/0000-0001-7954-3195

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