PubMed Health⌕ Search

Biomedical subjects

A Liljas

Publications and source records attributed to A Liljas.

At least 19 recordsLinked to original sources
PubMed1992

Structure of native and apo carbonic anhydrase II and structure of some of its anion-ligand complexes.

In order to obtain a better structural framework for understanding the catalytic mechanism of carbonic anhydrase, a number of inhibitor complexes of the enzyme were investigated crystallographically. The three-dimensional structure of free human carbonic anhydrase II was refined at pH 7.8 (1.54 A resolution) and at pH 6.0 (1.67 A resolution). The structure around the zinc ion was identical at both pH values. The structure of the zinc-free enzyme was virtually identical with that of the native enzyme, apart from a water molecule that had moved 0.9 A to fill the space that would be occupied by the zinc ion. The complexes with the anionic inhibitors bisulfite and formate were also studied at neutral pH. Bisulfite binds with one of its oxygen atoms, presumably protonized, to the zinc ion and replaces the zinc water. Formate, lacking a hydroxyl group, is bound with its oxygen atoms not far away from the position of the non-protonized oxygen atoms of the bisulfite complex, i.e. at hydrogen bond distance from Thr199 N and at a position between the zinc ion and the hydrophobic part of the active site. The result of these and other studies have implications for our view of the catalytic function of the enzyme, since virtually all inhibitors share some features with substrate, product or expected transition states. A reaction scheme where electrophilic activation of carbon dioxide plays an important role in the hydration reaction is presented. In the reverse direction, the protonized oxygen of the bicarbonate is forced upon the zinc ion, thereby facilitating cleavage of the carbon-oxygen bond. This is achieved by the combined action of the anionic binding site, which binds carboxyl groups, the side-chain of threonine 199, which discriminates between hydrogen bond donors and acceptors, and hydrophobic interaction between substrate and the active site cavity. The required proton transfer between the zinc water and His64 can take place through water molecules 292 and 318.

Anions↗
PubMed1992

Ribosomal proteins from Thermus thermophilus for structural investigations.

In parallel with crystallographic studies of ribosomes from Thermus thermophilus, a long-term program on the crystallization and structural investigations of ribosomal proteins from the same microorganism has been started at the Institute of Protein Research (Pushchino, Russia). At present, more than half of the individual ribosomal proteins from T thermophilus have been purified without denaturating agents on a preparative scale and some of them have been obtained in the crystalline form. X-ray structural analysis of two ribosomal proteins, L1 and S6, is being carried out jointly with the Institute of Molecular Biology (Moscow, Russia) and laboratory of professor A Liljas (Lund University, Sweden). L1 is the large protein of the large ribosomal subunit. It can bind not only to a specific site on the 23S rRNA, but also to the mRNA that codes for L1 and L11, thereby acting as a translational repressor for the synthesis of these proteins. The crystals of L1 are orthorhombic and diffract to about 2 A resolution. Native data and data for several heavy atom derivatives have been collected. S6 is a small acidic protein from the small ribosomal subunit. The crystals of S6 are orthorhombic and diffract to 2 A resolution. Native data and derivatives' data have been collected.

Amino Acid Sequence↗
PubMed1991

Crystals of protein S6 from the 30 S ribosomal subunit of Thermus thermophilus.

Crystals of protein S6 from the small ribosomal subunit of an extreme thermophile, Thermus thermophilus, have been obtained by the hanging-drop/vapor diffusion technique using methane pentanediol as a precipitant in the presence of potassium fluoride. The crystals belong to the space group C222 with cell parameters a = 106.7, b = 52.8, c = 41.0 A. They diffract to 2.0 A resolution.

Amino Acid Sequence↗
PubMed1991

Crystallization and preliminary X-ray investigation of a recombinant form of rat catechol O-methyltransferase.

Rat catechol O-methyltransferase cDNA was introduced into an E. coli expression vector pKEX14, which utilizes the inducible T7 promoter. Active and soluble recombinant catechol O-methyltransferase was produced in bacteria and purified to electrophoretic homogeneity by chromatographic procedures. The purified enzyme has been crystallized by the method of vapor diffusion using polyethylene glycol as precipitant. The space group is P3(1)21 or P3(2)21 with a = b = 51.3 A and c = 168.5 A and one molecule in the asymmetric unit. The crystals diffract beyond 3.2 A and are suitable for three-dimensional X-ray structure determination.

