Junichi Komoto

Variation of loop sequence alters stability of cytolethal distending toxin (CDT): Crystal structure of CDT from Actinobacillus actinomycetemcomitans

Cytolethal distending toxin (CDT) secreted by Actinobacillus actinomycetemcomitans induces cell cycle arrest of cultured cells in the G2 phase. The crystal structure of the natural form of A. actinomycetemcomitans DCT (aCDT) has been determined at 2.4 Å resolution. aCDT is a heterotrimer consisting of the three subunits, aCdtA, aCdtB, and aCdtC. Two crystallographically independent aCDTs form a dimer through interactions of the aCdtB subunits. The primary structure of aCDT has 94.3% identity with that of Haemophilus ducreyi CDT (hCDT), and the structure of aCDT is quite similar to that of hCDT reconstituted from the three subunits determined recently. However, the molecular packings in the crystal structures of aCDT and hCDT are quite different. A careful analysis of molecular packing suggests that variation of the amino acid residues in a nonconserved loop (181TSSPSSPERRGY192 of aCdtB and 181NSSSSPPERRVY192 of hCdtB) is responsible for the different oligomerization of very similar CDTs. The loop of aCdtB has a conformation to form a dimer, while the loop conformation of hCdtB prevents hCDT from forming a dimer. Although dimerization of aCDT does not affect toxic activity, it changes the stability of protein. aCDT rapidly aggregates and loses toxic activity in the absence of sucrose in a buffered solution, while hCDT is stable and retains toxic activity. Another analysis of crystal structures of aCDT and hCDT suggests that the receptor contact area is in the deep groove between CdtA and CdtC, and the characteristic “aromatic patch” on CdtA.

Effects of Site-directed Mutagenesis on Structure and Function of Recombinant Rat Liver S-Adenosylhomocysteine Hydrolase CRYSTAL STRUCTURE OF D244E MUTANT ENZYME

A site-directed mutagenesis, D244E, ofS-adenosylhomocysteine hydrolase (AdoHcyase) changes drastically the nature of the protein, especially the NAD+binding affinity. The mutant enzyme contained NADH rather than NAD+ (Gomi, T., Takata, Y., Date, T., Fujioka, M., Aksamit, R. R., Backlund, P. S., and Cantoni, G. L. (1990)J. Biol. Chem. 265, 16102–16107). In contrast to the site-directed mutagenesis study, the crystal structures of human and rat AdoHcyase recently determined have shown that the carboxyl group of Asp-244 points in a direction opposite to the bound NAD molecule and does not participate in any hydrogen bonds with the NAD molecule. To explain the discrepancy between the mutagenesis study and the x-ray studies, we have determined the crystal structure of the recombinant rat-liver D244E mutant enzyme to 2.8-Å resolution. The D244E mutation changes the enzyme structure from the open to the closed conformation by means of a ∼17° rotation of the individual catalytic domains around the molecular hinge sections. The D244E mutation shifts the catalytic reaction from a reversible to an irreversible fashion. The large affinity difference between NAD+ and NADH is mainly due to the enzyme conformation, but not to the binding-site geometry; an NAD+ in the open conformation is readily released from the enzyme, whereas an NADH in the closed conformation is trapped and cannot leave the enzyme. A catalytic mechanism of AdoHcyase has been proposed on the basis of the crystal structures of the wild-type and D244E enzymes.

Crystal structure of guanidinoacetate methyltransferase from rat liver: a model structure of protein arginine methyltransferase.

Guanidinoacetate methyltransferase (GAMT) is the enzyme that catalyzes the last step of creatine biosynthesis. The enzyme is found in abundance in the livers of all vertebrates. Recombinant rat liver GAMT has been crystallized with S-adenosylhomocysteine (SAH), and the crystal structure has been determined at 2.5 A resolution. The 36 amino acid residues at the N terminus were cleaved during the purification and the truncated enzyme was crystallized. The truncated enzyme forms a dimer, and each subunit contains one SAH molecule in the active site. Arg220 of the partner subunit forms a pair of hydrogen bonds with Asp134 at the guanidinoacetate-binding site. On the basis of the crystal structure, site-directed mutagenesis on Asp134, and chemical modification and limited proteolysis studies, we propose a catalytic mechanism of this enzyme. The truncated GAMT dimer structure can be seen as a ternary complex of protein arginine methyltransferase (one subunit) complexed with a protein substrate (the partner subunit) and the product SAH. Therefore, this structure provides insight into the structure and catalysis of protein arginine methyltransferases.

Monoclinic guanidinoacetate methyltransferase and gadolinium ion-binding characteristics.

Guanidinoacetate methyltransferase (GAMT) is the enzyme that catalyzes the last step of creatine biosynthesis. The enzyme is found in abundance in the livers of all vertebrates. Recombinant rat liver GAMT truncated at amino acid 37 from the N-terminus has been crystallized with S-adenosylhomocysteine (SAH) in a monoclinic modification and the crystal structure has been determined at 2.8 A resolution. There are two dimers in the crystallographic asymmetric unit. Each dimer has non-crystallographic twofold symmetry and is related to the other dimer by pseudo-4(3) symmetry along the crystallographic b axis. The overall structure of GAMT crystallized in the monoclinic modification is quite similar to the structure observed in the tetragonal modification [Komoto et al. (2002), J. Mol. Biol. 320, 223-235], with the exception of the loop containing Tyr136. In the monoclinic modification, the loops in three of the four subunits have a catalytically unfavorable conformation and the loop of the fourth subunit has a catalytically favorable conformation as observed in the crystals of the tetragonal modification. From the structures in the monoclinic and tetragonal modifications, we can explain why the Y136F mutant enzyme retains considerable catalytic activity while the Y136V mutant enzyme loses the catalytic activity. The crystal structure of a Gd derivative of the tetragonal modification has also been determined. By comparing the Gd-derivative structure with the native structures in the tetragonal and the monoclinic modifications, useful characteristic features of Gd-ion binding for application in protein crystallography have been observed. Gd ions can bind to proteins without changing the native protein structures and Gd atoms produce strong anomalous dispersion signals from Cu Kalpha radiation; however, Gd-ion binding to protein requires a relatively specific geometry.

Crystallization and preliminary x-ray diffraction studies of guanidinoacetate methyltransferase from rat liver.

Guanidinoacetate methyltransferase is the enzyme which catalyzes the last step of creatine biosynthesis. The enzyme is found ubiquitously and in abundance in the livers of all vertebrates. Recombinant rat-liver guanidinoacetate methyltransferase has been crystallized with guanidinoacetate and S-adenosylhomocysteine. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 54.8, b = 162.5, c = 56.1 A, beta = 96.8 (1) degrees at 93 K, and typically diffract beyond 2.8 A.