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Binding Site Preorganization and Ligand Discrimination in the Purine Riboswitch
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
2015 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 119, no 3, 773-782 p.Article 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.

Place, publisher, year, edition, pages
2015. Vol. 119, no 3, 773-782 p.
National Category
Physical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-252042DOI: 10.1021/jp5052358ISI: 000351329400016PubMedID: 25014157OAI: oai:DiVA.org:uu-252042DiVA: diva2:808794
Available from: 2015-04-29 Created: 2015-04-28 Last updated: 2017-12-04Bibliographically approved
In thesis
1. Computational Studies of Protein Synthesis on the Ribosome and Ligand Binding to Riboswitches
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. 64 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1549
Keyword
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: 2017-10-17

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Lind, ChristofferÅqvist, Johan

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