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Anandapadamanaban, Madhanagopal
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Publications (6 of 6) Show all publications
Anandapadamanaban, M. (2015). Structural insights into protein-protein interactions governing regulation in transcription initiation and ubiquitination. (Doctoral dissertation). Linköping: Linköping University Electronic Press
Open this publication in new window or tab >>Structural insights into protein-protein interactions governing regulation in transcription initiation and ubiquitination
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Virtually every aspect of the cellular processes in eukaryotes requires that the interactions between protein molecules are well coordinated in different regulatory pathways. Any protein dysfunction involved in these regulatory pathways might lead to various pathological conditions. Understanding the structural and functional peculiarities of these proteins molecular machineries will help in formulating structure-based drug design.

The first regulatory process studied here is the RNA polymerase-II mediated transcription of the eukaryotic protein-coding genes to produce mRNAs. This process requires the formation of the ‘transcription initiation’ by the assembly of Pre-Initiation Complex (PIC) formation at a core promoter region. Regulation at this initiation level is a key mechanism for the control of gene expression that governs cellular growth and differentiation. The transcription Factor IID (TFIID) is a conserved multiprotein general transcription factor with an essential role in  nucleating the PIC formation, composed of TATA Binding Protein (TBP) and about 14 TBP Associated Factors (TAFs). The here presented crystal structure (1.97Å) of TBP bound to TAND1 and TAND2 domains from TAF1 reveals a detailed molecular pattern of interactions involving both transcriptionally activating and repressing regions in TBP, thereby uncovering central principles for anchoring of TBP-binding motifs. Together with NMR and cellular analysis, this work provides the structural basis of competitive binding with TFIIA to modulate TBP in promoter recognition.

In eukaryotes, another fundamental mechanism in the regulation of cellular physiology is the posttranslational modification of substrate proteins by ubiquitin, termed ‘ubiquitination’. Important actors in this mechanism are the ubiquitin-ligases (E3s) that culminate the transfer of ubiquitin to the substrate and govern the specificity of this system. One E3 ligase in particular, TRIM21, defines a subgroup of the Tripartite Motif (TRIM) family, which belongs to the major RING-type of E3 ubiquitin ligases, and plays an important role in pathogenesis of autoimmunity by mediating ubiquitination of transcription factors. The crystal structure (2.86Å) of the RING domain from TRIM21 in complex with UBE2E1, an E2 conjugating enzyme, together with the NMR and SAXS analysis as well as biochemical functional analysis, reveals the molecular basis for the dynamic binding interfaces. The TRIM21 mode of ubiquitin recognition and activation for catalytic transfer of ubiquitin can be modeled onto the entire TRIM family.

Finally, we explored the concepts of conformational selection in proteins as a possible key component for protein-mediated transcriptional regulation. In this framework, MexR, a bacterial repressor of the MexAB-OprM efflux pump, and its mutant Arg21Trp were studied as an example for proteins presenting different conformations. The residue Arg21Trp mutation is clinically identified to cause of Multi-Drug Resistant (MDR) by attenuated DNA binding, and leads to the overexpression of the MexAB-OprM efflux pump. With the crystal structure (2.19Å) of MexR mutant Arg21Trp, in combination with MD-simulations and SAXS for both wild-type and mutant, we could unravel the atomic details of the wild-type conformations consisting in subsets of populations of DNA bound and unbound forms. Remarkably, the mutant Arg21Trp stabilize the DNA unbound state and shifts MexR in a pre-existing equilibrium, from a repressed to a derepressed state.

