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Lind, Christoffer
Publications (9 of 9) Show all publications
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
Lind, C., Esguerra, M. & Åqvist, J. (2017). A close-up view of codon selection in eukaryotic initiation. RNA Biology, 14(7), 815-819
Open this publication in new window or tab >>A close-up view of codon selection in eukaryotic initiation
2017 (English)In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 14, no 7, p. 815-819Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
TAYLOR & FRANCIS INC, 2017
Keywords
Codon selection, molecular dynamics simulation, ribosome, start codon, translation initiation
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-333423 (URN)10.1080/15476286.2017.1308998 (DOI)000407258600001 ()28340329 (PubMedID)
Available from: 2017-11-13 Created: 2017-11-13 Last updated: 2017-11-13Bibliographically approved
Lind, C. (2017). Computational Studies of Protein Synthesis on the Ribosome and Ligand Binding to Riboswitches. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Computational Studies of Protein Synthesis on the Ribosome and Ligand Binding to Riboswitches
2017 (English)Doctoral 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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. p. 64
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1549
Keywords
Binding free energy, Ribosome, Codon reading, Translation initiation, Translation termination, Mitochondrial translation, Release factor, Purine riboswitch, Molecular Dynamics, Free Energy Perturbation
National Category
Biochemistry and Molecular Biology Bioinformatics (Computational Biology)
Research subject
Biology with specialization in Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-328583 (URN)978-91-513-0051-1 (ISBN)
Public defence
2017-10-13, B41 BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2017-09-22 Created: 2017-08-27 Last updated: 2018-01-13
Lind, C., Oliveira, A. & Åqvist, J. (2017). Origin of the omnipotence of eukaryotic release factor 1. Nature Communications, 8, Article ID 1425.
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
Lind, C. & Åqvist, J. (2016). Principles of start codon recognition in eukaryotic translation initiation. Nucleic Acids Research, 44(17), 8425-8432
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
Sund, J., Lind, C. & Åqvist, J. (2015). Binding Site Preorganization and Ligand Discrimination in the Purine Riboswitch. Journal of Physical Chemistry B, 119(3), 773-782
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
Korkmaz, G., Lind, C., Åqvist, J. & Sanyal, S. (2014). Characterizing an engineered release factor capable of reading all three stop codons. Paper presented at Experimental Biology Meeting, APR 26-30, 2014, San Diego, CA. The FASEB Journal, 28(1), Article ID 569.2.
Open this publication in new window or tab >>Characterizing an engineered release factor capable of reading all three stop codons
2014 (English)In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 28, no 1, article id 569.2Article in journal, Meeting abstract (Other academic) Published
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-246840 (URN)000346646701432 ()
Conference
Experimental Biology Meeting, APR 26-30, 2014, San Diego, CA
Available from: 2015-03-10 Created: 2015-03-10 Last updated: 2017-12-04Bibliographically approved
Lind, C., Sund, J. & Åqvist, J. (2013). Codon-reading specificities of mitochondrial release factors and translation termination at non-standard stop codons. Nature Communications, 4
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
Aqvist, J., Lind, C., Sund, J. & Wallin, G. (2012). Bridging the gap between ribosome structure and biochemistry by mechanistic computations. Current opinion in structural biology, 22(6), 815-823
Open this publication in new window or tab >>Bridging the gap between ribosome structure and biochemistry by mechanistic computations
2012 (English)In: Current opinion in structural biology, ISSN 0959-440X, E-ISSN 1879-033X, Vol. 22, no 6, p. 815-823Article in journal (Refereed) Published
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.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-191777 (URN)10.1016/j.sbi.2012.07.008 (DOI)000312421000016 ()
Available from: 2013-01-15 Created: 2013-01-14 Last updated: 2017-12-06Bibliographically approved
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