Animals↗
PubMed1990

Characterization of the binding sites of protein L11 and the L10.(L12)4 pentameric complex in the GTPase domain of 23 S ribosomal RNA from Escherichia coli.

Ribonuclease and chemical probes were used to investigate the binding sites of ribosomal protein L11 and the pentameric complex L10.(L12)4 on Escherichia coli 23 S RNA. Protein complexes were formed with an RNA fragment constituting most of domains I and II or with 23 S RNA and they were investigated by an end-labelling method and a reverse transcriptase procedure, respectively. The results demonstrate that the two protein moieties bind at adjacent sites within a small RNA region. The L11 binding region overlaps with those of the modified peptide antibiotics thiostrepton and micrococcin and is constrained structurally by a three-helix junction while the L10.(L12)4 site is centred on an adjacent internal loop. The secondary structure of the whole region was determined in detail by the phylogenetic sequence comparison method, and the results for the L11 binding region, together with the experimental data, were used in a computer graphics approach to build a partial RNA tertiary structural model. The model provides insight into the topography of the L11 binding site. It also provides a structural rationale for the mutually co-operative binding of protein L11 with the antibiotics thiostrepton and micrococcin, and with the L10.(L12)4 protein complex.

Base Sequence↗
PubMed1990

Refined structure of the acetazolamide complex of human carbonic anhydrase II at 1.9 A.

The binding of acetazolamide to human carbonic anhydrase II (HCA II) has been investigated by X-ray crystallography. The atomic positions of the enzyme inhibitor complex have been refined at 1.9 A resolution using the least squares refinement program package PROLSQ. The crystallographic R-factor is 17.6%. The bound inhibitor is clearly resolved in the active site of the enzyme. The acetazolamide amine group is bound as a fourth ligand to the zinc ion, the other three are all histidine residues. In addition to van der Waals' interactions and the previously described binding of the sulphonamide group, the inhibitor forms a hydrogen bond from the carbonyl oxygen of the acetylamido group to the amino group of Gln 92.

Acetazolamide↗
PubMed1989

The flexible region of protein L12 from bacterial ribosomes studied by proton nuclear magnetic resonance.

The dimeric protein L7/L12 from bacterial ribosomes has a highly elongated and flexible structure. We have, using 1H NMR methods, analyzed the extent of the flexible region and also the size of the organized structures of the molecule. A number of mutants of the protein as well as monomeric and dimeric forms of the protein and a COOH-terminal fragment have been used for the identification of certain resonances. Thus, residues 37-50 were found to be highly mobile whereas the amino-terminal and COOH-terminal regions are organized into folded domains. The flexibility between the domains and its relation to functional properties of the protein are discussed.

Acetylation↗
PubMed1988

Structural comparison of the prokaryotic ribosomal proteins L7/L12 and L30.

The structures of two prokaryotic ribosomal proteins, the carboxyterminal half of L7/L12 from Escherichia coli (L12CTF) and L30 from Bacilus stearothermophilus display a remarkably similar fold in which alpha-helices pack onto one side of an antiparallel, three-stranded, beta-pleated sheet. A detailed comparison of the structures by least-squares methods reveals that more than two-thirds of the alpha carbons can be superimposed with a root mean square distance of 2.33 A. The principal difference is an extra alpha-helix in L12CTF. The sequences of the proteins display a distinct conservation in regions which are crucial to the common fold, in particular the hydrophobic core. It is proposed that the similarity is a result of divergent evolution.

Amino Acid Sequence↗
PubMed1988

Refined structure of human carbonic anhydrase II at 2.0 A resolution.

The structure of human erythrocytic carbonic anhydrase II has been refined by constrained and restrained structure-factor least-squares refinement at 2.0 A resolution. The conventional crystallographic R value is 17.3%. Of 167 solvent molecules associated with the protein, four are buried and stabilize secondary structure elements. The zinc ion is ligated to three histidyl residues and one water molecule in a nearly tetrahedral geometry. In addition to the zinc-bound water, seven more water molecules are identified in the active site. Assuming that Glu-106 is deprotonated at pH 8.5, some of the hydrogen bond donor-acceptor relations in the active site can be assigned and are described here in detail. The O gamma 1 atom of Thr-199 donates its proton to the O epsilon 1 atom of Glu-106 and can function as a hydrogen bond acceptor only in additional hydrogen bonds.

