Research GroupsCarlomagno Group
Integrative Structural Biology

INTEGRATED STRUCTURAL BIOLOGY

To be able to develop new drugs, it is necessary to uncover the molecular basis of the disease. Therefore, it is essential to understand which cellular processes are at the basis of the disease of interest and how they are regulated.

 

Our research focuses on the structural and functional biology of large bio-molecular complexes with enzymatic activity, including protein and RNA-protein (RNP) complexes. Our philosophy is to image the particle at several key stages during its functional cycle using an integrative approach that combines structural data at multiple levels of resolution. We use this “dynamic” structural imaging to elucidate the molecular basis of activity and regulation of the molecular complex. Subsequently, we exploit this knowledge to design small molecule binders that are able to modulate the enzymatic activity of the particle for either in vivo functional studies or drug development.

 

Structural biology, biophysics and in silico modelling are established approaches to uncover the functional and regulatory mechanisms of enzymes, consisting of both proteins and protein-nucleic acids complexes. Thanks to the recent hardware and software developments, electron microscopy (EM) holds the promise to overcome the need of crystallization for structure determination of any stable bio-molecular complex. Despite these progresses, understanding the dynamics of bio-molecular assemblies during their functional cycles, as well as the nature of transient interactions, continues to be a challenge. Dynamics and transient interactions are the basis of both enzyme processivity and regulation; in this context, structural biology techniques that can deal with conformational heterogeneity, structural changes or dynamic processes are important. Among them, nuclear magnetic resonance (NMR) spectroscopy, small angle scattering (SAS), electron paramagnetic resonance (EPR), cross-links detected by mass-spectrometry and fluorescence (FRET) are the most prominent.

My group has a world-class expertise in NMR spectroscopy and SAS, as well as their combination with EPR and X-ray crystallography. Recently, we have started building up expertise in the analysis of EM data acquired in collaboration. The full potentiality of structural biology lies on the complementarity of all available techniques. In line with this idea, my group develops integrative structure determination methods (Figure 1, Karaca et al., Nature Methods 2017).

Figure 1. Integrative computational tools can translate complementary sparse data at different levels of resolution into accurate structural information (RDC, NMR-detected Residual Dipolar Couplings; PCS, NMR-detected Pseudo Contact Shifts; PRE, NMR-detected Paramagnetic Relaxation Enhancement; EPR, Electron Paramagnetic Resonance; FRET, Foerster Resonance Energy Transfer; EM, Electron Microscopy; SAXS, Small Angle X-ray Scattering).

NMR is one of the main techniques in our portfolio, as it is applicable in solution and reports both structural and dynamic properties of individual atomic positions, without need of chemical modifications. Long considered unsuitable for high molecular weight molecules, recent hardware and methodology developments have allowed the application of NMR to complexes as large as 1 MDa. Using a combination of NMR spectroscopy, SAS and biochemical data, in 2013 we solved the structure(s) of the archaeal Box C/D sRNP enzyme (molecular weight ~ 400 kDa), which is responsible of 2’-O-ribose methylation of ribosomal RNA (Figure 2). A large conformational change was detected upon substrate binding, revealing an unexpected three-dimensional organization of the catalytic RNP complex and a mechanism for the regulation of methylation at different rRNA sites (Lapinaite et al. Nature, 2013). In the meantime, we have applied integrative structural biology to three other complexes in the context of RNA editing, histone modifications and peptide synthesis.

Figure 2. Structure of the RNA-methylating machinery Box C/D RNP loaded with substrates (Nature, 2013).

With the study of large RNP complexes, we entered the field of solid-state (ss) NMR. ssNMR is particularly suitable for large complexes, as the quality of the spectra is independent of the size of the molecule. Despite not preserving the same level of dynamics as in solution, the sample preparation for ssNMR does not require crystallization and conserves a high level of hydration. Pioneering the application of ssNMR to study RNA and RNA-protein complexes, we have determined the first de novo high-resolution structure of RNA by ssNMR data (Marchanka et al. Angew. Chemie. 2013; Marchanka et al. Nature Commun. 2015). We believe that there is an as yet unexplored potential of ssNMR spectroscopy in the field of RNPs.

At the BMWZ we are active in the following research areas:

1.     RNA editing, modification and metabolism.

2.     Protein allosteric regulation in cancer.

3.     Protein unfoldases in bacteria and eukaryotes.

4.     Non-ribosomal peptide synthetases.

5.     Methodology development. 

References

1. Karaca, Rodrigues, Graziadei, Bonvin, Carlomagno “An Integrative Framework for Structure Determination of Molecular Machines” Nature Methods 2017 14, 897–902

2. Lapinaite, Simon, Skjaerven, Rakwalska-Bange, Gabel, Carlomagno “The structure of the Box C/D enzyme reveals regulation of rRNA methylation” Nature 2013 502, 519-523

3. Marchanka, Simon, Carlomagno “A Suite of solid-state NMR experiments for RNA intranucleotide resonance assignment in a 21 kDa protein-RNA complex” Angewandte Chemie 2013 52, 9996-10001

4.  Marchanka, Simon, Althoff-Ospelt, Carlomagno “RNA structure determination by solid-state NMR spectroscopy” Nature Communications, 2015 doi: 10.1038/ncomms8024