De Novo Design And Characterization Of A Protein Binding An Abiotic Tetranuclear Zinc Cluster

The protein matrix finely tunes metal cofactor geometry and reactivity, despite the limited number of ligands.[1] De novo protein design is particularly appealing in gaining new insights on the concurring interactions providing specific functions.[2] While there has been considerable progress in designing proteins that bind mononuclear as well as multinuclear metal cofactors,[3-5] the design of tetranuclear clusters with oxygen-rich environments has not been accomplished. Here, we describe our recent work on the design, structural and spectroscopic characterization of tetra-Zn2+ carboxylate-bridged clusters. The distorted cube-like structure is deeply inserted into the hydrophobic core of a four-helix bundle scaffold, and largely stabilized by a fully connected hydrogen-bonded network, unprecedented in protein design, consisting of 16 polar side chains. By varying the metal-bridging and the interfacial residues in the bundle, we show that subtle changes (in the order of few angstroms) have a great impact on the coordination geometry, as well as oligomeric state and stability. The crystal structures of the first four analogues confirm the validity of the design process in two cases, and define general rules for subsequent designs.[6] Three of these newly designed proteins have been fully characterized by CD and NMR spectroscopies. Solution studies strongly suggest that the desired structure is achieved also in the apo state, providing evidence that the peptide is able to actively impart the designed geometry to the metal cluster.[7] In perspective, such abiotic tetra-Zn2+ cluster will be adopted as catalyst for both complex transesterification, and hydrolysis reactions.[8] It may also constitute the basis for the construction of the first de novo designed oxygen evolving complex, by replacing zinc with designated redox-active metals. [1] O. Maglio, F. Nastri, A. Lombardi, in Ioninc Interactions in Natural and Synthetic Macromolecules (Eds.: A. Ciferri, A. Perico), John Wiley & Sons, Inc., Hoboken, NJ, USA, 2012, pp. 361–450. [2] F. Yu, V. M. Cangelosi, M. L. Zastrow, M. Tegoni, J. S. Plegaria, A. G. Tebo, C. S. Mocny, L. Ruckthong, H. Qayyum, V. L. Pecoraro, Chem. Rev. 2014, 114, 3495–3578. [3] F. Nastri, M. Chino, O. Maglio, A. Bhagi-Damodaran, Y. Lu, A. Lombardi, Chem. Soc. Rev. 2016, 45, 5020–5054. [4] A. F. Peacock, Curr. Opin. Chem. Biol. 2016, 31, 160–165. [5] M. Chino, L. Leone, G. Zambrano, F. Pirro, D. D’Alonzo, V. Firpo, D. Aref, L. Lista, O. Maglio, F. Nastri, A. Lombardi, Biopolymers 2018, e23107. [6] S.Q. Zhang, M. Chino, L. Liu, Y. Tang, X. Hu, W. F. DeGrado, A. Lombardi, J. Am. Chem. Soc. 2018, 140, 1294–1304. [7] M. Chino, S.Q. Zhang, F. Pirro, L. Leone, O. Maglio, A. Lombardi, W. F. DeGrado, Biopolymers 2018, under review. [8] T. Ohshima, Chem. Pharm. Bull. (Tokyo) 2016, 64, 523–539.