High resolution refinement of β‐galactosidase in a new crystal form reveals multiple metal‐binding sites and provides a structural basis for α‐complementation

Abstract

The unrefined fold of Escherichia coli b-galactosidase based on a monoclinic crystal form with four independent tetramers has been reported previously. Here, we describe a new, orthorhombic form with one tetramer per asymmetric unit that has permitted refinement of the structure at 1.7 Å resolution. This high-resolution analysis has confirmed the original description 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 binding sites have been identified, one close to the active site. In a crevice on the protein surface, five linked five-membered solvent rings form a partial clathrate-like structure. Some other unusual aspects of the structure include seven apparent cis-peptide bonds, four of which are proline, and several internal salt-bridge networks. Deep solvent-filled channels and tunnels extend across the surface of the molecule and pass through the center of the tetramer. Because of these departures from a compact globular shape, the molecule is not well characterized by prior empirical relationships between the mass and surface area of proteins. The 50 or so residues at the amino terminus have a largely extended conformation and mostly lie across the surface of the protein. At the same time, however, segment 13-21 contributes to a subunit interface, and residues 29-33 pass through a "tunnel" formed by a domain interface. Taken together, the overall arrangement provides a structural basis for the phenomenon of a-complementation.

LacZ β‐galactosidase: Structure and function of an enzyme of historical and molecular biological importance

“…7 Subsequently the structure was refined to 1.7 A resolution in an orthorhombic crystal with a single tetramer in the asymmetric unit. 8 The latter form is technically superior and has been used for subsequent structural and functional studies.…”

“…7,8 The third (central) domain (residues 334-627) is a socalled triose phosphate isomerase (TIM) or a 8 b 8 barrel with the active site forming a deep pit at the C-terminal end of this barrel. As noted below, critical elements of the active site are also contributed by amino acids from elsewhere in the same polypeptide chain as well as from other chains within the tetramer.…”

“…8 In Figure 3(a), there is one two-fold axis of symmetry that is horizontal, another that is vertical and a third that is perpendicular to the page. The horizontal two-fold axis forms the socalled ''long'' interface, while the vertical symmetry axis forms the ''activating'' interface.…”

“…It is of interest that the p electron cloud of the benzyl group of Phe601 also becomes a ''ligand'' of the monovalent cation during the reaction. In addition, the O6 hydroxyl 8 of galactose replaces one of the waters whenever substrate, transition states, or the covalent intermediate are at the active site. Interactions such as this, between the monovalent ion and a hydroxyl, seem to be unique to b-galactosidases.…”

“…Although several Mg 2þ bind to each subunit, 8,29 only two directly affect activity. Some of the others may have structural importance.…”