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Title: Seeding for sirtuins: microseed matrix seeding to obtain crystals of human Sirt3 and Sirt2 suitable for soaking

Abstract

In the present study, microseed matrix seeding was successfully applied to obtain a large number of crystals of the human sirtuin isotypes Sirt2 and Sirt3. These crystals appeared predictably in diverse crystallization conditions, diffracted to a higher resolution than reported in the literature and were subsequently used to study the protein–ligand interactions of two indole inhibitors. Sirtuins constitute a family of NAD{sup +}-dependent enzymes that catalyse the cleavage of various acyl groups from the ∊-amino group of lysines. They regulate a series of cellular processes and their misregulation has been implicated in various diseases, making sirtuins attractive drug targets. To date, only a few sirtuin modulators have been reported that are suitable for cellular research and their development has been hampered by a lack of structural information. In this work, microseed matrix seeding (MMS) was used to obtain crystals of human Sirt3 in its apo form and of human Sirt2 in complex with ADP ribose (ADPR). Crystal formation using MMS was predictable, less error-prone and yielded a higher number of crystals per drop than using conventional crystallization screening methods. The crystals were used to solve the crystal structures of apo Sirt3 and of Sirt2 in complex with ADPR at anmore » improved resolution, as well as the crystal structures of Sirt2 in complex with ADPR and the indoles EX527 and CHIC35. These Sirt2–ADPR–indole complexes unexpectedly contain two indole molecules and provide novel insights into selective Sirt2 inhibition. The MMS approach for Sirt2 and Sirt3 may be used as the basis for structure-based optimization of Sirt2/3 inhibitors in the future.« less

Authors:
 [1]; ;  [2];  [1]
  1. Albert-Ludwigs-University Freiburg, Albertstrasse 25, 79104 Freiburg, Baden-Württemberg (Germany)
  2. Albert-Ludwigs-University Freiburg, Albertstrasse 21, 79104 Freiburg, Baden-Württemberg (Germany)
Publication Date:
OSTI Identifier:
22515178
Resource Type:
Journal Article
Journal Name:
Acta Crystallographica. Section F, Structural Biology Communications
Additional Journal Information:
Journal Volume: 71; Journal Issue: Pt 12; Other Information: PMCID: PMC4666478; PMID: 26625292; PUBLISHER-ID: cb5089; PUBLISHER-ID: S2053230X15019986; OAI: oai:pubmedcentral.nih.gov:4666478; Copyright (c) Rumpf et al. 2015; This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 2053-230X
Country of Publication:
United States
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CRYSTAL STRUCTURE; CRYSTALLIZATION; LIGANDS; MATRICES

Citation Formats

Rumpf, Tobias, Gerhardt, Stefan, Einsle, Oliver, and Jung, Manfred. Seeding for sirtuins: microseed matrix seeding to obtain crystals of human Sirt3 and Sirt2 suitable for soaking. United States: N. p., 2015. Web. doi:10.1107/S2053230X15019986.
Rumpf, Tobias, Gerhardt, Stefan, Einsle, Oliver, & Jung, Manfred. Seeding for sirtuins: microseed matrix seeding to obtain crystals of human Sirt3 and Sirt2 suitable for soaking. United States. https://doi.org/10.1107/S2053230X15019986
Rumpf, Tobias, Gerhardt, Stefan, Einsle, Oliver, and Jung, Manfred. 2015. "Seeding for sirtuins: microseed matrix seeding to obtain crystals of human Sirt3 and Sirt2 suitable for soaking". United States. https://doi.org/10.1107/S2053230X15019986.
@article{osti_22515178,
title = {Seeding for sirtuins: microseed matrix seeding to obtain crystals of human Sirt3 and Sirt2 suitable for soaking},
author = {Rumpf, Tobias and Gerhardt, Stefan and Einsle, Oliver and Jung, Manfred},
abstractNote = {In the present study, microseed matrix seeding was successfully applied to obtain a large number of crystals of the human sirtuin isotypes Sirt2 and Sirt3. These crystals appeared predictably in diverse crystallization conditions, diffracted to a higher resolution than reported in the literature and were subsequently used to study the protein–ligand interactions of two indole inhibitors. Sirtuins constitute a family of NAD{sup +}-dependent enzymes that catalyse the cleavage of various acyl groups from the ∊-amino group of lysines. They regulate a series of cellular processes and their misregulation has been implicated in various diseases, making sirtuins attractive drug targets. To date, only a few sirtuin modulators have been reported that are suitable for cellular research and their development has been hampered by a lack of structural information. In this work, microseed matrix seeding (MMS) was used to obtain crystals of human Sirt3 in its apo form and of human Sirt2 in complex with ADP ribose (ADPR). Crystal formation using MMS was predictable, less error-prone and yielded a higher number of crystals per drop than using conventional crystallization screening methods. The crystals were used to solve the crystal structures of apo Sirt3 and of Sirt2 in complex with ADPR at an improved resolution, as well as the crystal structures of Sirt2 in complex with ADPR and the indoles EX527 and CHIC35. These Sirt2–ADPR–indole complexes unexpectedly contain two indole molecules and provide novel insights into selective Sirt2 inhibition. The MMS approach for Sirt2 and Sirt3 may be used as the basis for structure-based optimization of Sirt2/3 inhibitors in the future.},
doi = {10.1107/S2053230X15019986},
url = {https://www.osti.gov/biblio/22515178}, journal = {Acta Crystallographica. Section F, Structural Biology Communications},
issn = {2053-230X},
number = Pt 12,
volume = 71,
place = {United States},
year = {Wed Nov 18 00:00:00 EST 2015},
month = {Wed Nov 18 00:00:00 EST 2015}
}