VII AUSE Congress and II ALBA User's Meeting - 2015

A structural overview of Pontin, Reptin and their complex(es)

17 Jun 2015, 12:40

20m

Maxwell auditorium (ALBA Synchrotron)

Oral presentations II ALBA User's Meeting Session 3 - VII AUSE Congress 2015

Speaker

Dr Pedro Matias (Instituto de Tecnologia Química e Biológica António Xavier - ITQB/UNL)

Description

Pontin, also known as RuvBL1 and Reptin, also known as RuvBL2 are highly conserved eukaryotic proteins belonging to the AAA+ family of ATPases, and closely related to the bacterial DNA helicase RuvB. They are ubiquitously expressed and have been associated with many cellular functions. In addition, a link was established between Pontin, Reptin and cancer [1]. The crystal structure of human Pontin was determined in our lab in 2006 [2]. It is hexameric, formed by ADP-bound monomers. Each monomer contains 3 domains: domains I and III are involved in ATP binding and hydrolysis and are structurally similar to equivalent domains in the bacterial homolog RuvB; a sequence insertion intercalated into domain I folds into domain II, unique among AAA+ proteins. 3D structures of Pontin/Reptin complexes were determined by other groups using electron microscopy [3-4]. In the human and yeast complexes, Pontin and Reptin form a dodecamer with two structurally distinct hexameric rings. It was suggested that both rings interact via domain II, but neither study settled the issue of whether the rings are homo- or hetero-oligomeric. However, a third EM study reported a single hetero-hexameric ring structure for the yeast Pontin/Reptin complex [5]. The first crystal structure of a Pontin/Reptin complex with a truncated domain II was published in 2011 by our lab [6]. The structure of a SeMet derivative revealed a dodecamer formed by two heterohexameric rings composed of alternating Pontin and Reptin monomers that interact via the retained part of domain II. More recently, the crystal structure of a full-length homologue from the thermophilic fungus *Chaetomium thermophilum* was published [7] and structures of larger complexes integrating Pontin/Reptin heterohexamers were obtained by EM [8-9]. The crystal structure of truncated Reptin was published in 2012 [10]. We have been working towards the crystal structure of full-length Reptin. To date, the best diffraction data were measured to 3.4 Å at ALBA XALOC and a preliminary structure obtained by MR. However, the location of domain II could not be ascertained and the search for better-diffracting crystals continues.

References

1. Huber O, Menard L, Haurie V, Nicou A, Taras D, Rosenbaum J. 2008. Pontin and reptin, two related ATPases with multiple roles in cancer. Cancer Res 68:6873-6876.

2. Matias PM, Gorynia S, Donner P, Carrondo MA. 2006. Crystal structure of the human AAA+ protein RuvBL1. J Biol Chem 281:38918-38929.

3. Puri T, Wendler P, Sigala B, Saibil H, Tsaneva IR. 2007. Dodecameric structure and ATPase activity of the human TIP48/TIP49 complex. J Mol Biol 366:179-192.

4. Torreira E, Jha S, Lopez-Blanco JR, Arias-Palomo E, Chacon P, Canas C, Ayora S, Dutta A, Llorca O. 2008. Architecture of the pontin/reptin complex, essential in the assembly of several macromolecular complexes. Structure 16:1511-1520.

5. Gribun A, Cheung KL, Huen J, Ortega J, Houry WA. 2008. Yeast Rvb1 and Rvb2 are ATP-dependent DNA helicases that form a heterohexameric complex. J Mol Biol 376:1320-1333.

6. Gorynia S, Bandeiras TM, Pinho FG, McVey CE, Vonrhein C, Round A, Svergun DI, Donner P, Matias PM, Carrondo MA. 2011. Structural and functional insights into a dodecameric molecular machine - the RuvBL1/RuvBL2 complex. J Struct Biol 176:279-291.

7. Lakomek K, Stoehr G, Tosi A, Schmailzl M, Hopfner KP. 2015. Structural basis for dodecameric assembly states and conformational plasticity of the full-length AAA+ ATPases Rvb1 . Rvb2. Structure 23:483-495.

8. Tosi A, Haas C, Herzog F, Gilmozzi A, Berninghausen O, Ungewickell C, Gerhold CB, Lakomek K, Aebersold R, Beckmann R, Hopfner KP. 2013. Structure and subunit topology of the INO80 chromatin remodeler and its nucleosome complex. Cell 154:1207-1219.

9. Nguyen VQ, Ranjan A, Stengel F, Wei D, Aebersold R, Wu C, Leschziner AE. 2013. Molecular architecture of the ATP-dependent chromatin-remodeling complex SWR1. Cell 154:1220-1231.

10. Petukhov M, Dagkessamanskaja A, Bommer M, Barrett T, Tsaneva I, Yakimov A, Queval R, Shvetsov A, Khodorkovskiy M, Kas E, Grigoriev M. 2012. Large-scale conformational flexibility determines the properties of AAA+ TIP49 ATPases. Structure 20:1321-1331.