Three-dimensional structures of mutant forms of E. coli inorganic pyrophosphatase with Asp-->Asn single substitution in positions 42, 65, 70, and 97.

The three-dimensional structures of four mutant E. coli inorganic pyrophosphatases (PPases) with single Asp-->Asn substitutions at positions 42, 65, 70, and 97 were solved at 1.95, 2.15, 2.10, and 2.20 A resolution, respectively. Asp-42-->Asn and Asp-65-->Asn mutant PPases were prepared as complexes with sulfate--a structural analog of phosphate, the product of enzymatic reaction. A comparison of mutant enzymes with native PPases revealed that a single amino acid substitution changes the position of the mutated residue as well as the positions of several functional groups and some parts of a polypeptide chain. These changes are responsible for the fact that mutant PPases differ from the native ones in their catalytic properties. The sulfate binding to the mutant PPase active site causes molecular asymmetry, as shown for the native PPase earlier. The subunit asymmetry is manifested in different positions of sulfate and several functional groups, as well as changes in packing of hexamers in crystals and in cell parameters.

Changes in E. coli inorganic pyrophosphatase structure induced by binding of metal activators.

The three-dimensional structures of E. coli inorganic pyrophosphatase (PPase) and its complexes with Mn2+ in a high affinity site and with Mg2+ in high and low affinity sites determined by authors in 1994-1996 at 1.9-2.2 A resolution are compared. Metal ion binding initiates the shifts of alpha-carbon atoms and of functional groups and rearrangement of non-covalent interaction system of hexameric enzyme molecule. As a result, the apoPPase with six equal subunits turns after Mg2+ binding into the structure with three types of subunits distinguished by structure and occupance of the low affinity Mg2+ site. Induced asymmetry reflects the subunit interactions and cooperativity between Mg2+ binding sites. These molecular rearrangements are structural basis to account for special features of the enzyme behavior and to propose one of the pathways for enzymatic activity regulation of constitutive PPases in vivo.

Crystal structure of Escherichia coli inorganic pyrophosphatase complexed with SO4(2-). Ligand-induced molecular asymmetry.

The three-dimensional structure of inorganic pyrophosphatase from Escherichia coli complexed with sulfate was determined at 2.2 A resolution using Patterson's search technique and refmed to an R-factor of 19.2%. Sulfate may be regarded as a structural analog of phosphate, the product of the enzyme reaction, and as a structural analog of methyl phosphate, the irreversible inhibitor. Sulfate binds to the pyrophosphatase active site cavity as does phosphate and this diminishes molecular symmetry, converting the homohexamer structure form (alpha3)2 into alpha3'alpha3". The asymmetry of the molecule is manifested in displacements of protein functional groups and some parts of the polypeptide chain and reflects the interaction of subunits and their cooperation. The significance of re-arrangements for pyrophosphatase function is discussed.

Crystal structure of holo inorganic pyrophosphatase from Escherichia coli at 1.9 A resolution. Mechanism of hydrolysis.

Crystalline holo inorganic pyrophosphatase from Escherichia coli was grown in the presence of 250 mM MgCl2. The crystal structure has been solved by Patterson search techniques and refined to an R-factor of 17.6% at 1.9 A resolution. The upper estimate of the root-mean-square error in atomic positions is 0.26 A. These crystals belong to space group P3(2)21 with unit cell dimensions a = b = 110.27 A and c = 78.17 A. The asymmetric unit contains a trimer of subunits, i.e., half of the hexameric molecule. In the central cavity of the enzyme molecule, three Mg2+ ions, each shared by two subunits of the hexamer, are found. In the active sites of two crystallographically independent subunits, two Mg2+ ions are bound. The second active site Mg2+ ion is missing in the third subunit. A mechanism of catalysis is proposed whereby a water molecule activated by a Mg2+ ion and Tyr 55 play essential roles.

Crystal structure of a coagulogen, the clotting protein from horseshoe crab: a structural homologue of nerve growth factor.

The clotting cascade system of the horseshoe crab (Limulus) is involved in both haemostasis and host defence. The cascade results in the conversion of coagulogen, a soluble protein, into an insoluble coagulin gel. The clotting enzyme excises the fragment peptide C from coagulogen, giving rise to aggregation of the monomers. The crystal structure of coagulogen reveals an elongated molecule that embraces the helical peptide C fragment. Cleavage and removal of the peptide C would expose an extended hydrophobic cove, which could interact with the hydrophobic edge of a second molecule, leading to a polymeric fibre. The C-terminal half of the coagulogen molecule exhibits a striking topological similarity to the neurotrophin nerve growth factor (NGF), providing the first evidence for a neurotrophin fold in invertebrates. Similarities between coagulogen and Spatzle, the Drosophila ligand of the receptor Toll, suggest that the neurotrophin fold might be considered more ancient and widespread than previously realized.

The 2.8 A crystal structure of Gla-domainless activated protein C.

The structure of the Gla-domainless form of the human anticoagulant enzyme activated protein C has been solved at 2.8 A resolution. The light chain is composed of two domains: an epidermal growth factor (EGF)-like domain modified by a large insert containing an additional disulfide, followed by a typical EGF-like domain. The arrangement of the long axis of these domains describes an angle of approximately 80 degrees. Disulfide linked to the light chain is the catalytic domain, which is generally trypsin-like but contains a large insertion loop at the edge of the active site, a third helical segment, a prominent cationic patch analogous to the anion binding exosite I of thrombin and a trypsin-like Ca[II] binding site. The arrangement of loops around the active site partially restricts access to the cleft. The S2 and S4 subsites are much more polar than in factor Xa and thrombin, and the S2 site is unrestricted. While quite open and exposed, the active site contains a prominent groove, the surface of which is very polar with evidence for binding sites on the primed side, in addition to those typical of the trypsin class found on the non-primed side.

Escherichia coli inorganic pyrophosphatase: site-directed mutagenesis of the metal binding sites.

Aspartic acids 65, 67, 70, 97 and 102 in the inorganic pyrophosphatase of Escherichia coli, identified as evolutionarily conserved residues of the active site, have been replaced by asparagine. Each mutation was found to decrease the k(app) value by approx. 2-3 orders of magnitude. At the same time, the Km values changed only slightly. Only minor changes take place in the pK values of the residues essential for both substrate binding and catalysis. All mutant variants have practically the same affinity to Mg2+ as the wild-type pyrophosphatase.