Chino, Marco (2014) ENGINEERING METAL BINDING SITES IN DE NOVO DESIGNED FOUR-HELIX BUNDLES. [Tesi di dottorato]

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A single polypeptide chain may access to an astronomical amount of conformers[1]. Nature selected only a trivial number of them through evolution, composing an alphabet of scaffolds, which can afford the complete set of chemical reactions needed to support life[2]. These structural templates are so stable to allow several mutations without disruption of the global folding even reaching the ability to bind several exogenous cofactors. In this perspective metal cofactors play a crucial role in regulation and catalysis of several processes. Nature is able to modulate the chemistry of metals adopting only few ligands and slightly different geometries[3]. Thus, understanding how the fine-tuning of the scaffolds hosting them imparts the wide spectrum of reactivity is of crucial interest both in the fields of structural biology and bioinorganic chemistry. Several scaffolds and metal binding motifs are object of intense work in the literature[4]. In this PhD work, we focused on the four-helix bundle as scaffold for metal binding sites in the context of protein de novo design, to obtain basic biochemical components for biosensing or catalysis. In order to accomplish our objectives we chose to expand the designable space of a well-characterized de novo designed family of metalloproteins, the DFs (Due-Ferro). DF1, the progenitor of the family, bears a dicarboxylate bridged dinuclear metal center in its hydrophobic core constituted by the four-helix bundle unit[5]. We investigated the opportunity to find new chemical properties[6,7] by loosening on the one side and tightening on the other the C2 symmetric environment of the metal cofactor, giving raise to two new class of de novo designed proteins: DF-Click and QF. Previous attempts to design asymmetrical DFs gave successful results[8,9], even resulting in catalytic activity towards different oxidation reaction[8] upon redesign of the metal binding site[7]. We implemented an alternative synthetic approach to generate new DF analogues by means of copper catalyzed azide-alkyne cycloaddition (CuAAC), also known as “Click Chemistry”[10]. Our design strategy led to a helix-loop-helix heterodimer, obtained through side chain chemical ligation, affording the DF-Click series. The design, synthesis and spectroscopic characterization of an analogue of this series named DF-Click1, which was inspired to the hydroxylase component of the bacterial multicomponent monooxygenases (BMMs)[11], was completed during the PhD research activity by means of solution NMR, circular dichroism, UV-Visible absorption spectroscopy. Further, as we were interested in the metal properties induced by an asymmetrical site, we also evaluated the opportunity to design a model with a higher degree of symmetry. We aimed to the synthesis of a D2 symmetric homotetramer able to bind a tetranuclear metal cofactor. We named this novel de novo designed protein class QF (Quattro Ferro). It consists of four helix strands each one bearing the ExxH binding motif as in helix 2 of DF1. It was inspired to the cubane like Mn-O cluster found in Photosystem II, where four manganese ions provide the oxidative equivalents necessary for the four electrons oxidation of water. During the PhD work, the design process, synthesis and spectroscopic analysis of four analogues were accomplished: W45Y27 and W45Y28, which derive from the ab initio design of a protein crystal with predetermined topology; QF6 and QF33, whose design derives from the serendipitous finding of an already deposited crystal structure of a GCN4 mutant with a similar crystal topology, but without the desired polarity. A combination of NMR and X-Ray analysis confirms the correctness of the design. Spectroscopic analysis intended to ascertain the metal binding stoichiometry and geometry is under course. References (1) Dill, K. A.; Chan, H. S. Nat. Struct. Mol. Biol. 1997, 4, 10. (2) Sillitoe, I.; Cuff, A. L.; Dessailly, B. H.; Dawson, N. L.; Furnham, N.; Lee, D.; Lees, J. G.; Lewis, T. E.; Studer, R. A.; Rentzsch, R.; Yeats, C.; Thornton, J. M.; Orengo, C. A. Nucleic Acids Res. 2012, 41, D490. (3) Bertini, I. Biological Inorganic Chemistry: Structure and Reactivity; University Science Books, 2007. (4) Lu, Y.; Berry, S. M.; Pfister, T. D. Chem. Rev. 2001, 101, 3047. (5) Maglio, O.; Nastri, F.; Martin de Rosales, R. T.; Faiella, M.; Pavone, V.; DeGrado, W. F.; Lombardi, A. Comptes Rendus Chim. 2007, 10, 703. (6) Faiella, M.; Andreozzi, C.; de Rosales, R. T. M.; Pavone, V.; Maglio, O.; Nastri, F.; DeGrado, W. F.; Lombardi, A. Nat Chem Biol 2009, 5, 882. (7) Reig, A. J.; Pires, M. M.; Snyder, R. A.; Wu, Y.; Jo, H.; Kulp, D. W.; Butch, S. E.; Calhoun, J. R.; Szyperski, T.; Solomon, E. I.; Degrado, W. F. Nat. Chem. 2012, 4, 900. (8) Kaplan, J.; DeGrado, W. F. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 11566. (9) Calhoun, J. R.; Kono, H.; Lahr, S.; Wang, W.; DeGrado, W. F.; Saven, J. G. J. Mol. Biol. 2003, 334, 1101. (10) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. Int. Ed. 2001, 40, 2004. (11) Tinberg, C. E.; Lippard, S. J. Acc. Chem. Res. 2011, 44, 280

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