Crystallographic Comparison of Tris-Thiolate Sites in Designed Proteins to Control Metal Geometries.

[摘要] De Novo three-stranded coiled coil proteins formed using TRI and CoilSer peptides are used to generate a Cys-rich site that arranges thiols to chelate metals into geometries resembling heavy metal sites in metalloregulatory systems. A series of high resolution X-ray diffraction structures was obtained to assess the Cys behavior upon metal binding. In the apo-structures, the three Cys ligands, preferentially orient toward the helical core, revealing a preorganized pocket to bind a trigonal pyramidal Pb(II)S3- and As(III)S3. This arrangement is predisposed for trigonal planar Hg(II)S3- and 4-coordinate Zn(II)S3O- structures, requiring significant thiol rotation to bind metals. The structures confirm the control of coordination number is achieved on Cd(II) and Hg(II) complexes through ligand modifications. The predisposition of Cys upon metal binding results in a mixture of 3- and 4-coordinate Cd(II), while the bulky Pen restricts thiol rotation, causing a shift in the metal plane that displaces water, generating a pure Cd(II)S3-. Substitution of Ala for Leu opens a cavity above the Cys site which allows for four waters to enter, forming the Cd(II)S3O- center. Additionally, inclusion of D-amino acid ligands compels metals into specific geometry. D-Leu, above the Cys site, reorients the side chain towards the coordination layer diminishing the space for water access yielding Cd(II)S3-; however, D-Leu below the metal site opens more space, allowing for equal Cd(II)S3O- and Cd(II)S3O2- structures. The substitution of D-Leu residues simultaneously above and below the Cys site results in a predominant Cd(II)S3O-. The chirality perturbation by D-Cys causes a shift of the sulfur plane down, thus making a larger cavity suitable for Cd(II)S3O-. Moreover, D-Pen binds Hg(II) in a linear fashion, while L-Pen can enforce an unusual trigonal Hg(II) center. These studies provide insights on how to control desired metal geometries in metalloproteins.

[发布机构] University of Michigan

[效力级别] Tris-thiolate sites in designed proteins

[关键词] De novo designed protein;Tris-thiolate sites in designed proteins;Control of metal geometries using three-stranded coiled coil peptides;Crystal structures of de novo designed peptides;D-amino acids in de novo protein design;Chemistry;Science;Biophysics