Taken together, these studies substantially broaden our knowledge at a molecular level in protein interactions that are involved in transcriptional regulation and ubiquitination, studied by a carefully selected combination of complementary structural methods spanning different resolutions and time scales.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2015. p. 73
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1694
National Category
Chemical Sciences Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-122468 (URN)10.3384/diss.diva-122468 (DOI)978-91-7685-984-1 (ISBN)
Public defence
2015-12-04, Planck, Fysikhuset, Campus Valla, Linköping, 09:30 (English)
Opponent
Supervisors
Available from: 2015-11-03 Created: 2015-11-03 Last updated: 2017-01-10Bibliographically approved
Dovega, R., Tsutakawa, S., Quistgaard, E. M., Anandapadmanaban, M., Löw, C. & Nordlund, P. (2014). Structural and Biochemical Characterization of Human PR70 in Isolation and in Complex with the Scaffolding Subunit of Protein Phosphatase 2A. PLoS ONE, 9(7), e0101846
Open this publication in new window or tab >>Structural and Biochemical Characterization of Human PR70 in Isolation and in Complex with the Scaffolding Subunit of Protein Phosphatase 2A
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2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 7, p. e0101846-Article in journal (Refereed) Published
Abstract [en]

Protein Phosphatase 2A (PP2A) is a major Ser/Thr phosphatase involved in the regulation of various cellular processes. PP2A assembles into diverse trimeric holoenzymes, which consist of a scaffolding (A) subunit, a catalytic (C) subunit and various regulatory (B) subunits. Here we report a 2.0 angstrom crystal structure of the free B /PR70 subunit and a SAXS model of an A/PR70 complex. The crystal structure of B /PR70 reveals a two domain elongated structure with two Ca2+ binding EF-hands. Furthermore, we have characterized the interaction of both binding partner and their calcium dependency using biophysical techniques. Ca2+ biophysical studies with Circular Dichroism showed that the two EF-hands display different affinities to Ca2+. In the absence of the catalytic C-subunit, the scaffolding A-subunit remains highly mobile and flexible even in the presence of the B /PR70 subunit as judged by SAXS. Isothermal Titration Calorimetry studies and SAXS data support that PR70 and the A-subunit have high affinity to each other. This study provides additional knowledge about the structural basis for the function of B containing holoenzymes.

Place, publisher, year, edition, pages
Public Library of Science, 2014
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-109375 (URN)10.1371/journal.pone.0101846 (DOI)000339040600073 ()25007185 (PubMedID)
Available from: 2014-08-15 Created: 2014-08-15 Last updated: 2017-12-05Bibliographically approved
Löw, C., Quistgaard, E. M., Kovermann, M., Anandapadmanaban, M., Balbach, J. & Nordlund, P. (2014). Structural basis for PTPA interaction with the invariant C-terminal tail of PP2A. Biological chemistry (Print), 395(7-8), 881-889
Open this publication in new window or tab >>Structural basis for PTPA interaction with the invariant C-terminal tail of PP2A
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2014 (English)In: Biological chemistry (Print), ISSN 1431-6730, E-ISSN 1437-4315, Vol. 395, no 7-8, p. 881-889Article in journal (Refereed) Published
Abstract [en]

Protein phosphatase 2A (PP2A) is a highly abundant heterotrimeric Ser/Thr phosphatase involved in the regulation of a variety of signaling pathways. The PP2A phosphatase activator (PTPA) is an ATP-dependent activation chaperone, which plays a key role in the biogenesis of active PP2A. The C-terminal tail of the catalytic subunit of PP2A is highly conserved and can undergo a number of posttranslational modifications that serve to regulate the function of PP2A. Here we have studied structurally the interaction of PTPA with the conserved C-terminal tail of the catalytic subunit carrying different posttranslational modifications. We have identified an additional interaction site for the invariant C-terminal tail of the catalytic subunit on PTPA, which can be modulated via posttranslational modifications. We show that phosphorylation of Tyr307(PP2A-C) or carboxymethylation of Leu309(PP2A-C) abrogates or diminishes binding of the C-terminal tail, whereas phosphorylation of Thr304(PP2A-C) is of no consequence. We suggest that the invariant C-terminal residues of the catalytic subunit can act as affinity enhancer for different PP2A interaction partners, including PTPA, and a different code of posttranslational modifications can favour interactions to one subunit over others.

