Structural analysis of nucleotide binding sites of antimicrobial ribonucleases
- Autores: José Antonio Blanco Barrera
- Directores de la Tesis: Ester Boix i Borràs (dir. tes.)
- Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2015
- Idioma: inglés
- Tribunal Calificador de la Tesis: Xavier Parés i Casasampera (presid.), Alícia Guasch i Mitjans (secret.), Douglas Laurents (voc.)
- Materias:
- Enlaces
- Resumen
This thesis encompasses the structural and functional analysis of antimicrobial ribonucleases.
Nucleotide-type ligand interaction sites have been analysed using RNase A as a reference protein. RNase complexes were analysed by statistical structural analysis and X-ray crystallography. Together with the catalytic triad, other secondary interaction subsites were also defined at the protein surface. The RNase A superfamily embraces ribonucleases with diverse functions not necessarily related to RNase activity. In particular, antimicrobial properties are ascribed to members with high isoelectric point values, which also explain their stability and high affinity to membrane components. However, a noticeably lower RNase activity is seen for cationic RNases owing to the lack of key residues important for the correct substrate alignment.
The different nucleotide-type substrate binding and recognition patterns were deduced from an overall structure complex statistics¿ analysis and compared to the particular traits of selected families of representative endoribonucleases . A large amount of mono- and dinucleotide protein complexes was analysed. The results provided a general model of protein- nucleotide interactions for cytotoxic endoribonucleases. The identification of amino acids and atoms frequently involved in the recognition interactions defined three-dimensional motifs for phosphate, ribose and bases in the RNase A superfamily. Together with the conserved catalytic triad at the active site, residue variability is commonly observed throughout the secondary binding subsites, in agreement with the RNase preferential binding patterns, the different alignment capability, substrate specificity and variable catalytic efficiency. Results were complemented with molecular modelling predictions and evolution comparisons by sequence alignment and structural overlapping. A final side-by-side comparison with the microbial RNase T1 superfamily has allowed an analysis of the common and particular features of substrate recognition processes, thereby building a general interaction architecture applicable to recognition for polyanionic biomolecules that may set a structural basis for the design of new drugs.
Additionally, structural studies by X-ray crystallography were carried out. Recombinant wild- type RNases (RNase A, RNase 3, RNase 6) and mutant variants were expressed and purified in a high-yield prokaryotic system andtheir crystal structures were solved. In particular, a crystallisation condition has been discovered for human RNase 6. We report here the first crystal structure of RNase 6, which sets the basis for further analysis of interactions with nucleotide molecules and other putative ligands. On the other hand, the structural analysis of an RNase A mutant (RNase A/H7H10), where the secondary phosphate binding site p2 has been converted into a second active site, in complex with 3¿-CMP, enabled the visualisation of induced neighbouring conformational changes. A second RNase A ¿ 3¿-CMP complex, obtained at atomic resolution, has enabled a more detailed comparative analysis with lower-resolution protein-nucleotide complexes together with a side-by-side study of subsite environments with the mutant complex, explaining the mutant catalytic properties. The additional visualisation of the protonation state of the active site residues has also provided information about the mechanism of catalysis. Following, two RNase 3/ECP active site mutants (ECP/H15A, ECP/H128N) were crystallised. Structural studies confirmed the conservation of the protein overall three dimensional structure together with previous kinetic experiments related to the abolished catalytic activity. Also, two native ECP crystals obtained by two distinct crystallization conditions were used for a comparison of the different unit cell packing, residue side chain variability and anion recognition sites. Finally, the structures of RNase A, RNase 3/ECP and RNase 6 with bound sulphate anions were compared, and their putative anion recognition sites characterized. The comparison of the binding subsites confirmed the higher affinity of RNase 3/ECP for sulphate/ phosphate anionsand may lead to the identification of protein regions prone to host nucleotides or heterosaccharide compounds.
