Characterization of metal binding by a designed protein: single ligand substitutions at a tetrahedral Cys2His2 site.
The tetrahedral Cys2His2 Zn(II)-binding site in the de novo designed protein Z alpha 4 [Regan, L., & Clarke, N. D. (1990) Biochemistry 29, 10878] has been studied by independently mutating each of the metal-binding ligands to alanine. The contribution of each ligand to the geometry and affinity of metal binding has been characterized using Co(II), Zn(II), and Cd(II). The results indicate that all four ligands contribute to high-affinity metal binding in Z alpha 4. Two of the four metal-site mutants retain the tetrahedral Zn(II)-binding geometry of Z alpha 4, with one water molecule presumed to bind in the vacant ligand position. These mutants provide the first examples of a demonstrated de novo tetrahedral three-coordinate site designed into a protein and as such are a first step toward the design of catalytic rather than structural Zn(II) sites. One of the metal-site mutants binds Zn(II) with either tetrahedral four-coordinate or five-coordinate geometry, while the last ligand-to-alanine substitution abolishes tetrahedral binding. The importance of ligand type for metal-binding in Z alpha 4 was investigated by characterizing two ligand-swap mutants in which a cysteine residue was replaced with a histidine. In both cases, tetrahedral metal binding was lost. Collectively, these results affirm the strategy used to design Z alpha 4 by showing that all designed liganding residues are participating in metal binding, and by suggesting that the tetrahedral geometry of the binding site is perturbed when the designed side chain ligands are replaced with alternate ligands.
Klemba
M
M
Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
Regan
L
L
D001665:Binding Sites; D002104:Cadmium / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003035:Cobalt / Q000737:chemistry; D008667:Metalloproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008670:Metals / Q000378:metabolism*; D008958:Models, Molecular; D016254:Mutagenesis, Insertional; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015032:Zinc / Q000737:chemistry
D002352:Carrier Proteins; D008667:Metalloproteins; D008670:Metals; D011994:Recombinant Proteins; C098390:protein Z alpha 4; D002104:Cadmium; D003035:Cobalt; D015032:Zinc