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  • 1.
    Aqvist, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Lind, Christoffer
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Sund, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Wallin, Goran
    Bridging the gap between ribosome structure and biochemistry by mechanistic computations2012In: Current opinion in structural biology, ISSN 0959-440X, E-ISSN 1879-033X, Vol. 22, no 6, p. 815-823Article in journal (Refereed)
    Abstract [en]

    The wealth of structural and biochemical data now available for protein synthesis on the ribosome presents major new challenges for computational biochemistry. Apart from technical difficulties in modeling ribosome systems, the complexity of the overall translation cycle with a multitude of different kinetic steps presents a formidable problem for computational efforts where we have only seen the beginning. However, a range of methodologies including molecular dynamics simulations, free energy calculations, molecular docking and quantum chemical approaches have already been put to work with promising results. In particular, the combined efforts of structural biology, biochemistry, kinetics and computational modeling can lead towards a quantitative structure-based description of translation.

  • 2.
    Ge, Xueliang
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Mandava, Chandra Sekhar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Lind, Christoffer
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Complementary charge-based interaction between the ribosomal-stalk protein L7/12 and IF2 is the key to rapid subunit association2018In: 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)
    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.

  • 3.
    Korkmaz, Gürkan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Lind, Christoffer
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Characterizing an engineered release factor capable of reading all three stop codons2014In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 28, no 1, article id 569.2Article in journal (Other academic)
  • 4.
    Lind, Christoffer
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Computational Studies of Protein Synthesis on the Ribosome and Ligand Binding to Riboswitches2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The ribosome is a macromolecular machine that produces proteins in all kingdoms of life. The proteins, in turn, control the biochemical processes within the cell. It is thus of extreme importance that the machine that makes the proteins works with high precision. By using three dimensional structures of the ribosome and homology modelling, we have applied molecular dynamics simulations and free-energy calculations to study the codon specificity of protein synthesis in initiation and termination on an atomistic level. In addition, we have examined the binding of small molecules to riboswitches, which can change the expression of an mRNA.

    The relative affinities on the ribosome between the eukaryotic initiator tRNA to the AUG start codon and six near-cognate codons were determined. The free-energy calculations show that the initiator tRNA has a strong preference for the start codon, but requires assistance from initiation factors 1 and 1A to uphold discrimination against near-cognate codons.

    When instead a stop codon (UAA, UGA or UAG) is positioned in the ribosomal A-site, a release factor binds and terminates protein synthesis by hydrolyzing the nascent peptide chain. However, vertebrate mitochondria have been thought to have four stop codons, namely AGA and AGG in addition to the standard UAA and UAG codons. Furthermore, two release factors have been identified, mtRF1 and mtRF1a. Free-energy calculations were used to determine if any of these two factors could bind to the two non-standard stop codons, and thereby terminate protein synthesis. Our calculations showed that the mtRF’s have similar stop codon specificity as bacterial RF1 and that it is highly unlikely that the mtRF’s are responsible for terminating at the AGA and AGG stop codons.

    The eukaryotic release factor 1, eRF1, on the other hand, can read all three stop codons singlehandedly. We show that eRF1 exerts a high discrimination against near-cognate codons, while having little preference for the different cognate stop codons. We also found an energetic mechanism for avoiding misreading of the UGG codon and could identify a conserved cluster of hydrophobic amino acids which prevents excessive solvent molecules to enter the codon binding site.

    The linear interaction energy method was used to examine binding of small molecules to the purine riboswitch and the FEP method was employed to explicitly calculate the LIE b-parameters. We show that the purine riboswitches have a remarkably high degree of electrostatic preorganization for their cognate ligands which is fundamental for discriminating against different purine analogs.

    List of papers
    1. Codon-reading specificities of mitochondrial release factors and translation termination at non-standard stop codons
    Open this publication in new window or tab >>Codon-reading specificities of mitochondrial release factors and translation termination at non-standard stop codons
    2013 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 4Article in journal (Refereed) Published
    Abstract [en]

    A key feature of mitochondrial translation is the reduced number of transfer RNAs and reassignment of codons. For human mitochondria, a major unresolved problem is how the set of stop codons are decoded by the release factors mtRF1a and mtRF1. Here we present three-dimensional structural models of human mtRF1a and mtRF1 based on their homology to bacterial RF1 in the codon recognition domain, and the strong conservation between mitochondrial and bacterial ribosomal RNA in the decoding region. Sequence changes in the less homologous mtRF1 appear to be correlated with specific features of the mitochondrial rRNA. Extensive computer simulations of the complexes with the ribosomal decoding site show that both mitochondrial factors have similar specificities and that neither reads the putative vertebrate stop codons AGA and AGG. Instead, we present a structural model for a mechanism by which the ICT1 protein causes termination by sensing the presence of these codons in the A-site of stalled ribosomes.

