Microbes use metalloregulatory proteins to control heavy metal detoxification using a thiolate-rich site; however, the specificity and selectivity of binding each metal is not well-understood. Here, our approach to investigate heavy metal interactions has been to use de novo three-stranded coiled coil (3SCC) designed proteins that predispose thiol residues in positions to chelate metals in geometries that turn out to be relevant for heavy metal sequestration in these metalloregulatory systems. The 3SCCs have been well-characterized and spectroscopically shown to form protein complexes with Hg(II), Pb(II), Zn(II) and As(III). As structural details of these designed peptides have remained elusive, in these studies a series of high resolution X-ray diffraction structures are obtained to assess whether the Cys environment in the apo-coordination is pre-organized or predisposed prior to metal binding into each geometry. The Cys residues in the apo-(CSL16C)3, determined to 1.43 Å, display alternate conformations. The combination of major rotamers where all Cys ligands are oriented at the core reveals a pre-organized thiolate-rich pocket, suitable to bind a metal center in a trigonal pyramidal geometry as confirmed by the obtained Pb(II)S3- and As(III)S3 complexes from the Pb(II)SZn(II)N(GRAND-CSL16CL30H)3+ (2.17Å resolution) and published As(III)(CSL9C)3 structures, respectively. The metal forms an endo-configuration in this three Cys environment, which is believed to be influenced by the preference of the direction of the lone pair within 3SCC. The apo-arrangement, on the other hand, is predisposed and requires significant thiol rotations when the metal is bound into a trigonal planar Hg(II)S3- in Hg(II)SZn(II)N(GRAND-CSL16CL30H)3+ (2.09 Å resolution) and a 4-coordinate Zn(II)S3O in Zn(II)S(GRAND-CSL12A16C)3- (1.89 Å resolution) structure. The crystallographic comparisons allow us to learn the basic principles of how 3SCC environments can achieve a variety of metal structures in which the knowledge gained can be applied to understand the selectivity of metal sequestration in metalloregulatory systems.

Understanding ligand complexation upon heavy metal binding into different geometries within de novo three-stranded coiled coil proteins

251st National Meeting (2016)

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