Place, publisher, year, edition, pages
Walter de Gruyter, 2014
Keywords
activation chaperone; phosphatase; posttranslational modification; signaling; X-ray structure
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-109228 (URN)10.1515/hsz-2014-0106 (DOI)000338844300017 ()25003389 (PubMedID)
Available from: 2014-08-12 Created: 2014-08-11 Last updated: 2017-12-05Bibliographically approved
Anandapadmanaban, M., Andrésen, C., Helander, S., Ohyama, Y., Siponen, M. I., Lundström, P., . . . Sunnerhagen, M. (2013). High-resolution structure of TBP with TAF1 reveals anchoring patterns in transcriptional regulation. Nature Structural & Molecular Biology, 20(8), 1008-+
Open this publication in new window or tab >>High-resolution structure of TBP with TAF1 reveals anchoring patterns in transcriptional regulation
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2013 (English)In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 20, no 8, p. 1008-+Article in journal (Refereed) Published
Abstract [en]

The general transcription factor TFIID provides a regulatory platform for transcription initiation. Here we present the crystal structure (1.97 angstrom) and NMR analysis of yeast TAF1 N-terminal domains TAND1 and TAND2 bound to yeast TBP, together with mutational data. We find that yeast TAF1-TAND1, which in itself acts as a transcriptional activator, binds TBPs concave DNA-binding surface by presenting similar anchor residues to TBP as does Mot1 but from a distinct structural scaffold. Furthermore, we show how TAF1-TAND2 uses an aromatic and acidic anchoring pattern to bind a conserved TBP surface groove traversing the basic helix region, and we find highly similar TBP-binding motifs also presented by the structurally distinct TFIIA, Mot1 and Brf1 proteins. Our identification of these anchoring patterns, which can be easily disrupted or enhanced, provides insight into the competitive multiprotein TBP interplay critical to transcriptional regulation.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 75 VARICK ST, 9TH FLR, NEW YORK, NY 10013-1917 USA, 2013
National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-96977 (URN)10.1038/nsmb.2611 (DOI)000322715300016 ()
Note

Funding Agencies|Swedish Research Council|621-2011-6028621-2012-5250621-2012-5136|VINNOVA|P32045-1|Swedish Cancer Foundation|11 0681|Swedish Child Cancer Foundation|PROJ09/092|Forum Scientium Award||Canadian Institutes for Health Research|MT-13611|Japan Society for the Promotion of Science|23370077|Knut and Alice Wallenberg foundation||Canada Research Chair||

Available from: 2013-09-05 Created: 2013-09-02 Last updated: 2017-12-06
Espinosa, A., Hennig, J., Ambrosi, A., Anandapadamanaban, M., Sandberg Abelius, M., Sheng, Y., . . . Wahren-Herlenius, M. (2011). Anti-Ro52 Autoantibodies from Patients with Sjögren's Syndrome Inhibit the Ro52 E3 Ligase Activity by Blocking the E3/E2 Interface. Journal of Biological Chemistry, 286(42), 36478-36491
Open this publication in new window or tab >>Anti-Ro52 Autoantibodies from Patients with Sjögren's Syndrome Inhibit the Ro52 E3 Ligase Activity by Blocking the E3/E2 Interface
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2011 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 42, p. 36478-36491Article in journal (Refereed) Published
Abstract [en]

