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Punekar, A. S. (2014). Ribosomal RNA Modification Enzymes: Structural and functional studies of two methyltransferases for 23S rRNA modification in Escherichia coli . (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Ribosomal RNA Modification Enzymes: Structural and functional studies of two methyltransferases for 23S rRNA modification in Escherichia coli
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Escherichia coli ribosomal RNA (rRNA) is post-transcriptionally modified by site-specific enzymes. The role of most modifications is not known and little is known about how these enzymes recognize their target substrates. In this thesis, we have structurally and functionally characterized two S-adenosyl-methionine (SAM) dependent 23S rRNA methyltransferases (MTases) that act during the early stages of ribosome assembly in E. coli.

RlmM methylates the 2'O-ribose of C2498 in 23S rRNA. We have solved crystal structures of apo RlmM at 1.9Å resolution and of an RlmM-SAM complex at 2.6Å resolution. The RlmM structure revealed an N-terminal THUMP domain and a C-terminal catalytic Rossmann-fold MTase domain. A continuous patch of conserved positive charge on the RlmM surface is likely used for RNA substrate recognition. The SAM-binding site is open and shallow, suggesting that the RNA substrate may be required for tight cofactor binding. Further, we have shown RlmM MTase activity on in vitro transcribed 23S rRNA and its domain V.

RlmJ methylates the exocyclic N6 atom of A2030 in 23S rRNA. The 1.85Å crystal structure of RlmJ revealed a Rossmann-fold MTase domain with an inserted small subdomain unique to the RlmJ family. The 1.95Å structure of the RlmJ-SAH-AMP complex revealed that ligand binding induces structural rearrangements in the four loop regions surrounding the active site. The active site of RlmJ is similar to N6-adenine DNA MTases. We have shown RlmJ MTase activity on in vitro transcribed 23S rRNA and a minimal substrate corresponding to helix 72, specific for adenosine. Mutagenesis experiments show that residues Y4, H6, K18 and D164 are critical for catalytic activity.

These findings have furthered our understanding of the structure, evolution, substrate recognition and mechanism of rRNA MTases.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. p. 65
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1107
Keywords
Escherichia coli, ribosome biogenesis, ribosome assembly, ribosomal RNA, peptidyltransferase center, domain V, post-transcriptional modification, methyltransferases, S-adenosyl-methionine, RlmM, Cm2498, RlmJ, m6A2030, X-ray crystallography, substrate recognition, substrate specificity, catalytic mechanism, evolution
National Category
Structural Biology Biochemistry and Molecular Biology
Research subject
Biology with specialization in Structural Biology; Biochemistry
Identifiers
urn:nbn:se:uu:diva-212394 (URN)978-91-554-8834-5 (ISBN)
Public defence
2014-02-14, B42, Biomedical Center, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2014-01-22 Created: 2013-12-10 Last updated: 2014-02-10
Hultqvist, G., Haq, R., Punekar, A., Chi, C., Engström, Å., Bach, A., . . . Jemth, P. (2013). Energetic pathway sampling in a protein interaction domain. Structure, 21, 1193-1202
Open this publication in new window or tab >>Energetic pathway sampling in a protein interaction domain
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2013 (English)In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 21, p. 1193-1202Article in journal (Other academic) Published
Abstract [en]

The affinity and specificity of protein-ligand interactions are influenced by energeticcrosstalk within the protein domain. However, the molecular details of such intradomain allostery are still unclear. Here, we have experimentally detected and computationally predicted interactionpathways in the postsynaptic density 95/discs large/zonula occludens 1 (PDZ)-peptide ligand model system using wild-type and circularly permuted PDZ proteins. The circular permutant introduced small perturbations in the tertiary structure and a concomitant rewiring of allosteric pathways, allowing us to describe how subtle changes may reshape energetic signaling. The results were analyzed in the context of other members of the PDZ family, which were found to contain distinct interaction pathways for different peptide ligands. The data reveal a fascinating scenario whereby several energetic pathways are sampled within one single domain and distinct pathways are activated by specific protein ligands. 

