X-ray structure of Pur-α reveals a Whirly-like fold and an unusual nucleic-acid binding surface

Edited by Brian W. Matthews, University of Oregon, Eugene, OR, and approved September 15, 2009
November 3, 2009
106 (44) 18521-18526

Abstract

The PUR protein family is a distinct and highly conserved class that is characterized by its sequence-specific RNA- and DNA-binding. Its best-studied family member, Pur-α, acts as a transcriptional regulator, as host factor for viral replication, and as cofactor for mRNP localization in dendrites. Pur-α-deficient mice show severe neurologic defects and die after birth. Nucleic-acid binding by Pur-α is mediated by its central core region, for which no structural information is available. We determined the x-ray structure of residues 40 to 185 from Drosophila melanogaster Pur-α, which constitutes a major part of the core region. We found that this region contains two almost identical structural motifs, termed “PUR repeats,” which interact with each other to form a PUR domain. DNA- and RNA-binding studies confirmed that PUR domains are indeed functional nucleic-acid binding domains. Database analysis show that PUR domains share a fold with the Whirly class of nucleic-acid binding proteins. Structural analysis combined with mutational studies suggest that a PUR domain binds nucleic acids through two independent surface regions involving concave β-sheets. Structure-based sequence alignment revealed that the core region harbors a third PUR repeat at its C terminus. Subsequent characterization by small-angle x-ray scattering (SAXS) and size-exclusion chromatography indicated that PUR repeat III mediates dimerization of Pur-α. Surface envelopes calculated from SAXS data show that the Pur-α dimer consisting of repeats I to III is arranged in a Z-like shape. This unexpected domain organization of the entire core domain of Pur-α has direct implications for ssDNA/ssRNA and dsDNA binding.

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Data Availability

Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 3K44).

Acknowledgments.

We thank Verena Aumiller, Thomas Carell, Klaus Förstemann, Daniela Hüls, Andrea Hildebrand, Sigrun Jaklin, Marisa Müller, Johannes Soeding, Sabine Ströbl, and Gregor Witte for their help; Stephen K. Burley for his support during the early stage of this project; Dirk Kostrewa for his continuous support during structure determination; and the crystallization facility of the Max Planck Institute for Biochemistry (Martinsried, Germany). We acknowledge the European Molecular Biology Laboratory/Deutsches Elektronen Synchrotron and European Synchrotron Radiation Facility for provision of synchrotron radiation facilities and thank Dmitry Svergun at DESY-X33 for support and Adam Round and Petra Pernot for assistance in using beamline ID14–3. This work was supported by the Helmholtz Association (VG-NH 142 to D.N.) and the Deutsche Forschungsgemeinschaft (FOR855 and SFB646 to D.N.). Stéphane Roche is a fellow of the Human Frontiers Science Program Organization.

