d‐Cysteine Ligands Control Metal Geometries within De Novo Designed Three‐Stranded Coiled Coils

Abstract

While metal ion binding to naturally occurring L-amino acid proteins is well documented, understanding the impact of the opposite chirality (D) amino acids on the structure and stereochemistry of metals is in its infancy. We examine the effect of a D-configuration cysteine within a designed L-amino acid three-stranded coiled coil in order to enforce a precise coordination number on a metal center. The D-chirality does not alter the native fold, but the side-chain reorientation modifies the sterics of the metal binding pocket. L-Cys side-chains within the coiled-coil have previously been shown to rotate substantially from their preferred positions in the apo structure to create a binding site for a tetra-coordinate metal ion. However, here we show by x-ray crystallography that D-Cys side chains are pre-organized with suitable geometry to bind such a ligand. This is confirmed by comparison of the Zn(II)Cl(CSL16DC)32− to the published Zn(II)(H2O)(GRAND-CSL12AL16LC)3−1. Moreover, spectroscopic analysis indicates that the Cd(II) geometry observed using L-Cys ligands (a mixture of 3- and 4- coordinate Cd(II)) is altered to a single 4-coordinate specie when D-Cys is present. This work opens a new avenue for the control of metal site environment in man-made proteins, by simply altering the binding ligand with its mirror imaged D-configuration. Thus, the use of non-coded amino acids in a metal’s coordination sphere promises to be a powerful tool for controlling the properties of future metalloproteins.

How Outer Coordination Sphere Modifications Can Impact Metal Structures in Proteins: A Crystallographic Evaluation

“…The Hg II ‐S bond distance in the Hg II S Zn(II) N (GRAND‐CSL16CL30H) 3 + crystal structure (PDB code: 5KB1; PDB=Protein Data Bank) has been reported to be 2.38 Å, which is in good agreement with an X‐ray absorption result for Hg II (TRIL16C) 3 − (2.43 Å) . At the same time, the extended X‐ray absorption fine structure (EXAFS) result for the Cd II −S bond length for the trigonal planar Cd II (TRIL16Pen) 3 − is 2.46 Å, which leads one to predict the trigonal planar structures of Hg II S 3 and Cd II S 3 are similar. Thus, regardless of the metal size difference, the crystallographic Hg II S 3 structures could be used to explain general characteristics of Cd II S 3 .…”

“…The Hg II -S bond distance in the Hg II S Zn(II) N (GRAND-CSL16CL30H) 3 + crystal structure (PDB code:5 KB1;P DB = Protein Data Bank) has been reported to be 2.38 , [13] which is in good agreement with an X-ray absorption result for Hg II (TRIL16C) 3 À (2.43 ). [7] At the same time, the extended X-ray absorption fine structure (EXAFS) result for the Cd II ÀSb ond length for the trigonal planar Cd II (TRIL16Pen) 3 À is 2.46 , [29] which leads one to predict the trigonal planar structures of Hg II S 3 and Cd II S 3 are similar.T hus, regardless of the metal size difference, the crystallographic Hg II S 3 structures could be used to explain general characteristics of Cd II S 3 .I nt his study,w e have also achieved av ariety of crystal structures based on the sequences designed for Cd II studies. We have obtained the Hg II (GRAND-CSL12AL16C) 3 À ,r epresenting the TRIL12AL16C environment, toa nalyze the effect of Ala (12th position) above the metal site in comparison with the 12 Leu packing of the known Hg II S Zn(II) N (GRAND-CSL16CL30H) 3 + .…”

Incorporation of second coordination sphere d-amino acids alters Cd(II) geometries in designed thiolate-rich proteins

“…These units considerably expand polypeptide templates that possibly pave ways toward designing novel peptide architectures. Until our work, no studies have been reported that use d -amino acids to engineer metal environments or to control metal geometries [4, 5, 27]. …”

Modifying the Steric Properties in the Second Coordination Sphere of Designed Peptides Leads to Enhancement of Nitrite Reductase Activity

“…Previous studies comparing the coordination number of Cd II bound in TRI systems revealed that while a mixture of CdS 3 and CdS 3 O species were formed in Cd(TRIL16C) 3 − , only CdS 3 O was obtained in Cd( TRI L12AL16C) 3 − or Cd( TR L16 D C) 3 −. [17, 18] Furthermore, the introduction of greater steric encumbrance in the Cd( TRI L12 D LL16C) 3 − construct resulted in exclusive CdS 3 while providing greater proximal space to the metal binding site in Cd(GRCSL16CL19 D L) 3 − led to the formation of CdS 3 O 2 . [19] These studies demonstrated that modification of steric bulk around the metal-binding site could control solvent/substrate access to the metal.…”