Kerstin Fridborg

Structure of Satellite tobacco necrosis virus at 3.0 Å resolution

Abstract The structure of Satellite tobacco necrosis virus (STNV) has been determined to 3.0 A resolution by X-ray crystallography. Electron density maps were obtained with phases based on one heavy-atom derivative and several cycles of phase refinement using the 60-fold non-crystallographic symmetry in the particle. A model for one protein subunit was built using a computer graphics display. The subunit is constructed mainly of a β-roll structure forming two β-sheets, each of four antiparallel strands. The N-termini of the subunits form bundles of three α-helices extending into the RNA region of the virus at the 3-fold axis. The topology of the polypeptide chain is the same as, and the conformation clearly similar to, that of the shell domains of the Tomato bushy stunt virus (TBSV) and Southern bean mosaic virus (SBMV) protein subunits. The subunit packing in the T = 1 STNV structure is, however, significantly different from the packing of these T = 3 viruses: parts of some of the structural elements facing the RNA in TBSV and SBMV are utilized for subunit-subunit contacts in STNV. No RNA structure is obvious in the present icosahedrally averaged electron density maps. The protein surface facing the RNA contains mainly hydrophilic residues, especially lysine and arginine.

The crystal structure of bacteriophage Qβ at 3.5 å resolution

Abstract Background: The capsid protein subunits of small RNA bacteriophages form a T=3 particle upon assembly and RNA encapsidation. Dimers of the capsid protein repress translation of the replicase gene product by binding to the ribosome binding site and this interaction is believed to initiate RNA encapsidation. We have determined the crystal structure of phage Qβ with the aim of clarifying which factors are the most important for particle assembly and RNA interaction in the small phages. Results The crystal structure of bacteriophage Qβ determined at 3.5 a resolution shows that the capsid is stabilized by disulfide bonds on each side of the flexible loops that are situated around the fivefold and quasi-sixfold axes. As in other small RNA phages, the protein capsid is constructed from subunits which associate into dimers. A contiguous ten-stranded antiparallel β sheet facing the RNA is formed in the dimer. The disulfide bonds lock the constituent dimers of the capsid covalently in the T=3 lattice. Conclusion The unusual stability of the Qβ particle is due to the tight dimer interactions and the disulfide bonds linking each dimer covalently to the rest of the capsid. A comparison with the structure of the related phage MS2 shows that although the fold of the Qβ coat protein is very similar, the details of the protein–protein interactions are completely different. The most conserved region of the protein is at the surface, which, in MS2, is involved in RNA binding.

Crystal structure of human erythrocyte carbonic anhydrase C: III. Molecular structure of the enzyme and of one enzyme-inhibitor complex at 5.5 Å resolution☆

Abstract The structures of human carbonic anhydrase C and of the complex between the enzyme and the inhibitor acetoxymercurisulphanilamide have been determined by X-ray diffraction using four heavy-atom derivatives. The enzyme molecule is an ellipsoid with the approximate dimensions 40 A × 45 A × 55 A. At the active site the molecule has a large cavity, at the bottom of which the zinc atom is bound. One part of the cavity is a narrow slit where the sulphonamide inhibitor attach and bind to the zinc. The distance between the zinc atom and the only SH-group of the enzyme is about 14 A. The most likely polypeptide chain-folding and the helical content are discussed.

Urea-PETT compounds as a new class of HIV-1 reverse transcriptase inhibitors. 3. Synthesis and further structure-activity relationship studies of PETT analogues

The further development of allosteric HIV-1 RT inhibitors in the urea analogue series of PETT (phenylethylthiazolylthiourea) derivatives is described here. The series includes derivatives with an ethyl linker (1-5) and racemic (6-16) and enantiomeric (17-20) cis-cyclopropane compounds. The antiviral activity was determined both at the RT level and in cell culture on both wild-type and mutant forms of HIV-1. Most compounds have anti-HIV-1 activity on the wt in the nanomolar range. Resistant HIV-1 was selected in vitro for some of the compounds, and the time for resistant HIV-1 to develop was longer for urea-PETT compounds than it was for reference compounds. Preliminary pharmacokinetics in rats showed that compound 18 is orally bioavailable and penetrates well into the brain. The three-dimensional structure of complexes between HIV-1 RT and two enantiomeric compounds (17 and 18) have been determined. The structures show similar binding in the NNI binding pocket. The propionylphenyl moieties of both inhibitors show perfect stacking to tyrosine residues 181 and 188. The cyclopropyl moiety of the (+)-enantiomer 18 exhibits optimal packing distances for the interactions with leucine residue 100 and valine residue 179.

