Transient interactions between macromoleculesprotein-protein and protein-rna

Abstract

The characterization of molecular interaction networks in living cells is of great relevance to understand biological systems as a whole. In this context, the identification of binding surfaces, in addition to binding affinity data or thermodynamics/kinetics of specific interactions provide valuable information about these systems. Despite their critical role in a broad range of processes, transient intermolecular interactions remain poorly understood and the methods to characterize them are quite limited. NMR is one of the most suitable techniques to detect transient interactions and fully characterize them in vitro. Furthermore, there exist other experimental approaches which can be used to complement NMR data or even to support them, providing a complete view of the binding event and its relevance in a biological context. The main goal of this thesis is to characterize transient binding interactions and elucidate their relevance in different biological systems (protein-protein or protein-RNA complexes). In general, different NMR methods are applied to obtain the key information about the systems. To further evaluate the NMR results, other approaches as ITC, SPR and in cell assays are used. In this thesis, transient interactions have been studied each in a different system. ¿ Malarial MSP119 protein binding to cupredoxins has been studied as an example of protein-protein interactions. Proteins from the cupredoxins family have demonstrated antimalarial activity and potential as therapeutic agents. We have shown here that the cupredoxin Rc interacts with MSP119 with micromolar Kd, forming a well-defined complex. The interaction occurs on the same surface as that targeted by inhibitory antibodies, suggesting that a similar mechanism could take place. Its strength is dependent on the presence and, to a lesser extent, on the oxidation state of the metal center, which is consistent with the need for an intact interaction surface and charge conservation. Furthermore, we have observed the inhibition of P. falciparum parasitemia in infected cells where holo-Rc is present as a result of the interaction with MSP119. However, negligible effect on parasite growth could be observed for apo-Rc and Pc forms. ¿ Human tumour suppressor TIA-1 binding to RNA, has been studied as an example of protein-nucleic acid interactions. The different contribution of each TIA-1 protein domain to the RNA/DNA recognition seems to be related to the specific RNA sequence. The contribution of the third RRM domain of TIA-1 and its homologs to the RNA recognition process has been hinted in recent studies which suggest a key role for RRM3 in RNA binding and mRNA transcript selection. In the current study, the specific binding to certain RNA sequences by RRM3 domain has been determined and its role in regulating and enhancing the protein binding to C-rich RNA stretches is suggested. Interestingly, RRM3 shows a preference for ACUCC, which substantially increases the TIA-1 specificity for this kind of sequences over the U-rich ones. In addition, our study reveals that TIA-1 RRM3 domain acts as a pH-dependent molecular switch. Given that TIA-1 shuttles between the nucleus and the cytoplasm, such changes may modulate the overall binding to RNA.