Keywords
intradomain allostery, PDZ domain, protein binding, circular permutant
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-185579 (URN)10.1016/j.str.2013.05.010 (DOI)000321681600016 ()
Available from: 2012-12-10 Created: 2012-11-26 Last updated: 2017-10-16Bibliographically approved
Punekar, A. S. & Selmer, M. (2013). Purification, crystallization and preliminary X-ray diffraction analysis of the 23S rRNA methyltransferase RlmJ from Escherichia coli. Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, 69, 1001-1003
Open this publication in new window or tab >>Purification, crystallization and preliminary X-ray diffraction analysis of the 23S rRNA methyltransferase RlmJ from Escherichia coli
2013 (English)In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 69, p. 1001-1003Article in journal (Refereed) Published
Abstract [en]

Methyltransferase RlmJ uses the cofactor S-adenosylmethionine to methylate the exocyclic nitrogen N6 of nucleotide A2030 in 23S rRNA during ribosome assembly in Escherichia coli. RlmJ with a C-terminal hexahistidine tag was overexpressed in E. coli and purified as a monomer using Ni2+-affinity and size-exclusion chromatography. The recombinant RlmJ was crystallized using the sitting-drop vapour-diffusion method and a full data set was collected to 1.85 angstrom resolution from a single apo crystal. The crystals belonged to space group P2(1), with unit-cell parameters a = 46.9, b = 77.8, c = 82.5 angstrom, beta = 104 degrees. Data analysis suggested two molecules per asymmetric unit and a Matthews coefficient of 2.20 angstrom(3) Da(-1).

National Category
Structural Biology
Identifiers
urn:nbn:se:uu:diva-208048 (URN)10.1107/S1744309113020289 (DOI)000323719700010 ()
Available from: 2013-09-24 Created: 2013-09-23 Last updated: 2017-12-06Bibliographically approved
Punekar, A. S., Liljeruhm, J., Shepherd, T. R., Forster, A. C. & Selmer, M. (2013). Structural and functional insights into the molecular mechanism of rRNA m6A methyltransferase RlmJ. Nucleic Acids Research, 41(20), 9537-9548
Open this publication in new window or tab >>Structural and functional insights into the molecular mechanism of rRNA m6A methyltransferase RlmJ
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2013 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 41, no 20, p. 9537-9548Article in journal (Refereed) Published
Abstract [en]

RlmJ catalyzes the m(6)A2030 methylation of 23S rRNA during ribosome biogenesis in Escherichia coli. Here, we present crystal structures of RlmJ in apo form, in complex with the cofactor S-adenosyl-methionine and in complex with S-adenosyl-homocysteine plus the substrate analogue adenosine monophosphate (AMP). RlmJ displays a variant of the Rossmann-like methyltransferase (MTase) fold with an inserted helical subdomain. Binding of cofactor and substrate induces a large shift of the N-terminal motif X tail to make it cover the cofactor binding site and trigger active-site changes in motifs IV and VIII. Adenosine monophosphate binds in a partly accommodated state with the target N6 atom 7 Å away from the sulphur of AdoHcy. The active site of RlmJ with motif IV sequence 164DPPY167 is more similar to DNA m(6)A MTases than to RNA m(6)2A MTases, and structural comparison suggests that RlmJ binds its substrate base similarly to DNA MTases T4Dam and M.TaqI. RlmJ methylates in vitro transcribed 23S rRNA, as well as a minimal substrate corresponding to helix 72, demonstrating independence of previous modifications and tertiary interactions in the RNA substrate. RlmJ displays specificity for adenosine, and mutagenesis experiments demonstrate the critical roles of residues Y4, H6, K18 and D164 in methyl transfer.