Supporting Information

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Supporting Information

References

1
GL Gallia, EM Johnson, K Khalili, Puralpha: A multifunctional single-stranded DNA- and RNA-binding protein. Nucleic Acids Res 28, 3197–3205 (2000).
2
MK White, EM Johnson, K Khalili, Multiple roles for Puralpha in cellular and viral regulation. Cell Cycle 8, 1–7 (2009).
3
Y Kanai, N Dohmae, N Hirokawa, Kinesin transports RNA: Isolation and characterization of an RNA-transporting granule. Neuron 43, 513–525 (2004).
4
S Ohashi, et al., Identification of mRNA/protein (mRNP) complexes containing Pur-alpha, mStaufen, fragile X protein, and myosin Va and their association with rough endoplasmic reticulum equipped with a kinesin motor. J Biol Chem 277, 37804–37810 (2002).
5
P Jin, et al., Pur alpha binds to rCGG repeats and modulates repeat-mediated neurodegeneration in a Drosophila model of fragile X tremor/ataxia syndrome. Neuron 55, 556–564 (2007).
6
K Khalili, et al., Puralpha is essential for postnatal brain development and developmentally coupled cellular proliferation as revealed by genetic inactivation in the mouse. Mol Cell Biol 23, 6857–6875 (2003).
7
NN Chen, K Khalili, Transcriptional regulation of human JC polyomavirus promoters by cellular proteins YB-1 and Pur alpha in glial cells. J Virol 69, 5843–5848 (1995).
8
AL Brass, et al., Identification of host proteins required for HIV infection through a functional genomic screen. Science 319, 921–926 (2008).
9
R Konig, et al., Global analysis of host-pathogen interactions that regulate early-stage HIV-1 replication. Cell 135, 49–60 (2008).
10
CP Krachmarov, LG Chepenik, S Barr-Vagell, K Khalili, EM Johnson, Activation of the JC virus Tat-responsive transcriptional control element by association of the Tat protein of human immunodeficiency virus 1 with cellular protein Pur alpha. Proc Natl Acad Sci USA 93, 14112–14117 (1996).
11
LG Chepenik, AP Tretiakova, CP Krachmarov, EM Johnson, K Khalili, The single-stranded DNA binding protein, Pur-alpha, binds HIV-1 TAR RNA and activates HIV-1 transcription. Gene 210, 37–44 (1998).
12
DC Daniel, et al., Coordinate effects of human immunodeficiency virus type 1 protein Tat and cellular protein Puralpha on DNA replication initiated at the JC virus origin. J Gen Virol 82, 1543–1553 (2001).
13
LL Conte, C Chothia, J Janin, The atomic structure of protein-protein recognition sites. J Mol Biol 285, 2177–2198 (1999).
14
S Dasgupta, GH Iyer, SH Bryant, CE Lawrence, JA Bell, Extent and nature of contacts between protein molecules in crystal lattices and between subunits of protein oligomers. Proteins 28, 494–514 (1997).
15
L Holm, S Kaariainen, P Rosenstrom, A Schenkel, Searching protein structure databases with DaliLite v. 3. Bioinformatics 24, 2780–2781 (2008).
16
MA Schumacher, E Karamooz, A Zikova, L Trantirek, J Lukes, Crystal structures of T. brucei MRP1/MRP2 guide-RNA binding complex reveal RNA matchmaking mechanism. Cell 126, 701–711 (2006).
17
D Desveaux, J Allard, N Brisson, J Sygusch, A new family of plant transcription factors displays a novel ssDNA-binding surface. Nat Struct Biol 9, 512–517 (2002).
18
AD Bergemann, ZW Ma, EM Johnson, Sequence of cDNA comprising the human pur gene and sequence-specific single-stranded-DNA-binding properties of the encoded protein. Mol Cell Biol 12, 5673–5682 (1992).
19
MJ Wortman, EM Johnson, AD Bergemann, Mechanism of DNA binding and localized strand separation by Pur alpha and comparison with Pur family member, Pur beta. Biochim Biophys Acta 1743, 64–78 (2005).
20
M Muller, et al., Formation of She2p tetramers is required for mRNA binding, mRNP assembly, and localization. RNA, 2009).
21
J Brandsen, et al., C-terminal domain of transcription cofactor PC4 reveals dimeric ssDNA binding site. Nat Struct Biol 4, 900–903 (1997).
22
N Darbinian, GL Gallia, K Khalili, Helix-destabilizing properties of the human single-stranded DNA- and RNA-binding protein Puralpha. J Cell Biochem 80, 589–595 (2001).
23
L Lambert, UF Muller, AE Souza, HU Goringer, The involvement of gRNA-binding protein gBP21 in RNA editing-an in vitro and in vivo analysis. Nucleic Acids Res 27, 1429–1436 (1999).
24
UF Muller, L Lambert, HU Goringer, Annealing of RNA editing substrates facilitated by guide RNA-binding protein gBP21. EMBO J 20, 1394–1404 (2001).
25
J Koller, et al., Trypanosoma brucei gBP21. An arginine-rich mitochondrial protein that binds to guide RNA with high affinity. J Biol Chem 272, 3749–3757 (1997).
26
A Heuck, et al., Monomeric myosin V uses two binding regions for the assembly of stable translocation complexes. Proc Natl Acad Sci USA 104, 19778–19783 (2007).
27
S Doublie, Preparation of selenomethionyl proteins for phase determination. Methods in Enzymology, eds W Charles, J Carter, RM Sweet (Academic Press, San Diego) Vol 276, 523–537 (1997).
28
W Kabsch, Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J Appl Crystallogr 26, 795–800 (1993).
29
N Collaborative Computational Project, The CCP4 suite: Programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 50, 760–763 (1994).
30
SR Ness, RA de Graaff, JP Abrahams, NS Pannu, CRANK: New methods for automated macromolecular crystal structure solution. Structure 12, 1753–1761 (2004).
31
K Cowtan, The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr D Biol Crystallogr 62, 1002–1011 (2006).
32
P Emsley, K Cowtan, Coot: Model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60, 2126–2132 (2004).
33
GN Murshudov, AA Vagin, EJ Dodson, Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53, 240–255 (1997).
34
TC Terwilliger, Automated structure solution, density modification and model building. Acta Crystallogr D 58, 1937–1940 (2002).
35
AA Vaguine, J Richelle, SJ Wodak, SFCHECK: A unified set of procedures for evaluating the quality of macromolecular structure-factor data and their agreement with the atomic model. Acta Crystallogr D 55, 191–205 (1999).
36
EF Pettersen, et al., UCSF Chimera–a visualization system for exploratory research and analysis. J Comput Chem 25, 1605–1612 (2004).
37
J Sambrook, DW Russell Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Third Edition, Cold Spring Harbor, New York, 2001).
38
PV Konarev, MV Petoukhov, VV Volkov, DI Svergun, ATSAS2.1, a program package for small-angle scattering data analysis. J Appl Cryst 39, 277–286 (2006).