Crystal structure studies on human erythrocyte carbonic anhydrase C. (II).

Several heavy-atom derivatives of human carbonic anhydrase form C have been prepared. Mercury atoms have been placed in at least four different sites. Two of the heavy-atom derivatives were obtained using sulphonamide inhibitors. The zinc atom, necessary for enzyme activity, has been removed and replaced by a mercury atom by means of reactions in the enzyme crystals. The positions of the heavy atoms and degrees of substitution in these positions were refined by a least squares procedure. For all derivatives, the mercury content found by X-ray investigation has been correlated with the values obtained by activation analyses. By using the native enzyme and four different isomorphous heavy-atom derivatives, X-ray analysis has led to an electron density projection of the unit cell of the protein. The same derivatives will be used to determine the three-dimensional structure of the enzyme (this work is in progress to 5.5 A resolution). Chemical and crystallographical evidence has led to approximate positions of the zinc atom and of the only SH-group in the enzyme molecule. The minimum Zn-S distance is 10 A. In sulphonamide derivatives one small electron density peak (probably a sulphonamide group) is situated close to the zinc position.

Crystallographic studies of RNA hairpins in complexes with recombinant MS2 capsids: implications for binding requirements.

The coat protein of bacteriophage MS2 is known to bind specifically to an RNA hairpin formed within the MS2 genome. Structurally this hairpin is built up by an RNA double helix interrupted by one unpaired nucleotide and closed by a four-nucleotide loop. We have performed crystallographic studies of complexes between MS2 coat protein capsids and four RNA hairpin variants in order to evaluate the minimal requirements for tight binding to the coat protein and to obtain more information about the three-dimensional structure of these hairpins. An RNA fragment including the four loop nucleotides and a two-base-pair stem but without the unpaired nucleotide is sufficient for binding to the coat protein shell under the conditions used in this study. In contrast, an RNA fragment containing a stem with the unpaired nucleotide but missing the loop nucleotides does not bind to the protein shell.

Structure of human carbonic anhydrase B: I. Crystallization and heavy atom modifications

Abstract Human carbonic anhydrase B has been crystallized from 2.3 m -ammonium sulphate solution at pH 8.7. A method for reproducible crystallization is presented. The crystals are suitable for high-resolution X-ray diffraction studies. They belong to the orthorhombic space group P 212121 with cell dimensions a = 81.5 A , b = 73.6 A , c = 37.1 A . The position of the essential zinc ion has been established from two projections. The zinc ion has been replaced by mercury to form one of the heavy atom derivatives.

Crystallization and preliminary X-ray diffraction studies of the bacteriophage Qβ

Crystals of bacteriophage Qbeta have been obtained by the vapor-diffusion technique. The crystals diffract to at least 3.5 A resolution. The crystal space group is C222(1) with the unit-cell parameters a = 478, b = 296, c = 477 A, alpha = beta = gamma = 90 degrees. The unit cell contains four virus particles. A pattern of systematic extinctions has been used to deduce the packing of the particles in the cell. A limited data set to 3.9 A resolution has been collected, and the predicted position has been confirmed by the self-rotation and the Patterson functions.

Structure and function of carbonic anhydrase: comparative studies of sulphonamide binding to human erythrocyte carbonic anhydrases B and C

Soon after the discovery of carbonic anhydrase from bovine erythrocytes (E.C.4.2.1.1), which catalyses the reaction by Meldrum and Roughton (1932) it was shown by Mann and Keilin (1940) that certain sulphonamides were specific and potent inhibitors of this enzyme. It has been well documented (see Maren, 1967, for an extensive review on inhibition and physiology, and Lindskog et al. 1971, and Coleman, 1971, for general reviews on carbonic anhydrases) that aromatic and heterocyclic sulphonamides with unsubstituted R—SO2 NH2 groups constitute a class of powerful inhibitors, while aliphatic sulphonamides are poor inhibitors of the carbonic anhydrases.

Crystallization of P2 myelin protein

Single crystals of bovine P2 myelin protein have been grown in polyethylene glycol 4000 by the hanging-drop vapor diffusion method. Crystals belonging to space group P2(1)2(1)2(1) with cell dimensions a = 91.8 A, b = 99.5 A, c = 56.5 A (1 A = 0.1 nm). The diffraction pattern extends to better than 2.3 A resolution.