Crystal structures of the copper and nickel complexes of RNase A: Metal-induced interprotein interactions and identification of a novel copper binding motif

Abstract

We report the crystal structures of the copper and nickel complexes of RNase A. The overall topology of these two complexes is similar to that of other RNase A structures. However, there are significant differences in the mode of binding of copper and nickel. There are two copper ions per molecule of the protein, but there is only one nickel ion per molecule of the protein. Significant changes occur in the interprotein interactions as a result of differences in the coordinating groups at the common binding site around His-105. Consequently, the copper-and nickel-ion-bound dimers of RNase A act as nucleation sites for generating different crystal lattices for the two complexes. A second copper ion is present at an active site residue His-119 for which all the ligands are from one molecule of the protein. At this second site, His-119 adopts an inactive conformation (B) induced by the copper. We have identified a novel copper binding motif involving the ␣-amino group and the N-terminal residues.Metal ions are responsible for a wide range of functions, including nucleophilic catalysis in enzymes such as carbonic anhydrase and carboxypeptidase, electron transfer in proteins such as rubredoxin and the cytochromes, and stabilization of protein structure in many proteins from zinc finger to alcohol dehydrogenase (1, 2) and in gene regulation (3,4). Cu 2ϩ and Zn 2ϩ are essential trace elements present in many species (5, 6), whereas Ni 2ϩ enters the cell by chronic exposure (7) and is a known carcinogen (8). Copper functions as a cofactor in copper-zinc superoxide dismutase and plays an important role in the defense against oxygen-derived free radicals that have deleterious effects on biological macromolecules (4). Because of the many biological roles played by these metal ions, efforts have been recently directed to engineer de novo metal binding sites to impart a desired property to a protein. For example, metal binding sites have been engineered to aid protein purification (9), to enhance protein stability (10, 11) and regulation of catalytic activity (12, 13), and to introduce metal ions into novel protein scaffolds (14,15). In this regard structural studies of metalloproteins have greatly aided in engineering of novel metal binding sites into proteins.However, presence of these transition metals in abnormal levels due to metabolic disorders or due to chronic exposure has been linked to DNA damage and eventually to cell death. For example, for Wilson and Menkes diseases, there is an aberrant copper metabolic pathway leading to copper accumulation (16). In Alzheimer disease, decreased levels of a metallothionein-like protein has been observed leading to an increase in the levels of zinc (17, 18). Inability of metalloproteins to perform normal functions has been directly attributed to the absence of metal ions leading to a conformational change, as shown by crystallographic, spectroscopic, and biochemical studies (19-23). But so far there has been limited evidence to elucidate how metals, upon binding, can forc...

A New Type of Metal Recognition by Human T Cells

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A novel paradigm for metal-induced ring flipping in the copper(II) complex of 1,2-bis( N -methylbenzimidazol-2 ′ -yl)benzene triflate

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