Georg E. Schulz

Substrate positions and induced-fit in crystalline adenylate kinase

The binding positions of ATP and AMP in pig muscle adenylate kinase (EC 2.7.4.3) have been located by X-ray diffraction analysis. For this purpose crystals have been soaked with solutions containing substrates and substrate analogues. Two adenosine pockets and the region of the phosphates have been identified. In combination with other experimental data the pockets have been assigned to the AMP site and the ATP site, respectively. Moreover, the results suggest that the known conformations of adenylate kinase reflect an induced-fit of the enzyme: conformation B being related to the free enzyme E and conformation A being related to E∗, the enzyme species after a substrate-induced conformational change.

Three-dimensional structure of glutathione reductase at 2 Å resolution

Abstract An electron density map of the FAD-containing enzyme glutathione reductase from human erythrocytes was produced at 2 A resolution using the multi-isomorphous-replacement method. The chemically determined amino acid sequence could be fitted unambiguously to this map. The enzyme has a molecular weight of 104,800 and consists of two identical subunits. Each of them can be subdivided into four domains and a flexible segment of 18 residues at the N-terminus. A subunit contains 11 α-helices comprising 31% of all residues and 32% β-structure in five pleated sheets. An intersubunit disulfide bridge, which is not expected for an intracellular enzyme, was detected in the crystal. The heavy atom binding sites, the subunit interface, and the intermolecular contacts in the crystal are discussed.

Comparison of the three-dimensional protein and nucleotide structure of the FAD-binding domain of p-hydroxybenzoate hydroxylase with the FAD- as well as NADPH-binding domains of glutathione reductase.

The chain fold of the FAD-binding domain of p-hydroxybenzoate hydroxylase resembles the chain folds of the two nucleotide-binding domains of glutathione reductase. This fold consists of a four-stranded parallel beta-sheet sandwiched between a three-stranded antiparallel beta-sheet and alpha-helices. The nucleotides bind in similar positions relative to this chain fold. The best superposition of the folds has been established and geometrically quantified, giving rise to an equivalencing scheme for 110 residue positions, of which only four residues are identical in all three domains. It is discussed whether this chain fold is also present in a number of other FAD-binding proteins with known sequence. After the second strand of the parallel beta-sheet both FAD-binding domains contain long chain excursions, which make intimate contacts to rather distant parts of the respective molecules. In the environment of the isoalloxazine rings we observe interesting similarities. In both enzymes the si-face of this ring is covered by polypeptide, and only the re-face is accessible for the cofactor NADPH. Furthermore, there is a long alpha-helix in each enzyme, which points with its N-terminal start to the O-2 alpha region of isoalloxazine. These helices are spatially in the same position with respect to the isoalloxazine ring but are at quite different positions along the polypeptide chain. Since they can stabilize a negative charge around O-2 alpha, they may be important for the catalytic processes.

FAD-binding site of glutathione reductase

Abstract The FAD-binding site in dimeric glutathione reductase has been elucidated by application of sequence and X-ray analyses in parallel. The geometry was derived from a multiple isomorphous replacement map of 2 A resolution. FAD binds in a rather elongated conformation with the flavin portion in the centre of one subunit and the adenine portion extending to its surface. The FMN moiety of FAD is completely buried in the protein, whereas the AMP moiety is partly accessible from the solvent. There are three strong dipoles in the vicinity of flavin, Lys66:Glu201 at N-5, Arg291:W-3:Asp331 close to C-8α, and His467: Glu472 near C-2, with the positive partners always pointing to the flavin moiety. The substrate, NADPH, releases its reduction equivalents to the re-face of flavin. Flavin passes them on to the redoxactive disulphide bridge Cys58: Cys63 at its si-face. This bridge has an unusual conformation. The negative charges of the pyrophosphate portion of FAD are not compensated by positively charged sidechains of the protein. On the contrary, there are two carboxylates nearby; Glu50 binding to the ribose, and Asp331 contacting the ribitol and one phosphate. Since electroneutrality is required, it is likely that at least one of the observed globules of electron density at the pyrophosphate is a cation. Adenine binds in a shallow nonpolar pocket with N-1A. N-3A, N-6αA forming hydrogen bonds to the protein. The conformation of FAD is compared with those of other protein-bound nucleotides. Sequence similarities with three other FAD-binding proteins are discussed.

Two conformations of crystalline adenylate kinase.

