Functional mechanisms of complex enzymes involved in RNA metabolism and methodology development for drug design
Figure 1: Structure of the RNA-methylating machinery Box C/D RNP shows that only one pair of proteins (blue) can add methyl groups to the RNA (red) at a time (Lapinate et al., 2013).
Figure 2: Schematic representation of the principle of the INPHARMA NOEs.
The Carlomagno group uses NMR spectroscopy in combination with biochemical and biophysical techniques to study the structure and dynamics of biomolecular complexes.
Previous and current research
Our group focuses on studying: i) structure-activity relationships of RNP complexes involved in RNA processing; and ii) the interaction of small drugs with cellular receptors.
Our work aims at describing the features of RNA-protein recognition in RNP complex enzymes and at characterising the structural basis for their function. Recently, we investigated the nucleolar multimeric Box C/D RNP complex responsible for the methylation of the 2’-O-position in rRNA. During the biosynthesis and processing of the pre-rRNA transcripts, post-transcriptional modifications of ribonucleotides occur in functionally important regions, such as at intersubunit interfaces, decoding and peptidyltransferase centers. Among the possible modifications, 2’-O-ribose methylation was shown to protect RNA from ribonucleolytic cleavage, stabilise single base pairs, serve as chaperone, and impact folding at high temperatures. We solved the structures of the 400 kDa enzyme in solution. A large conformational change is detected upon substrate binding, revealing an unexpected 3D organisation of the catalytic RNP (figure 1). In addition, the structure revealed an unsuspected mechanism of sequentially controlled methylation at dual sites of the rRNA, which might have important implications for ribosome biogenesis.
Conformational switches occur in macromolecular receptors at all cellular levels, dependent on the presence of small organic molecules that are able to trigger or inhibit specific cellular processes. In a second area of research, we develop both computational and experimental tools to access the structure of large receptors in complex with function regulators. We are the developers of INPHARMA, a novel approach to structure-based drug design that does not require crystallographic structures of the receptor-drug complex (figure 2). We apply our methods to study the functional mechanisms of anti-cancer drug-leads, designed as inhibitors of kinases, proteasome and membrane receptors.
Future projects and goals
My team uses a multidisciplinary approach combining nuclear magnetic resonance spectroscopy (NMR), and biochemical, biophysical and computational methods. Our philosophy is to tackle the structure of high molecular weight complexes, whose large size impedes a detailed structural description by NMR only, with an array of different complementary methodologies, such as segmental and specific labelling of both proteins and RNAs, small angle scattering (SAS), electron microscopy (EM), electron paramagnetic resonance (EPR), fluorescence resonance energy transfer (FRET), mutational analysis and biochemical experiments (e.g. cross-link). With our complementary approach it is possible to examine RNP particles in solution, in their native environment, where they preserve both their structure and dynamic properties.