Toward the de Novo Design of a Catalytically Active Helix Bundle: A Substrate-Accessible Carboxylate-Bridged Dinuclear Metal Center

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• 作者：Luigi Di Costanzo ; Herschel Wade ; Silvano Geremia ; Lucio Randaccio ; Vincenzo Pavone ; William F. DeGrado ; and Angela Lombardi
• 刊名：Journal of the American Chemical Society
• 出版年：2001
• 出版时间：December 26, 2001
• 年：2001
• 卷：123
• 期：51
• 页码：12749 - 12757
• 全文大小：501K
• 年卷期：v.123,no.51(December 26, 2001)
• ISSN：1520-5126

文摘

De novo design of proteins provides an attractive approach to uncover the essential features requiredfor their functions. Previously, we described the design and crystal structure determination of a di-Zn(II) complexof "due-ferri-1" (DF1), a protein patterned after the diiron-dimanganese class of redox-active proteins[Lombardi, A.; Summa, C.; Geremia, S.; Randaccio, L.; Pavone, V.; DeGrado, W. F. Proc. Natl. Acad. Sci.U.S.A. 2000, 97, 6298-6305]. The overall structure of DF1, which contains a carboxylate-bridged dinuclearmetal site, agrees well with the intended design. However, access to this dimetal site is blocked by a pair ofhydrophobic leucine residues (L13 and L13'), which prevent facile entry of metal ions and small molecules.We have now taken the next step in the eventual construction of a catalytically active metalloenzyme byengineering an active site cavity into DF1 through the replacement of these two leucine residues with smallerresidues. The crystal structure of the dimanganous form of L13A-DF1 indeed shows a substrate access channelto the dimetal center. In the crystal structure, water molecules and a ligating dimethyl sulfoxide molecule,which forms a monatomic bridge between the metal ions, occupy the cavity. Furthermore, the diferric form ofa derivative of L13A-DF1, DF2, is shown to bind azide, acetate, and small aromatic molecules.