Abstract Pig muscle adenylate kinase (EC2.7.4.3) can exist in three crystal forms, which are interconvertible. For crystal form A the enzyme structure is known in atomic detail. We report the X-ray diffraction analysis of crystal form B at 4.7 A resolution and a comparison with the A form. During the transition from A to B the packing arrangement of the molecules changes slightly. Moreover, the individual molecule undergoes an appreciable conformational change: by displacing a chain segment of seven residues and two adjacent α-helices a hydrophobic pocket is opened deep in the cleft near the centre of the molecule. Concomitantly the β-pleated sheet is enlarged by about four hydrogen bonds in the B form. Several lines of evidence indicate that the observed conformational change is an intrinsic property of the molecule and is not induced by crystal packing forces.

Gene duplication in glutathione reductase

Abstract The two nucleotide-binding domains of the flavo-enzyme glutathione reductase have similar chain folds. In order to evaluate whether the observed similarity is significant or not, a mean distance between both chains after best overlay was calculated. Insertions and deletions were taken into account. The significance of the observed similarity was then derived from the corresponding mean distance by evaluating the probability that such a distance is found by chance. This probability was determined from the distribution of mean distances between randomly generated chain folds. Care was taken to ensure that the simulated chain folds fit natural ones as well as possible. The resulting significance for the two domains of glutathione reductase is of the order of 10 6 , which indicates an evolutionary relationship; that is, a gene duplication.

Low resolution structure of partially trypsin-degraded polypeptide elongation factor, EF-Tu, from Escherichia coli

The low resolution structure of a trypsin-modified form of elongation factor EF-Tu from Escherichia coli has been determined by X-ray crystallographic methods. The crystals belong to space group P 2 1 2 1 2 1 with two molecules in the asymmetric unit. The phase determination was based on three isomorphous heavy-atom derivatives. The quality of the resulting electron density map at 6 A was sufficient to identify the molecules. The two molecules in the asymmetric unit are related by a non-crystallographic 2-fold rotation. A molecular model was derived by averaging the electron density of the two molecules at equivalent points. Its overall dimensions are 75 A × 50 A × 35 A. The molecule consists of a compact globular head of dimensions 45 A × 40 A × 40 A and a curled tail of diameter 25 A and length 55 A. There is a second connection between head and tail, probably an α -helix, such that the molecule forms a ring. The large groove in the centre could accommodate a RNA double helix. The head has a high α -helical content whereas the tail seems to be helix-free. A molecular weight of 43,000 was derived from the electron density map indicating that no major part of the molecule is missing. Possible interactions between EF-Tu and transfer RNA are discussed.

Halothane binds in the adenine-specific niche of crystalline adenylate kinase

In the past, the action of anaesthetics was generally interpreted in terms of rather unspecific hydrophobic interactions between drugs and biological structures (for a review see ref. [1 ] ). Recent results, however, suggest the existence of specific binding sites for compounds like xenon [2,3] and halothane [4,5]. The nature of halothane receptors might be elucidated by investigating the molecular basis of malignant hyperthermia [6]. This fatal syndrome which some individuals develop in response to halothane anaesthesi~ is characterized, among other findings, by muscle rigidity and a continuous rise of the body temperature. In several cases halothane-induced malignant hyperthermia was found to be associated with adenylate kinase (EC 2.7.4.3) deficiency [7]. Furthermore, a direct effect of the anaesthetic on the structure and function of this enzyme, which catalyzes the reaction ATP + AMP. ~2ADP, was demonstrated by various methods [8-10] . These observations prompted us to investigate the binding of halothane to crystals of adenylate kinase by X-ray diffraction analysis. It was found that halothane does not bind to all accessible hydrophobic regions of the protein, but only to the niche which has been identified as the binding sit~ of the adenine moiety of AMP[ 11].

Low resolution structure of adenylate kinase

Abstract A low resolution model of adenylate kinase has been derived from a 6 A electron density map. The molecular shape can be described approximately as an oblate ellipsoid with dimensions 40 A × 40 A × 30 A. The molecule is composed of two globular units separated by a 10 A deep cleft. In contrast to the bigger unit, the smaller globule appears to contain a high amount of α-helical structure. The location of the active centre is discussed. The crystals used for X-ray diffraction analysis belong to one of the enantiomorphic trigonal space groups P 3 1 21 or P 3 2 21, with one molecule in the asymmetric unit. The phase determination was based on four isomorphous heavy atom derivatives. Frequent transitions between different crystal forms complicate the analysis.

Crystals of partially trypsin-digested elongation factor Tu

Abstract Limited tryptic digestion of elongation factor Tu from Escherichia coli and Bacillus stearothermophilus at room temperature produces a small number of scissions without concomitant loss of GDP binding activity. The small number of large tryptic fragments produced are not separated by gel filtration under non-denaturing conditions and they coelute with the GDP binding activity. Crystals of the trypsin-treated elongation factor Tu from E. coli obtained from polyethylene glycol solutions are apparently identical to the pseudotetragonal crystals previously reported (Sneden et al., 1973).