Seeding for sirtuins: microseed matrix seeding to obtain crystals of human Sirt3 and Sirt2 suitable for soaking
- Albert-Ludwigs-University Freiburg, Albertstrasse 25, 79104 Freiburg, Baden-Württemberg (Germany)
- Albert-Ludwigs-University Freiburg, Albertstrasse 21, 79104 Freiburg, Baden-Württemberg (Germany)
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.
- OSTI ID:
- 22515178
- Journal Information:
- Acta Crystallographica. Section F, Structural Biology Communications, Vol. 71, 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); ISSN 2053-230X
- Country of Publication:
- United States
- Language:
- English
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