Georg Wiegand

Crystallographic refinement and atomic models of two different forms of citrate synthase at 2.7 and 1.7 A resolution.

The structures of pig heart and chicken heart citrate synthase have been determined by multiple isomorphous replacement and restrained crystallographic refinement for two crystal forms, a tetragonal form at 2·7 A and a monoclinic form at 1·7 A resolution, with crystallographic R-values of 0·199 and 0·192, respectively. The structure determination involved a novel application of restrained crystallographic refinement, in that the refinement of incomplete models was necessary in order to completely determine the course of the polypeptide chain. The recently determined amino acid sequence (Bloxham et al., 1981) has been fitted to the models. The molecule has substantially different conformations in the two crystal forms, and there is evidence that a conformational change is required for enzymatic activity. The molecule is a dimer of identical subunits with 437 amino acid residues each. The conformation is all α-helix, with 40 helices per dimer packing tightly to form a globular molecule. Many of the helices are kinked in various ways or bent smoothly over a large angle. Several of the helices show an unusual antiparallel packing. Each subunit is clearly divided into a large and a small domain. The two crystal forms differ by the relative arrangement of the two domains. The tetragonal form represents an open configuration with a deep cleft between the two domains, the monoclinic form is closed. The structural change from the open to the closed form can be described by an 18 ° rotation of the small domain relative to the large domain. Crystallographic analyses were performed with the product citrate bound in both crystal forms, with coenzyme A (CoA) and a citryl-CoA analogue bound to the monoclinic form. These studies establish the CoA and the citrate binding sites, and the conformations of the two product molecules in atomic detail. The subunits are extensively interdigitated, with one subunit making significant contributions to both the citrate and the CoA binding sites of the other subunit. The adenine moiety of CoA is bound to the small domain, and the pantothenic arm is bound to the large domain. The citrate molecule is bound in a cleft between the large domain. The citrate molecule is bound in a cleft between the large and small domain, with the si carboxymethylene group facing the SH arm of coenzyme A. In the monoclinic form, the cysteamine part of CoA shields the bound citrate completely from the solution. Partial reaction of CoA-SH and aspartate 375 to form aspartyl-CoA, and citrate to form citryl-CoA may occur in the crystals. The conformation of CoA is compact, characterized by an internal hydrogen bond O-52 … N-7 and a tightlybound water molecule O-51 … HOH … O-20.

Activation of Bacillus licheniformis α-amylase through a disorder→order transition of the substrate-binding site mediated by a calcium–sodium–calcium metal triad

BACKGROUND The structural basis as to how metals regulate the functional state of a protein by altering or stabilizing its conformation has been characterized in relatively few cases because the metal-free form of the protein is often partially disordered and unsuitable for crystallographic analysis. This is not the case, however, for Bacillus licheniformis alpha-amylase (BLA) for which the structure of the metal-free form is available. BLA is a hyperthermostable enzyme which is widely used in biotechnology, for example in the breakdown of starch or as a component of detergents. The determination of the structure of BLA in the metal-containing form, together with comparisons to the apo enzyme, will help us to understand the way in which metal ions can regulate enzyme activity. RESULTS We report here the crystal structure of native, metal-containing BLA. The structure shows that the calcium-binding site which is conserved in all alpha-amylases forms part of an unprecedented linear triadic metal array, with two calcium ions flanking a central sodium ion. A region around the metal triad comprising 21 residues exhibits a conformational change involving a helix unwinding and a disorder-->order transition compared to the structure of metal-free BLA. Another calcium ion, not previously observed in alpha-amylases, is located at the interface between domains A and C. CONCLUSIONS We present a structural description of a major conformational rearrangement mediated by metal ions. The metal induced disorder-->order transition observed in BLA leads to the formation of the extended substrate-binding site and explains on a structural level the calcium dependency of alpha-amylases. Sequence comparisons indicate that the unique Ca-Na-Ca metal triad and the additional calcium ion located between domains A and C might be found exclusively in bacterial alpha-amylases which show increased thermostability. The information presented here may help in the rational design of mutants with enhanced performance in biotechnological applications.

Crystal structure determination, refinement and the molecular model of the α-amylase inhibitor Hoe-467A

The crystal and molecular structure of the alpha-amylase inhibitor Hoe-467A has been determined and refined at high resolution. The polypeptide chain is folded in two triple-stranded sheets, which form a barrel. The topology of folding is as found in the immunoglobulin domains. The amino acid triplet Trp18-Arg19-Tyr20 has an exceptional conformation and position in the molecule and is possibly involved in inhibitory activity.