Ro52 (TRIM21) is an E3 ligase of the tripartite motif family that negatively regulates proinflammatory cytokine production by ubiquitinating transcription factors of the interferon regulatory factor family. Autoantibodies to Ro52 are present in patients with lupus and Sjögren's syndrome, but it is not known if these autoantibodies affect the function of Ro52. To address this question, the requirements for Ro52 E3 ligase activity were first analyzed in detail. Scanning a panel of E2 ubiquitin-conjugating enzymes, we found that UBE2D1–4 and UBE2E1–2 supported the E3 ligase activity of Ro52 and that the E3 ligase activity of Ro52 was dependent on its RING domain. We also found that the N-terminal extensions in the class III E2 enzymes affected their interaction with Ro52. Although the N-terminal extension in UBE2E3 made this E2 enzyme unable to function together with Ro52, the N-terminal extensions in UBE2E1 and UBE2E2 allowed for a functional interaction with Ro52. Anti-Ro52-positive patient sera and affinity-purified anti-RING domain autoantibodies inhibited the E3 activity of Ro52 in ubiquitination assays. Using NMR, limited proteolysis, ELISA, and Ro52 mutants, we mapped the interactions between Ro52, UBE2E1, and anti-Ro52 autoantibodies. We found that anti-Ro52 autoantibodies inhibited the E3 ligase activity of Ro52 by sterically blocking the E2/E3 interaction between Ro52 and UBE2E1. Our data suggest that anti-Ro52 autoantibodies binding the RING domain of Ro52 may be actively involved in the pathogenesis of rheumatic autoimmune disease by inhibiting Ro52-mediated ubiquitination.

Place, publisher, year, edition, pages
American Society for Biochemistry and Molecular Biology, 2011
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-53170 (URN)10.1074/jbc.M111.241786 (DOI)000296538300033 ()
Note

Funding agencies|Swedish Research Council||Swedish Foundation for Strategic Research||VINNOVA||CeNano||Swedish Cancer Society||Karolinska Institutet||Linkoping University||King Gustaf Vs 80-Year Foundation||Heart-Lung Foundation||Stockholm County Council||Gustafsson Foundation||Soderberg Foundation||National Cancer Institute of Canada||Swedish Rheumatism Association||Wallenberg Foundation||

Available from: 2010-01-18 Created: 2010-01-18 Last updated: 2017-12-12Bibliographically approved
Anandapadamanaban, M., Kyriakidis, N., Espinosa, A., Round, A. R., Trewhella, J., Wahren-Herlenius, M., . . . Sunnerhagen, M.Structure of a TRIM21 - UBE2El complex reveals the specificity of E2 and ubiquitin recognition by TRIM E3 RINGs.
Open this publication in new window or tab >>Structure of a TRIM21 - UBE2El complex reveals the specificity of E2 and ubiquitin recognition by TRIM E3 RINGs
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

TRIM21, a RlNG-containing E3 ubiquitin-ligase of the TRIM   protein family, is a major autoantigen in SLE and Sjögren's syndrome as well as a modifier of interferon regulatory factors, thereby regulating innate immune signalling. We herein report the 2.86 Å crystal structure ofhuman TRIM211-91 comprising the RING domain (residues 16-55), in complex with the human E2 conjugating UBE2El enzyme (also denoted UbcH6). The crystal structure, joint with analysis by NMR and SAXS as well as structure-directed mutations and functional assays provides a detailed view of the specificity-determining contacts that support specific E2 recognition in the TRIM family. A detailed comparison of our structure with known E2 bound ubiquitin complexes, supported by biochemical analyses, reveals the molecular basis for TRIM21 interactions with donor ubiquitin that activates catalytic ubiquitin transfer. Finally, our structure convincingly demonstrates the placement of the Ub-targeted Lys61 of the adjacent TRIM211- 91 close to the catalytically active UBE2El cysteine, and how the Lys61 amide is activated fora nucleophilic attack by hydrogen-bondeffected deshielding by conserved acidic residues at the E2 active site. In all, our structural findings provide molecular details ofthe selectivity involved in TRIM21 interactions with its cognate UBE2E1 enzyme and how TRIM21 positions ubiquitin in a catalytic conformation for ubiquitin transfer, and presents a snapshot of the Ub ligation step on a specific target residue of TRIM211-91 as an auto-ubiquitinated pseudo-substrate at high concentration. Increased structural and functional understanding of the TRIM mediated ubiquitination will aid development ofnovel therapeutic approaches in the entire TRIM family ofproteins.

National Category
Chemical Sciences Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-122466 (URN)
Available from: 2015-11-03 Created: 2015-11-03 Last updated: 2015-11-13Bibliographically approved
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