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Exploring structural dynamics of metal binding sites from CyaY protein family William A. Agudelo,* Diego S. Vazquez,* Martín E. Noguera, F. Luis González Flecha, Mariano C. González Lebrero and Javier Santos School of Pharmacy and Biochemistry, University of Buenos Aires, Junín 956, 1113, Argentine. *This authors contributed equally to this work Background The characterization of conformational changes that take place in proteins upon ligand binding is a key to understand the mechanisms involved in ligand recognition, selectivity and affinity [1]. Protein binding sites are defined by the amino acid sequence, the backbone topology, and molecular motions. Within this last, an important part is the side-chains dynamics. Proteins from the CyaY family are highly conserved among the species and some of them seem to play a role in iron homeostasis [2]. A putative metal binding consensus sequence from this family, EExxED, was grafted to a well-known amphipathic peptide as scaffold, giving the sequence Nt-LSKGQLEEFLEDNLAY-Ct named pIBS, from putative Iron Binding Site. This peptide was experimentally studied, finding that iron is able to interact with the random-coil ensemble raising and stabilizing a helical conformation. On the other hand, in the absence of the acid motif there is no helical conformation induction. It is expected that the distribution of acid residues side-chains rotamers differs between the metal-peptide complexes and the apo helical-restricted peptide, then is it possible an octahedral metal binding site with backbone helical conformation? To answer this, χ-angle rotamer distributions, coordination geometries, bulk-water/peptide interactions, and charge polarization were studied for the apo-peptide (restricted to the helical conformation) and the different peptide-metal complexes (restricted and not-restricted). Methodology The canonical α-helix model was designed using the 9 residues sequence Ac-LE2E3FLE6D7NY-NH2. In order to find binding configurations and to explore side chain configurations, docking [1] and classical all-atom AMBER12?GPU [2] simulations were perfomed. Magnesium ion was used as a metal model since it has the octahedral coordination geometry as a broad number of cations. Once binding configurations were obtained, QM/MM molecular dynamics were performed for thermodynamic calculations and binding site characterization. Results The spatially closely acid cluster (see Figure 1) toggles between two tridentate conformations involving EExxE and ExxED residues, being the latter the best ranked from an energetic point of view. Preliminary results indicate that the ExxED motif can adopt an octahedral geometry that not only implies the side chains, but also one metal coordination position was occupied by the E3 backbone carbonyl oxygen. This binding geometry agrees with the helical conformation expected and experimentally confirmed by circular dichroism in Fe3+, Cd2+ and Al3+ binding experiments. Figure 1 Molecular dynamics snapshot of the octahedral spatial layout of acid residues and bulk waters around Mg2+. Acknowledgements This work was supported by grants ANPCyT, UBACyT and CONICET. Reference [1] Gaudreault F et al.: Side-chain rotamer changes upon ligand binding: common, crucial, correlate with entropy and rearrange hydrogen bonding. Bioinformatics, 2012, 28: i423?i430. [2] Bencze K Z et al.: The Structure and Function of Frataxin. Crit Rev Biochem Mol Biol. 2006, 41 (5): 269-291 [3] Morris G M et al.: AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J. Comput. Chem. 2009, 30:2785-2791. [4] Case D A et al.: AMBER 12, University of California, San Francisco, 2012.