Anders Liljas

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.

The crystallography beamline I711 at MAX II.

A new X-ray crystallographic beamline is operational at the MAX II synchrotron in Lund. The beamline has been in regular use since August 1998 and is used both for macro- and small molecule diffraction as well as powder diffraction experiments. The radiation source is a 1.8 T multipole wiggler. The beam is focused vertically by a bendable mirror and horizontally by an asymmetrically cut Si(111) monochromator. The wavelength range is 0.8-1.55 A with a measured flux at 1 A of more than 10(11) photons s(-1) in 0.3 mm x 0.3 mm at the sample position. The station is currently equipped with a Mar345 imaging plate, a Bruker Smart 1000 area CCD detector and a Huber imaging-plate Guinier camera. An ADSC 210 area CCD detector is planned to be installed during 2000.

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.

Crystal structure of the ribosomal protein S6 from Thermus thermophilus.

The amino acid sequence and crystal structure of the ribosomal protein S6 from the small ribosomal subunit of Thermus thermophilus have been determined. S6 is a small protein with 101 amino acid residues. The 3D structure, which was determined to 2.0 A resolution, consists of a four‐stranded anti‐parallel beta‐sheet with two alpha‐helices packed on one side. Similar folding patterns have been observed for other ribosomal proteins and may suggest an original RNA‐interacting motif. Related topologies are also found in several other nucleic acid‐interacting proteins and based on the assumption that the structure of the ribosome was established early in the molecular evolution, the possibility that an ancestral RNA‐interacting motif in ribosomal proteins is the evolutionary origin for the nucleic acid‐interacting domain in large classes of ribonucleic acid binding proteins should be considered.

The ribosomal protein L8 is a complex of L7/L12 and L10

Most of the electrophoretic components from the 50s subunit of E. coli ribosomes that can be identified by two dimensional electrophoresis [ 1,2] have been purified and characterized in several laboratories (for a review see [3] ). However, several of the putative proteins were not identified as unique species in this laboratory [4]. One in particular, L8, was recently found to behave as though it might be an aggregate containing L7/L12 and LlO [ 51. We have now completed an analysis of the electrophoretic behaviour of L8, and show that it can be converted into the two well characterized components L7/L12 and LIO. The reverse process, namely, reconstituting I_8 from its constituent components has also been accomplished. The data persuade us that there is no unique protein species corresponding to L8 in the E. coli ribosome. The stability and specificity of the aggregate formed between L7/L12 and LlO suggest that these proteins are immediate neighbors bound to each other in the intact SOS subunit.

4 Lactate Dehydrogenase

Publisher Summary This chapter discusses the lactate dehydrogenase. Lactic acid is the end product of anaerobic glycolysis in muscle tissue has been known for all of this century. Cell-free extracts able to catalyze the oxidation of lactate to pyruvate. The five different permutations of two different polypeptide chains readily explained the electrophoretic patterns. The distribution of these two polypeptide chains was dependent on whether the extract originated in aerobic tissue, such as heart or in anaerobic tissue as in skeletal muscle. The NAD + binding structure found in L-lactate dehydrogenase (LDH) occurs frequently in other dehydrogenases and other proteins. In LDH the problem of catalysis is presented in stark simplicity. The complications of metal ions, linked substrate phosphorylation, or of ammonia uptake are absent. LDH is the only simpler dehydrogenase where both structure and sequence are known at present. The concept of multiple molecular forms of LDH has stimulated many investigations into the nature, function, and control of isozymes. There are only two major structural genes and there is a complex variety of other LDH genes, which can be expressed in some tissues at certain stages of development.

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.

The structure of elongation factor G in complex with GDP: conformational flexibility and nucleotide exchange

BACKGROUND Elongation factor G (EF-G) catalyzes the translocation step of translation. During translocation EF-G passes through four main conformational states: the GDP complex, the nucleotide-free state, the GTP complex, and the GTPase conformation. The first two of these conformations have been previously investigated by crystallographic methods. RESULTS The structure of EF-G-GDP has been refined at 2.4 A resolution. Comparison with the nucleotide-free structure reveals that, upon GDP release, the phosphate-binding loop (P-loop) adopts a closed conformation. This affects the position of helix CG, the switch II loop and domains II, IV and V. Asp83 has a conformation similar to the conformation of the corresponding residue in the EF-Tu/EF-Ts complex. The magnesium ion is absent in EF-G-GDP. CONCLUSIONS The results illustrate that conformational changes in the P-loop can be transmitted to other parts of the structure. A comparison of the structures of EF-G and EF-Tu suggests that EF-G, like EF-Tu, undergoes a transition with domain rearrangements. The conformation of EF-G-GDP around the nucleotide-binding site may be related to the mechanism of nucleotide exchange.

Crystal Structure of a Superantigen Bound to MHC Class II Displays Zinc and Peptide Dependence

The three‐dimensional structure of a bacterial superantigen, Staphylococcus aureus enterotoxin H (SEH), bound to human major histocompatibility complex (MHC) class II (HLA‐DR1) has been determined by X‐ray crystallography to 2.6 Å resolution (1HXY). The superantigen binds on top of HLA‐DR1 in a completely different way from earlier co‐crystallized superantigens from S.aureus. SEH interacts with high affinity through a zinc ion with the β1 chain of HLA‐DR1 and also with the peptide presented by HLA‐DR1. The structure suggests that all superantigens interacting with MHC class II in a zinc‐dependent manner present the superantigen in a common way. This suggests a new model for ternary complex formation with the T‐cell receptor (TCR), in which a contact between the TCR and the MHC class II is unlikely.

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.