Binding Sites↗
PubMed1988

Crystallographic studies of inhibitor binding sites in human carbonic anhydrase II: a pentacoordinated binding of the SCN- ion to the zinc at high pH.

The binding of four inhibitors--mercuric ion, 3-acetoxymercuri-4-aminobenzenesulfonamide (AMS), acetazolamide (Diamox), and thiocyanate ion--to human carbonic anhydrase II (HCA II) has been studied with X-ray crystallography. The binding of mercury to HCA II at pH 7.0 has been investigated at 3.1 A resolution. Mercuric ions are observed at both nitrogens in the His-64 ring. One of these sites is pointing toward the zinc ion. The only other binding site for mercury is at Cys-206. The binding of the two sulfonamide inhibitors AMS and Diamox, has been reinvestigated at 2.0 and 3.0 A, respectively. Only the nitrogen of the sulfonamide group binds to the zinc ion replacing the hydroxyl ion. The sulfonamide oxygen closest to the zinc ion is 3.1 A away. Thus the tetrahedral geometry of the zinc is retained, refuting earlier models of a pentacoordinated zinc. The structure of the thiocyanate complex has been investigated at pH 8.5 and the structure has been refined at 1.9 A resolution using the least-squares refinement program PROLSQ. The crystallographic R factor is 17.6%. The zinc ion is pentacoordinated with the anion as well as a water molecule bound in addition to the three histidine residues. The nitrogen atom of the SCN- ion is 1.9 A from the zinc ion but shifted 1.3 A with respect to the hydroxyl ion in the native structure and at van der Waals' distance from the O gamma l atom of Thr-199. This is due to the inability of the O gamma l atom of Thr-199 to serve as a hydrogen bond donor, thus repelling the nonprotonated nitrogen. The SCN- molecule reaches into the deep end of the active site cavity where the sulfur atom has displaced the so-called "deep" water molecule of the native enzyme. The zinc-bound water molecule is 2.2 A from the zinc ion and 2.4 A from the SCN- nitrogen. In addition, this water is hydrogen bonded to the O gamma l atom of Thr-199 and to another water molecule. We have observed that solvent and inhibitor molecules have three possible binding sites on the zinc ion and their significance for the catalysis and inhibition of HCA II will be discussed. All available crystallographic data are consistent with a proposed catalytic mechanism in which both the OH moiety and one oxygen of the substrate HCO3- ion are ligated to the zinc ion.

Acetazolamide↗
PubMed1987

Structure of the C-terminal domain of the ribosomal protein L7/L12 from Escherichia coli at 1.7 A.

The structure of a C-terminal fragment of the ribosomal protein L7/L12 from Escherichia coli has been refined using crystallographic data to 1.7 A resolution. The R-value is 17.4%. Six residues at the N terminus are too disordered in the structure to be localized. These residues are probably part of a hinge in the complete L7/L12 molecule. The possibility that a 2-fold crystallographic axis is a molecular 2-fold axis is discussed. A patch of invariant residues on the surface of the dimer is probably involved in functional interactions with elongation factors.

Amino Acid Sequence↗
PubMed1987

The structure and dynamics of ribosomal protein L12.

The protein L12 in bacterial ribosomes is essential for the proper function of a number of factors involved in protein synthesis. The protein is mostly described in terms of a rigid structure despite the repeated observation of high flexibility. This paper gives a review of the structure and flexibility of L12 in relation to its function.

Protein Conformation↗
PubMed1986

Crystallization of and preliminary X-ray data for bovine carbonic anhydrase III.

Crystals of bovine carbonic anhydrase III have been grown in a solution of polyethylene glycol. The crystals are monoclinic, space group P2(1), with the unit cell parameters a = 50.6 A, b = 44.7 A, c = 56.9 A, and beta = 90.3 degrees. The asymmetric unit contains 1 molecule. The diffraction pattern extends beyond 2.0-A resolution.

Animals↗
PubMed1978

Guanosinetriphosphatase activity dependent on elongation factor Tu and ribosomal protein L7/L12.

Incubation of electrophoretically pure samples of the Escherichia coli 50S ribosomal protein L7/L12 together with elongation factor Tu leads to the hydrolysis of GTP. Addition of elongation factor Ts stimulates this reaction. Elongation factor G cannot replace elongation factor Tu for the ribosome-free GTPase reaction dependent on L7/L12. The data suggest that elongation factor Tu and the protein L7/L12 interact directly at the ribosomal A site.

Bacterial Proteins↗