Synergic approach to XAFS analysis for the identification of most probable binding motifs for mononuclear zinc sites in metalloproteins

In the present work a data analysis approach, based on XAFS data, is proposed for the identification of most probable binding motifs of unknown mononuclear zinc sites in metalloproteins. This approach combines multiple-scattering EXAFS analysis performed within the rigid-body refinement scheme, non-muffin-tin ab initio XANES simulations, average structural information on amino acids and metal binding clusters provided by the Protein Data Bank, and Debye-Waller factor calculations based on density functional theory. The efficiency of the method is tested by using three reference zinc proteins for which the local structure around the metal is already known from protein crystallography. To show the applicability of the present analysis to structures not deposited in the Protein Data Bank, the XAFS spectra of six mononuclear zinc binding sites present in diverse membrane proteins, for which we have previously proposed the coordinating amino acids by applying a similar approach, is also reported. By comparing the Zn K-edge XAFS features exhibited by these proteins with those pertaining to the reference structures, key spectral characteristics, related to specific binding motifs, are observed. These case studies exemplify the combined data analysis proposed and further support its validity.