Seminar by Prof. Teresa Carlomagno

Leibniz University Hannover

Complexes in RNA metabolism and gene expression regulation: a view by integrative structural biology

The development of novel therapeutic strategies relies on the understanding of molecular mechanisms and molecular communication networks in diseases. In turn, an accurate description of molecular mechanisms requires structural information in an environment where the molecular machinery preserves its dynamic features. Our group focuses on studying large molecular machines in solution using an integrative structural biology approach developed around NMR spectroscopy.

Recent technical advancements have overcome the molecular-weight limits of traditional NMR methods. The most prominent example is the use of isoleucine, leucine and valine (ILV-) methyl groups as probes, which can provide inter-subunit distances even for molecular machines as large as hundreds of kDa.

During the past decade, a handful of integrative computational tools have been introduced that can accurately translate such sparse information into structures [1]. Though, as most of these tools were developed in a case-specific manner, there is still an ongoing demand for a standardized integrative computational method. Here we present a general integrative modeling framework that can deal with molecules of different nature (e.g. proteins, nucleic acids) and make use of diverse distance and/or proximity information to determine the structures of supramolecular assemblies [2]. Our framework includes (i) extension of the well-known integrative computational tool HADDOCK, such that it can address supramolecular complexes having >>6 components; (ii) proof-of-concept
incorporation of various (CSP, NOE, PRE) ILV-methyl distances, which is demonstrated on a realistic benchmark spanning from 85 kDa to 450 kDa; (iii) a new approach to score different conformers of large assemblies with respect to the experimental data; (iv) a statistical analysis to evaluate the fitness of the data.

This approach will be demonstrated on the example of protein-protein and protein-RNA complexes, whose structure is obtained by a powerful combination of solution-state NMR and small angle neutron scattering (SANS).

In the second part of the talk, I will show the application of solid-state NMR (ssNMR) to study large RNA-protein complexes. I will present the ssNMR-based structure of the 26mer box C/D RNA in complex with the protein L7Ae [3], together with the experimental strategy that we used to obtain it. Lastly I will present first ssNMR spectra for the RNA part of a 400 kDa RNP complex.

REFERENCES

[1] A. Lapinaite, B. Simon, L. Skjaerven, M. Rakwalska-Bange, F.Gabel, T. Carlomagno The structure of the Box C/D enzyme reveals regulation of rRNA methylation Nature 502, 519-523 (2013).

[2] E. Karaca, J.P.G.L.M. Rodrigues, A. Graziadei, A.M.J.J. Bonvin, T. Carlomagno An Integrative Framework for Structure Determination of Molecular Machines Nature Methods 14, 897–902 doi:10.1038/nmeth.4392 (2017).

[3] A. Marchanka, B. Simon, G. Althoff-Ospelt, T. Carlomagno RNA structure determination by solid-state NMR spectroscopy Nature Communications, DOI: 10.1038/ncomms8024 (2015)