Yukio Mitsui

The structural and sequence homology of a family of microbial ribonucleases

Abstract The crystal structures of five microbial ribonucleases have been determined in the last 2 years. They have been compared by the laboratories concerned in a collaborative exercise. Homology in the structure and sequence is present in the C-terminal half of the polypeptide chain, which is folded as a four-stranded, anti-parallel β-sheet containing the catalytic residues which are consistent with the idea of a diverging family of enzymes. The prokaryotic and eukaryotic enzymes are distinguished by a different design around the catalytic histidine. While the tertiary structures (particularly in the N-terminal half of the chain) in the two classes of ribonucleases are also notably different there are also large variations in the folding within the prokaryotic group of enzymes.

A preliminary investigation on the molecular structure of rice dwarf virus ribonucleic acid

X-Ray diffraction and infrared absorption measurements have been made of a ribonucleic acid obtained from rice dwarf virus which was isolated from the infected rice leaves. On a fibre diagram obtained at 75% relative humidity, 52 independent reflections were observed. All of them are indexed on a hexagonal lattice with a = 40·0 A and c = 30·5 A. It was found that the unit cell contains three molecules, each of which is displaced along the c-axis direction by c /3 from the neighbours. Intensities of the reflections were used to calculate the cylindrically symmetrical Patterson function. From this Patterson function it was concluded that the ribose-phosphate chains form two intertwined helices one of which is displaced along the helix axis by 13·0 A from the other. Each helix has its axis parallel to the c -axis, a pitch of 30·5 A, and ten nucleotide residues per turn. On the basis of the infrared dichroism observed for the 1225 cm −1 and 1084 cm −1 bands, the orientation of the PO 2 − group has been determined. The O…..O line makes an angle of about 70° and the bisector of 2 − group in the A and B forms of DNA.

Crystal structure of a bacterial protein proteinase inhibitor (Streptomyces subtilisin inhibitor) at 2·6 Å resolution

The crystal structure of a bacterial protein proteinase inhibitor (Streptomyces subtilisin inhibitor) was solved at 2·6 A resolution. Each subunit of the dimeric inhibitor has a five-stranded antiparallel β-sheet and two short α-helices. The subunit-subunit interface formed by a stack of two β-sheets provided by the two subunits resembles the dimer-dimer interface of concanavalin A. Conformation of the reactive site around the scissible bond Met73-Val74 seems very rigid. Between bovine pancreatic trypsin inhibitor (Kunitz) and the Streptomyces inhibitor, the reactive site conformations are almost identical with each other from the P2 to P2′ residues, while between the soybean trypsin inhibitor (Kunitz) and the Streptomyces inhibitor they are similar from the P2 to P1′ residues. There are overall similarities in conformation extending from the P3 to P2′ residues between the Streptomyces inhibitor and a hypothetical substrate presumed (Robertus et al., 1972b) to be bound to subtilisin BPN′ in a productive binding mode. Apart from the reactive site, there seems to be no structural relationship among the Streptomyces, bovine pancreatic and soybean inhibitors, suggesting their convergent evolution from separate ancestral proteins.

Crystal structure at 2.6 Å resolution of the complex of subtilisin BPN′ with Streptomyces subtilisin inhibitor

The crystal structure of the complex of a bacterial alkaline serine proteinase, subtilisin BPN, with its proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor) was solved at 2.6 A resolution. Compared with other similar complexes involving serine proteinases of the trypsin family, the present structure is unique in several respects. (1) In addition to the usual antiparallel beta-sheet involving the P1, P2 and P3 residues of the inhibitor, the P4, P5 and P6 residues form an antiparallel beta-sheet with a previously unnoticed chain segment (residues 102 through 104, which was named the S4-6 site) of subtilisin BPN. (2) The S4-6 site does not exist in serine proteinases of the trypsin family, whether of mammalian or microbial origin. (3) Global induced-fit movement seems to occur on SSI: a channel-like structure in SSI where hydrophobic side-chains are sandwiched between two lobes becomes about 2 A wider upon complexing with subtilisin. (4) The complex is most probably a Michaelis complex, as in most of the other complexes. (5) The main role of the secondary contact region of SSI seems to be to support the reactive site loop (primary contact region). Steric homology of the two contact regions between the inhibitors of the SSI family and the pancreatic secretory trypsin inhibitor-ovomucoid inhibitor family is so high that it seems to indicate divergent evolutionary processes and to support the general notion as to the relationship of prokaryotic and eukaryotic genes put forward by Doolittle (1978).

Side-chain interaction between the α-helices of poly-γ-benzyl-l- and d-glutamates☆

Abstract X-Ray diffraction and infrared absorption measurements have been made of a number of fibers obtained from mixtures of poly-γ-benzyl- l - and d -glutamates. An extensive examination has been made especially of the structural difference previously found between a fiber of an equimolar mixture of PBLG + PBDG and a fiber of pure PBLG. It has been found that the arrangement of the benzyl group at the end of the side-chain is much more regular in the equimolar mixture than that in pure PBLG. The X-ray data obtained are explained by considering that, in the fiber of PBLG + PBDG, the main-chain forms an α-helix which has 3.58 residues per turn, and that some of the benzyl groups are arranged along a helix with a different screw symmetry from the main-chain helix. Such an arrangement of the benzyl groups is considered to be caused by a side-chain-side-chain interaction and to give rise to three intense “extra” layer lines observed at 10.9, 5.0 and 3.6 A.

