Abstract
Available high-resolution crystal structures for the family of β-trefoil proteins in the structural databank were queried for buried waters. Such waters were classified as either: (a) unique to a particular domain, family, or superfamily or (b) conserved among all β-trefoil folds. Three buried waters conserved among all β-trefoil folds were identified. These waters are related by the threefold rotational pseudosymmetry characteristic of this protein architecture (representing three instances of an identical structural environment within each repeating trefoil-fold motif). The structural properties of this buried water are remarkable and include: residing in a cavity space no larger than a single water molecule, exhibiting a positional uncertainty (i.e., normalized B-factor) substantially lower than the average Cα atom, providing essentially ideal H-bonding geometry with three solvent-inaccessible main chain groups, simultaneously serving as a bridging H-bond for three different β-strands at a point of secondary structure divergence, and orienting conserved hydrophobic side chains to form a nascent core-packing group. Other published work supports an interpretation that these interactions are key to the formation of an efficient folding nucleus and folded thermostability. The fundamental threefold symmetric structural element of the β-trefoil fold is therefore, surprisingly, a buried water molecule.