National Category
Structural Biology
Identifiers
urn:nbn:se:uu:diva-211566 (URN)10.1093/nar/gkt719 (DOI)000326746400036 ()23945937 (PubMedID)
Available from: 2013-11-26 Created: 2013-11-26 Last updated: 2019-01-25Bibliographically approved
Punekar, A. S., Shepherd, T. R., Liljeruhm, J., Forster, A. C. & Selmer, M. (2012). Crystal structure of RlmM, the 2'O-ribose methyltransferase for C2498 of Escherichia coli 23S rRNA. Nucleic Acids Research, 40(20), 10507-20
Open this publication in new window or tab >>Crystal structure of RlmM, the 2'O-ribose methyltransferase for C2498 of Escherichia coli 23S rRNA
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2012 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 20, p. 10507-20Article in journal (Refereed) Published
Abstract [en]

RlmM (YgdE) catalyzes the S-adenosyl methionine (AdoMet)-dependent 2'O methylation of C2498 in 23S ribosomal RNA (rRNA) of Escherichia coli. Previous experiments have shown that RlmM is active on 23S rRNA from an RlmM knockout strain but not on mature 50S subunits from the same strain. Here, we demonstrate RlmM methyltransferase (MTase) activity on in vitro transcribed 23S rRNA and its domain V. We have solved crystal structures of E. coli RlmM at 1.9 Å resolution and of an RlmM-AdoMet complex at 2.6 Å resolution. RlmM consists of an N-terminal THUMP domain and a C-terminal catalytic Rossmann-like fold MTase domain in a novel arrangement. The catalytic domain of RlmM is closely related to YiiB, TlyA and fibrillarins, with the second K of the catalytic tetrad KDKE shifted by two residues at the C-terminal end of a beta strand compared with most 2'O MTases. The AdoMet-binding site is open and shallow, suggesting that RNA substrate binding may be required to form a conformation needed for catalysis. A continuous surface of conserved positive charge indicates that RlmM uses one side of the two domains and the inter-domain linker to recognize its RNA substrate.

National Category
Structural Biology
Identifiers
urn:nbn:se:uu:diva-187880 (URN)10.1093/nar/gks727 (DOI)000310970700054 ()22923526 (PubMedID)
Available from: 2012-12-11 Created: 2012-12-11 Last updated: 2019-01-25Bibliographically approved
Hultqvist, G., Punekar, A., Morrone, A., Chi, C., Engström, Å., Selmer, M., . . . Jemth, P. (2012). Tolerance of Protein Folding to a Circular Permutation in a PDZ Domain. PLoS ONE, 7(11), e50055
Open this publication in new window or tab >>Tolerance of Protein Folding to a Circular Permutation in a PDZ Domain
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 11, p. e50055-Article in journal (Refereed) Published
Abstract [en]

Circular permutation is a common molecular mechanism for evolution of proteins. However, such re-arrangement of secondary structure connectivity may interfere with the folding mechanism causing accumulation of folding intermediates, which in turn can lead to misfolding. We solved the crystal structure and investigated the folding pathway of a circularly permuted variant of a PDZ domain, SAP97 PDZ2. Our data illustrate how well circular permutation may work as a mechanism for molecular evolution. The circular permutant retains the overall structure and function of the native protein domain. Further, unlike most examples in the literature, this circular permutant displays a folding mechanism that is virtually identical to that of the wild type. This observation contrasts with previous data on the circularly permuted PDZ2 domain from PTP-BL, for which the folding pathway was remarkably affected by the same mutation in sequence connectivity. The different effects of this circular permutation in two homologous proteins show the strong influence of sequence as compared to topology. Circular permutation, when peripheral to the major folding nucleus, may have little effect on folding pathways and could explain why, despite the dramatic change in primary structure, it is frequently tolerated by different protein folds.

Keywords
Circular permutant, protein folding, PDZ domain
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-185577 (URN)10.1371/journal.pone.0050055 (DOI)000311821000173 ()
Available from: 2012-11-26 Created: 2012-11-26 Last updated: 2017-12-07Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-6541-9529

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