Information & Authors

Information

Published in

The cover image for PNAS Vol.106; No.44
Proceedings of the National Academy of Sciences
Vol. 106 | No. 44
November 3, 2009
PubMed: 19846792

Classifications

Data Availability

Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 3K44).

Submission history

Received: July 17, 2009
Published online: November 3, 2009
Published in issue: November 3, 2009

Keywords

  1. crystal structure
  2. DNA binding
  3. RNA binding
  4. fragile X-associated tremor
  5. ataxia syndrome

Acknowledgments

We thank Verena Aumiller, Thomas Carell, Klaus Förstemann, Daniela Hüls, Andrea Hildebrand, Sigrun Jaklin, Marisa Müller, Johannes Soeding, Sabine Ströbl, and Gregor Witte for their help; Stephen K. Burley for his support during the early stage of this project; Dirk Kostrewa for his continuous support during structure determination; and the crystallization facility of the Max Planck Institute for Biochemistry (Martinsried, Germany). We acknowledge the European Molecular Biology Laboratory/Deutsches Elektronen Synchrotron and European Synchrotron Radiation Facility for provision of synchrotron radiation facilities and thank Dmitry Svergun at DESY-X33 for support and Adam Round and Petra Pernot for assistance in using beamline ID14–3. This work was supported by the Helmholtz Association (VG-NH 142 to D.N.) and the Deutsche Forschungsgemeinschaft (FOR855 and SFB646 to D.N.). Stéphane Roche is a fellow of the Human Frontiers Science Program Organization.

Notes

This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0907990106/DCSupplemental.

Authors

Affiliations

Almut Graebsch
Institute of Structural Biology, Helmholtz Zentrum München, German Research Center for Environmental Health, Marchionini-Strasse 25, Munich, 81377, Germany; and
Department of Chemistry and Biochemistry, Gene Center Munich and Center for Integrated Protein Science CIPSM, Ludwig-Maximilians-University Munich, Feodor-Lynen-Strasse 25, Munich, 81377, Germany
Stéphane Roche
Institute of Structural Biology, Helmholtz Zentrum München, German Research Center for Environmental Health, Marchionini-Strasse 25, Munich, 81377, Germany; and
Department of Chemistry and Biochemistry, Gene Center Munich and Center for Integrated Protein Science CIPSM, Ludwig-Maximilians-University Munich, Feodor-Lynen-Strasse 25, Munich, 81377, Germany
Dierk Niessing1 [email protected]
Institute of Structural Biology, Helmholtz Zentrum München, German Research Center for Environmental Health, Marchionini-Strasse 25, Munich, 81377, Germany; and
Department of Chemistry and Biochemistry, Gene Center Munich and Center for Integrated Protein Science CIPSM, Ludwig-Maximilians-University Munich, Feodor-Lynen-Strasse 25, Munich, 81377, Germany

Notes

1
To whom correspondence should be addressed. E-mail: [email protected]
Author contributions: A.G., S.R., and D.N. designed research; A.G. performed research; A.G. and S.R. contributed new reagents/analytic tools; A.G., S.R., and D.N. analyzed data; and A.G. and D.N. wrote the paper.

Competing Interests

The authors declare no conflict of interest.

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    X-ray structure of Pur-α reveals a Whirly-like fold and an unusual nucleic-acid binding surface
    Proceedings of the National Academy of Sciences
    • Vol. 106
    • No. 44
    • pp. 18429-18873

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