    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-217675 (URN)10.1038/ncomms3940 (DOI)000329396700004 ()
    Available from: 2014-02-05 Created: 2014-02-04 Last updated: 2017-12-06Bibliographically approved
    2. Binding Site Preorganization and Ligand Discrimination in the Purine Riboswitch
    Open this publication in new window or tab >>Binding Site Preorganization and Ligand Discrimination in the Purine Riboswitch
    2015 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 119, no 3, p. 773-782Article in journal (Refereed) Published
    Abstract [en]

    The progress of RNA research has suggested a wide variety of RNA molecules as possible targets for pharmaceutical drug molecules. Structure-based computational methods for predicting binding modes and affinities are now important tools in drug discovery, but these methods have mainly been focused on protein targets. Here we employ molecular dynamics free-energy perturbation calculations and the linear interaction energy method to analyze the energetics of ligand binding to purine riboswitches. Calculations are carried out for 14 different purine complexes with the guanine and adenine riboswitches in order to examine their ligand recognition principles. The simulations yield binding affinities in good agreement with experimental data and rationalize the selectivity of the riboswitches for different ligands. In particular, it is found that these receptors have an unusually high degree of electrostatic preorganization for their cognate ligands, and this effect is further quantified by explicit free-energy calculations, which show that the standard electrostatic linear interaction energy parametrization is suboptimal in this case. The adenine riboswitch specifically uses the electrostatic preorganization to discriminate against guanine by preventing the formation of a G-U wobble base pair.

    National Category
    Physical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-252042 (URN)10.1021/jp5052358 (DOI)000351329400016 ()25014157 (PubMedID)
    Available from: 2015-04-29 Created: 2015-04-28 Last updated: 2017-12-04Bibliographically approved
    3. Principles of start codon recognition in eukaryotic translation initiation
    Open this publication in new window or tab >>Principles of start codon recognition in eukaryotic translation initiation
    2016 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 17, p. 8425-8432Article in journal (Refereed) Published
    Abstract [en]

    Selection of the correct start codon during initiation of translation on the ribosome is a key event in protein synthesis. In eukaryotic initiation, several factors have to function in concert to ensure that the initiator tRNA finds the cognate AUG start codon during mRNA scanning. The two initiation factors eIF1 and eIF1A are known to provide important functions for the initiation process and codon selection. Here, we have used molecular dynamics free energy calculations to evaluate the energetics of initiator tRNA binding to different near-cognate codons on the yeast 40S ribosomal subunit, in the presence and absence of these two initiation factors. The results show that eIF1 and eIF1A together cause a relatively uniform and high discrimination against near-cognate codons. This works such that eIF1 boosts the discrimination against a first position near-cognate G-U mismatch, and also against a second position A-A base pair, while eIF1A mainly acts on third codon position. The computer simulations further reveal the structural basis of the increased discriminatory effect caused by binding of eIF1 and eIF1A to the 40S ribosomal subunit.

    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-312122 (URN)10.1093/nar/gkw534 (DOI)000386158800039 ()27280974 (PubMedID)
    Funder
    Knut and Alice Wallenberg Foundation, KAW 2011.0081Swedish Research CouncilSwedish National Infrastructure for Computing (SNIC)
    Available from: 2017-01-04 Created: 2017-01-04 Last updated: 2017-11-29Bibliographically approved
    4. Origin of the omnipotence of eukaryotic release factor 1
    Open this publication in new window or tab >>Origin of the omnipotence of eukaryotic release factor 1
    2017 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 8, article id 1425Article in journal (Refereed) Published
    Abstract [en]

    Termination of protein synthesis on the ribosome requires that mRNA stop codons are recognized with high fidelity. This is achieved by specific release factor proteins that are very different in bacteria and eukaryotes. Hence, while there are two release factors with overlapping specificity in bacteria, the single omnipotent eRF1 release factor in eukaryotes is able to read all three stop codons. This is particularly remarkable as it is able to select three out of four combinations of purine bases in the last two codon positions. With recently determined 3D structures of eukaryotic termination complexes, it has become possible to explore the origin of eRF1 specificity by computer simulations. Here, we report molecular dynamics free energy calculations on these termination complexes, where relative eRF1 binding free energies to different cognate and near-cognate codons are evaluated. The simulations show a high and uniform discrimination against the near-cognate codons, that differ from the cognate ones by a single nucleotide, and reveal the structural mechanisms behind the precise decoding by eRF1.

    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-328582 (URN)10.1038/s41467-017-01757-0 (DOI)000414869900011 ()29127299 (PubMedID)
    Funder
    Swedish Research CouncilKnut and Alice Wallenberg FoundationSwedish National Infrastructure for Computing (SNIC)
    Available from: 2017-08-27 Created: 2017-08-27 Last updated: 2018-02-26Bibliographically approved
  • 5.
    Lind, Christoffer
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Esguerra, Mauricio
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    A close-up view of codon selection in eukaryotic initiation2017In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 14, no 7, p. 815-819Article in journal (Refereed)
    Abstract [en]

    When given an option to choose among a set of alternatives and only one selection is right, one might stop and reflect over which one is best. However, the ribosome has no time to stop and make such reflections, proteins need to be produced and very fast. Eukaryotic translation initiation is an example of such a conundrum. Here, scanning for the correct codon match must be fast, efficient and accurate. We highlight our recent computational findings, which show how the initiation machinery manages to recognize one specific codon among many possible challengers, by fine-tuning the energetic landscape of base-pairing with the aid of the initiation factors eIF1 and eIF1A. Using a recent 3-dimensional structure of the eukaryotic initiation complex we have performed simulations of codon recognition in atomic detail. These calculations provide an in-depth energetic and structural view of how discrimination against near-cognate codons is achieved by the initiation complex.

