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

A challenging objective of de novo metalloprotein design is to control of the outer coordination spheres of an active site to fine tune metal properties. The well-defined three stranded coiled coils, TRI and CoilSer peptides, are used to address this question. Substitution of Cys for Leu yields a thiophilic site within the core. Metals such as HgII , PbII , and AsIII result in trigonal planar or trigonal pyramidal geometries; however, spectroscopic studies have shown that CdII forms three-, four- or five-coordinate CdII S3 (OH2 )x (in which x=0-2) when the outer coordination spheres are perturbed. Unfortunately, there has been little crystallographic examination of these proteins to explain the observations. Here, the high-resolution X-ray structures of apo- and mercurated proteins are compared to explain the modifications that lead to metal coordination number and geometry variation. It reveals that Ala substitution for Leu opens a cavity above the Cys site allowing for water excess, facilitating CdII S3 (OH2 ). Replacement of Cys by Pen restricts thiol rotation, causing a shift in the metal-binding plane, which displaces water, forming CdII S3 . Residue d-Leu, above the Cys site, reorients the side chain towards the Cys layer, diminishing the space for water accommodation yielding CdII S3 , whereas d-Leu below opens more space, allowing for equal CdII S3 (OH2 ) and CdII S3 (OH2 )2 . These studies provide insights into how to control desired metal geometries in metalloproteins by using coded and non-coded amino acids.