Crystallographic Identification of Metal-Binding Sites in Escherichia coli Inorganic Pyrophosphatase^,

Abstract

We report refined crystal structures of the hexameric soluble inorganic pyrophosphatase from Escherichia coli (E-PPase) to R-factors of 18.3% and 17.1% at 2.2 and 2.3 angstroms, respectively. Both structures contain two independent monomers in the asymmetric unit of an R32 cell. The difference between the structures is that the latter contains 1.5 Mg2+ ions per monomer. One metal ion binds to the "tight" metal-binding site identified by equilibrium dialysis studies, and is coordinated to Asp65, Asp70, and Asp102. The other metal ion, shared between two monomers at a hitherto unidentified metal-binding site in the dyad interface between trimers, is coordinated through water molecules to Asp26s and Asn24s from two monomers. The hexamers with metal bound to them are more tightly associated than the ones without metal bound to them. Combined with our other mechanistic and structural data, the results suggest that, at high metal concentrations, E-PPase may bind at least 4.5 metals per monomer: two in the active site before binding substrate, two with substrate, and 0.5 in the dyad interface. Glu20 interacts via a water molecule with Asp70 and appears in the related yeast PPase structure (Heikinheimo, manuscript in preparation) to be involved in binding the second metal ion. Magnesium ion therefore stabilizes the hexamer form through both direct and indirect effects. The direct effect is by tighter association at the subunit interface; the indirect effect occurs because magnesium stabilizes the correct conformation of the loop between Glu20 and Ile32, a loop involved a trimer-trimmer interactions. Our results thus provide a structural explanation for the solution studies that show that the E20D variant is partially hexameric and that the hexamer form can be stabilized by binding magnesium ion.

Directed Mutagenesis Studies of the Metal Binding Site at the Subunit Interface of Escherichia coli Inorganic Pyrophosphatase

“…We assumed previ- (15), whereas crystals grown at 250 mM MgCl 2 contain Mg 2ϩ at all three sites (17). Also, the site that binds Mn 2ϩ most tightly is located in the active site and exhibits a 1 mol/mol binding stoichiometry (11).…”

“…Ј contains several water molecules one of which is replaced by Mg 2ϩ when the crystals are soaked in a decimolar concentration of a magnesium salt (15,17). As a result of the Mg 2ϩ binding, the side chains of the Asn 24 residues reorient, and the trimers move toward each other by about 0.4 Å.…”

“…Brinen et al (22) were able to engineer a metal binding site at the interface formed by trypsin and its protein inhibitor ecotin. However, in all known instances the intersubunit metal ion is coordinated directly to protein residues except for E-PPase where it is connected through water molecules (15,17). On the other hand, waterfilled cavities often occur at protein interfaces (23) and in protein interiors (24).…”

Improving the X-ray resolution by reversible flash-cooling combined with concentration screening, as exemplified with PPase

“…At ambient temperature, PPase crystals usually diffract to 2.0±1.8 A Ê resolution (Kankare et al, 1996;Harutyunyan et al, 1997;Avaeva et al, 1998). Using¯ash-cooling with mother liquor containing 27±30%(w/v) glycerol providing cryoprotection led to a minor improvement in resolution, to 1.8±1.7 A Ê .…”

Temperature and Metal Ions-Dependent Activity of the Family I Inorganic Pyrophosphatase from the Swine Roundworm Ascaris suum

“…The two families of PPases, however, have no sequence similarity at amino acid level and have distinct catalytic features [10,13,14,19]. Among the Family I PPases, the most well characterized PPases are those from E. coli [8,12] and yeast [4,11,15]. The enzymes have shown strong metal iondependency, with Mg 2+ conferring the highest PP i -hydrolysis activity [4], and sensitive to inhibition by Ca 2+ [17].…”