High resolution refinement of beta-galactosidase in a new crystal form reveals multiple metal-binding sites and provides a structural basis for alpha-complementation

The unrefined fold of Escherichia coli beta-galactosidase based on a monocl inic crystal form with four independent tetramers has been reported previou sly. Here, we describe a new, orthorhombic form with one tetramer per asymm etric unit that has permitted refinement of the structure at 1.7 Angstrom r esolution. This high-resolution analysis has confirmed the original descrip tion of the structure and revealed new details. An essential magnesium ion, identified at the active site in the monoclinic crystals, is also seen in the orthorhombic form. Additional putative magnesium binding sites are also seen. Sodium ions are also known to affect catalysis, and five putative bi nding sites have been identified, one close to the active site. In a crevic e on the protein surface, five linked five-membered solvent rings form a pa rtial clathrate-like structure. Some other unusual aspects of the structure include seven apparent cis-peptide bonds, four of which are proline, and s everal internal salt-bridge networks. Deep solvent-filled channels and tunn els extend across the surface of the molecule and pass through the center o f the tetramer. Because of these departures From a compact globular shape, the molecule is not well characterized by prior empirical relationships bet ween the mass and surface area of proteins. The 50 or so residues at the am ino terminus have a largely extended conformation and mostly lie across the surface of the protein. At the same time, however, segment 13-21 contribut es to a subunit interface, and residues 29-33 pass through a "tunnel" forme d by a domain interface. Taken together, the overall arrangement provides a structural basis for the phenomenon of alpha-complementation.