  • 6.
    Lind, Christoffer
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Oliveira, Ana
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Origin of the omnipotence of eukaryotic release factor 12017In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 8, article id 1425Article in journal (Refereed)
    Abstract [en]

    Termination of protein synthesis on the ribosome requires that mRNA stop codons are recognized with high fidelity. This is achieved by specific release factor proteins that are very different in bacteria and eukaryotes. Hence, while there are two release factors with overlapping specificity in bacteria, the single omnipotent eRF1 release factor in eukaryotes is able to read all three stop codons. This is particularly remarkable as it is able to select three out of four combinations of purine bases in the last two codon positions. With recently determined 3D structures of eukaryotic termination complexes, it has become possible to explore the origin of eRF1 specificity by computer simulations. Here, we report molecular dynamics free energy calculations on these termination complexes, where relative eRF1 binding free energies to different cognate and near-cognate codons are evaluated. The simulations show a high and uniform discrimination against the near-cognate codons, that differ from the cognate ones by a single nucleotide, and reveal the structural mechanisms behind the precise decoding by eRF1.

  • 7.
    Lind, Christoffer
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Sund, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Codon-reading specificities of mitochondrial release factors and translation termination at non-standard stop codons2013In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 4Article in journal (Refereed)
    Abstract [en]

    A key feature of mitochondrial translation is the reduced number of transfer RNAs and reassignment of codons. For human mitochondria, a major unresolved problem is how the set of stop codons are decoded by the release factors mtRF1a and mtRF1. Here we present three-dimensional structural models of human mtRF1a and mtRF1 based on their homology to bacterial RF1 in the codon recognition domain, and the strong conservation between mitochondrial and bacterial ribosomal RNA in the decoding region. Sequence changes in the less homologous mtRF1 appear to be correlated with specific features of the mitochondrial rRNA. Extensive computer simulations of the complexes with the ribosomal decoding site show that both mitochondrial factors have similar specificities and that neither reads the putative vertebrate stop codons AGA and AGG. Instead, we present a structural model for a mechanism by which the ICT1 protein causes termination by sensing the presence of these codons in the A-site of stalled ribosomes.

  • 8.
    Lind, Christoffer
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Principles of start codon recognition in eukaryotic translation initiation2016In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 17, p. 8425-8432Article in journal (Refereed)
    Abstract [en]

    Selection of the correct start codon during initiation of translation on the ribosome is a key event in protein synthesis. In eukaryotic initiation, several factors have to function in concert to ensure that the initiator tRNA finds the cognate AUG start codon during mRNA scanning. The two initiation factors eIF1 and eIF1A are known to provide important functions for the initiation process and codon selection. Here, we have used molecular dynamics free energy calculations to evaluate the energetics of initiator tRNA binding to different near-cognate codons on the yeast 40S ribosomal subunit, in the presence and absence of these two initiation factors. The results show that eIF1 and eIF1A together cause a relatively uniform and high discrimination against near-cognate codons. This works such that eIF1 boosts the discrimination against a first position near-cognate G-U mismatch, and also against a second position A-A base pair, while eIF1A mainly acts on third codon position. The computer simulations further reveal the structural basis of the increased discriminatory effect caused by binding of eIF1 and eIF1A to the 40S ribosomal subunit.

  • 9.
    Sund, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Lind, Christoffer
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Binding Site Preorganization and Ligand Discrimination in the Purine Riboswitch2015In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 119, no 3, p. 773-782Article in journal (Refereed)
    Abstract [en]

    The progress of RNA research has suggested a wide variety of RNA molecules as possible targets for pharmaceutical drug molecules. Structure-based computational methods for predicting binding modes and affinities are now important tools in drug discovery, but these methods have mainly been focused on protein targets. Here we employ molecular dynamics free-energy perturbation calculations and the linear interaction energy method to analyze the energetics of ligand binding to purine riboswitches. Calculations are carried out for 14 different purine complexes with the guanine and adenine riboswitches in order to examine their ligand recognition principles. The simulations yield binding affinities in good agreement with experimental data and rationalize the selectivity of the riboswitches for different ligands. In particular, it is found that these receptors have an unusually high degree of electrostatic preorganization for their cognate ligands, and this effect is further quantified by explicit free-energy calculations, which show that the standard electrostatic linear interaction energy parametrization is suboptimal in this case. The adenine riboswitch specifically uses the electrostatic preorganization to discriminate against guanine by preventing the formation of a G-U wobble base pair.

1 - 9 of 9
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