Local and overall conformations of DNA double helices with the A·T base pairs

Raman spectra have been observed of two different poly[d(A-T)].poly[d(A-T)] fibers, whose X-ray diffractions were confirmed to be purely of A and B forms. A number of spectral differences were found between the A and B forms of this DNA duplex, and they were ascribed to local conformational differences in the adenosine, thymidine and phosphodiester portions. The ascription was made on the basis of a separate series of Raman examinations on six crystals involving adenosine or thymidine, and fifteen other nucleotide crystals, whose structures are all known by previous crystallographic works. By taking these structure-spectrum correlations thus obtained into account, a Raman spectroscopic investigation was made of a few double-helical DNAs in aqueous solutions. It has been concluded that both poly[d(A-T)].poly[d(A-T)] and poly(dA).poly(dT) have a C2endo-anti adenosine, C2endo-anti thymidine, a b-type mainchain (beta = 160 +/- 15 degrees, gamma = 45 +/- 15 degrees, delta = 140 +/- 10 degrees) and an a2-type mainchain (beta = 210 +/- 10 degrees, gamma = 45 +/- 15 degrees, delta = 140 +/- 10 degrees) not only in low-salt medium but also in 6.6 M CsF solutions, where beta, gamma and delta are the torsion angles around O5-C5, C5-C4 and C4-C3 axes, respectively. Poly(rA).poly(dT), on the other hand, was considered to have a heteronomous duplex structure, in which the poly(rA) strand has a C3endo-anti adenosine and a1-type mainchain (beta = 175 +/- 25 degrees, gamma = 45 +/- 15 degrees, delta = 80 +/- 10 degrees) whereas the poly(dT) strand has a C2endo-anti thymidine and b-type mainchain.

Studies on the binding of adenylyl-3', 5'-cytidine to ribonuclease.

The interaction of adenylyl-3,5-cytidine (ApC) with ribonuclease-A (RNAase-A) was studied by steady-state kinetics and ultraviolet difference spectroscopy. X-ray difference Fourier synthesis at 4 A resolution was also used to study the binding of ApC to RNAase-S. Unlike well-studied compounds like uridylyl-3,5-adenosine, ApC binds in an unique way: (1) the cytidine moiety is bound to the B1 and R1 sites, (2) the adenosine moiety protrudes to the solution and is not fixed spatially and (3) the phosphate group is bound to the non-specific site (the Po site) previously postulated (Sawada, F. and Irie, M. (1969) J. Biochem. (Tokyo) 66, 415--418) as the binding site for the 5-phosphate of uridine 2,5-diphosphate or uridine 3,5-diphosphate. This conclusion is consistent with that derived for adenylyl-3,5 -4-thiouridine based on CD difference spectroscopy (White, M.D., Keren-Zur, M. and Lapidot, Y. (1977) Nucleic Acid Res. 4, 843--851). The Po site is most likely the epsilon-amino group of Lys 66.

Crystal structure of the complex of subtilisin BPN' with its protein inhibitor Streptomyces subtilisin inhibitor. The structure at 4.3 Angstroms resolution.

Abstract The crystal structure of the complex of subtilisin BPN′ (EC 3.4.21.14) with its protein inhibitor (Streptomyces subtilisin inhibitor) was solved at 4.3 A resolution, thus establishing the following. (1) Two subtilisin BPN′ molecules (2E) associate with one dimeric inhibitor molecule (I2) to form the complex molecule E2I2. (2) The conformation of neither the inhibitor nor subtilisin BPN′ undergoes any detectable change at this resolution upon complex formation. (3) The inhibitor binds to subtilisin to form an antiparallel β-sheet, as in the case of trypsin/ trypsin inhibitor complexes. (4) The scissible bond of the inhibitor is between Met73′ and Val74′, as proposed earlier (Ikenaka et al., 1974). (5) The protein inhibitor and the substrates bind to subtilisin BPN′ in essentially the same way.

X-ray crystallographic and chromatographic characterization of the crystals of Ca2+-calmodulin complexed with bee venom melittin.

Crystals of calmodulin complexed with both Ca2+ and melittin, a peptide from bee venom, have been grown from 2-methyl-2,4-pentanediol solution by using the hanging drop method of vapour diffusion. The crystals belong to space group P2(1)2(1)2(1) with a = 97.3(9) A, b = 56.5(0) A, c = 33.4(9) A and Z = 4. Analyses of the dissolved crystals by high performance liquid chromatography show that the crystals contain a 1:1 complex of calmodulin and melittin. An asymmetric unit contains one such complex and the solvent content of the crystals is 47.5% (v/v).

Preliminary X-ray studies of the interaction of salmon sperm DNA with spermine

Abstract The interaction of spermine with salmon sperm DNA was studied by X-ray diffraction methods. In the fibers of the complex, DNA was in the B-configuration with ten nucleotide pairs per turn (34 A) of the helix at 92% or higher relative humidity, while, at 75% and lower relative humidity, it was at least partly in the A-configuration, which was not observed in a similar experiment by Suwalsky et al. (1969) with calf thymus DNA. The A to B transition of the complex fibers was reversible.