NMR Spectroscopy of large complexes

Group Leader: Remco Sprangers

Phone: +49 (0)7071 - 601 1330

E-mail:

Introduction

The primary scientific goal of the research group is to understand the relationship between protein motions and protein function. This is especially relevant for most enzymes that have to undergo structural rearrangements to perform biological tasks. In the lab we focus on understanding the mechanism behind the bio-molecular complexes that play a role in the degradation of mRNA.

mRNA degradation

All organisms require a reliable mechanism to turn genes on and off. This regulation of gene expression underlies cellular processes ranging from the response to environmental signals to the development of multi-cellular organisms and cell-cell communication. Understandably, the cell tightly controls gene expression at every step from DNA to protein. Recent work has given new insights into these control mechanisms and revealed dedicated pathways that target mRNA for degradation, thereby efficiently turning genes off.

After export to the cytoplasm, mRNA is protected from degradation by a 5’ cap structure and a 3’ poly adenine tail. In the deadenylation dependent mRNA decay pathway, the polyA tail is gradually shortened by exonucleases. This ultimately attracts the degradation machinery that rapidly degrades the mRNA in both in the 5’ to 3’ direction and in the 3’ to 5’ direction. Additional mechanisms, including the nonsense mediated decay pathway, bypass the need for deadenylation and can remove the mRNA from the transcriptional pool independently. Interestingly, the same enzymes are responsible for the actual degradation of the mRNA independent of the pathway taken (see figure).

Figure 1: Simplified mechanism of mRNA decay. Dotted arrows indicate (indirect) interactions. Details of the pathways vary between different eukaryotes. PAB: poly-A binding protein.

Crystal structures for some of the complexes that play a role in mRNA decay have been solved, including the DCP1:DCP2 decapping complex, the DCPS scavenger decapping complex and the multi-component exosome complex. Our understanding of enzyme function is, however, limited to a static 3 dimensional fold of one of the many conformations that these proteins can adopt. To fully understand how molecular motions lead to catalytic activity a complete picture of the protein dynamics is required. In addition, the catalytic activity of these enzymes must be tightly regulated to prevent premature degradation of mRNA and to ensure maximum activity as soon as an mRNA has been identified as a substrate. As such, intermolecular interactions that modulate catalytic activity, e.g. by restricting molecular motions are of foremost interest.

Figure 2: Examples of protein decapping enzymes that can adept different conformations. Left: the Dcp1:Dcp2 complex (She et. al. Mol Cell 2008, She et. al. Nat Struct Mol Biol. 2006; 2QKM, 2A6T). Right: DcpS in the open and closed state (Gu et. al. Mol Cell 2004; 1XMM, 1XML)

NMR Spectroscopy

Nuclear Magnetic Resonance (NMR) spectroscopy is the method of choice to detect and quantify biologically important motions in a near cellular environment. Using dedicated experiments we can probe motions ranging from the pico- to nanosecond time-scale up to the second time-scale, with atomic resolution. In addition, we can monitor how the molecular motions are affected by the interaction with adaptor complexes.

Traditionally, the application of NMR spectroscopy has been limited to complexes below 25 kDa in molecular weight. Recent advances have, however extended this molecular weight regime into the hundreds of kDa and in favorable cases over 1 MDa. This makes the large complexes involved in mRNA decay amenable to detailed studies of molecular dynamics. On the campus of the Max Planck Institute for Developmental Biology in Tübingen we now have 2 brand new NMR spectrometers installed (600 MHz and 800 MHz).

In summary, we hope to obtain a complete picture of protein dynamics and the modulation thereof. These aspects are fundamental to the understanding of the delicate balance between mRNA synthesis and degradation that allows the cell to regulate cellular processes by rapidly switching specific genes on or off.

Figure 3: 600 and 800 MHz NMR machines at the Tuebingen Campus, Germany

Vacancies

The lab has space for diploma students, please e-mail remco.sprangers and include a brief cover letter and CV.

Students interested in joining the lab for a PhD are encouraged to apply through the PhD program, the International Max Planck Research School (IMPRS) or to e-mail a cover letter and CV directly to remco.sprangers. Currently, we are looking for candidates with a strong background in biochemistry, structural biology and/or biophysics.

For more information, please have a look at the institutes vacancies page and at the poster.

Selected Publications

Fromm SA, Truffault V, Kamenz J, Braun JE, Hoffmann NA, Izaurralde E, Sprangers R. The structural basis of Edc3- and Scd6-mediated activation of the Dcp1:Dcp2 mRNA decapping complex. EMBO J. 2011 Nov 15.

Mund M, Neu A, Ullmann J, Neu U, Sprangers R. Structure of the LSm657 Complex: An Assembly Intermediate of the LSm1-7 and LSm2-8 Rings. J Mol Biol. 2011 Oct 6.

Religa TL, Sprangers R, Kay LE. Dynamic regulation of archaeal proteasome gate opening as studied by TROSY NMR. Science 2010 Apr 2;328(5974):98-102.

Sprangers R, Kay LE. Probing supramolecular structure from measurement of methyl (1)H-(13)C residual dipolar couplings. J Am Chem Soc. 2007 Oct 24;129(42):12668-9.

Sprangers R, Velyvis A, Kay LE. Solution NMR of supramolecular complexes: providing new insights into function. Nat Methods. 2007 Sep;4(9):697-703.

Sprangers R, Kay LE. Quantitative dynamics and binding studies of the 20S proteasome by NMR. Nature. 2007 Feb 8;445(7128):618-22.

Sprangers R, Gribun A, Hwang PM, Houry WA, Kay LE. Quantitative NMR spectroscopy of supramolecular complexes: dynamic side pores in ClpP are important for product release.
Proc Natl Acad Sci U S A. 2005 Nov 15;102(46):16678-83.

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