(485b) An Interior Water Is Essential for Maintaining the Structure of Fkbp12

Globular proteins often contain structurally well-resolved internal water molecules. Previously we reported results of a molecular dynamics study which suggested that a buried water (Wat3) may play an important role in modulating local and global conformations of the FK506 binding protein-12 (FKBP12) (Park and Saven, Proteins 60, 450-463 (2005)). In particular, simulations of wild type and mutant FKBP12 showed that disrupting a hydrogen bond to Wat3 by mutating E60 to either A or Q would cause the W59 side chain to rotate to a new conformation. The side chain in the new conformation protrudes into the ligand binding and would clash with the bound FK506, suggesting that the E60-Wat3 interaction is functionally important. To test whether the loss of a single hydrogen bond to Wat3 can indeed have the predicted structural consequences, we have determined 0.92 ? 1.29 Å structures of wild type FKBP12 and its two mutants (E60A, E60Q) by x-ray crystallography. The mutant structures show that the shape of the ligand binding pocket changes as predicted when the water hydrogen bond to the side chain of residue 60 is lost by a mutation, although the water molecule does not interact directly with any of the amino acids of the binding pocket. The corroboration between simulation and experiment demonstrates that the essential features of protein-water interactions can be reliably modeled at a molecular level. The structural coupling between Wat3 and the protein matrix seen in these structures presents the first example where distant parts of a single protein are structurally correlated by a network containing a water molecule. The current study strongly suggests that buried water molecules constitute a noncovalent extension to the protein structure.