A novel strategy for inhibition of α-amylases: yellow meal worm α-amylase in complex with the Ragi bifunctional inhibitor at 2.5 å resolution

Abstract Background: α -Amylases catalyze the hydrolysis of α -D-(1,4)-glucan linkages in starch and related compounds. There is a wide range of industrial and medical applications for these enzymes and their inhibitors. The Ragi bifunctional α -amylase/trypsin inhibitor (RBI) is the prototype of the cereal inhibitor superfamily and is the only member of this family that inhibits both trypsin and α -amylases. The mode of inhibition of α -amylases by these cereal inhibitors has so far been unknown. Results: The crystal structure of yellow meal worm α -amylase (TMA) in complex with RBI was determined at 2.5 a resolution. RBI almost completely fills the substrate-binding site of TMA. Specifically, the free N terminus and the first residue (Ser1) of RBI interact with all three acidic residues of the active site of TMA (Asp 185, Glu222 and Asp287). The complex is further stabilized by extensive interactions between the enzyme and inhibitor. Although there is no significant structural reorientation in TMA upon inhibitor binding, the N-terminal segment of RBI, which is highly flexible in the free inhibitor, adopts a 3 10 -helical conformation in the complex. RBIs trypsin-binding loop is located opposite the α -amylase-binding site, allowing simultaneous binding of α -amylase and trypsin. Conclusions: The binding of RBI to TMA constitutes a new inhibition mechanism for α -amylases and should be general for all α -amylase inhibitors of the cereal inhibitor superfamily. Because RBI inhibits two important digestive enzymes of animals, it constitutes an efficient plant defense protein and may be used to protect crop plants from predatory insects.

Kinetic Stabilization of Bacillus Licheniformis Alpha-Amylase Through Introduction of Hydrophobic Residues at the Surface

It is generally assumed that in proteins hydrophobic residues are not favorable at solvent-exposed sites, and that amino acid substitutions on the surface have little effect on protein thermostability. Contrary to these assumptions, we have identified hyperthermostable variants of Bacillus licheniformis α-amylase (BLA) that result from the incorporation of hydrophobic residues at the surface. Under highly destabilizing conditions, a variant combining five stabilizing mutations unfolds 32 times more slowly and at a temperature 13 °C higher than the wild-type. Crystal structure analysis at 1.7 Å resolution suggests that stabilization is achieved through (a) extension of the concept of increased hydrophobic packing, usually applied to cavities, to surface indentations, (b) introduction of favorable aromatic-aromatic interactions on the surface, (c) specific stabilization of intrinsic metal binding sites, and (d) stabilization of a β-sheet by introducing a residue with high β-sheet forming propensity. All mutated residues are involved in forming complex, cooperative interaction networks that extend from the interior of the protein to its surface and which may therefore constitute “weak points” where BLA unfolding is initiated. This might explain the unexpectedly large effect induced by some of the substitutions on the kinetic stability of BLA. Our study shows that substantial protein stabilization can be achieved by stabilizing surface positions that participate in underlying cooperatively formed substructures. At such positions, even the apparently thermodynamically unfavorable introduction of hydrophobic residues should be explored.

Crystallization and preliminary X-ray data of well-ordered crystals from pig heart citrate synthase

Citrate synthase catalyzes the first reaction of the citric acid cycle and fills a prominent role in cellular regulation. The enzyme catalyzes the enolase, ligase and hydrolase activities and forms with the two substrates oxalacetate and acetyl-CoA citrate. The question of whether citryl-CoA is an intermediate is not completely settled. However, the work of Eggerer et al. makes it a likely intermediate [l] , and the stereochemical course of the reaction has been brilliantly elucidated [2]. Oxalacetate forms with citrate synthase a binary complex, demonstrated by ultraviolet difference spectra, and protects the citrate synthase activity against urea denaturation [3] . Pig heart citrate synthase requires no cofactors or metal ions for activity [4]. Until now there have been no definitive investigations which amino acid residues are essential for the catalytic activity. But the enzyme activity is inhibited by reagents that modify amino groups. Sulfhydryl groups do not participate in the active site [5,6]. However, histidine residues seem to be involved in the enzymatic citrate synthesis [7]. ATP is a competitive inhibitor of acetyl-CoA at least in vitro [8]. Although the primary structure of citrate synthase is not known, the investigation of its tertiary and quaternary structure by X-ray crystallography seems feasible. The first crystalline preparation of pig heart citrate synthase was described by Ochoa and Stern [9] This communication describes the growing of large crystals of pig heart citrate synthase suitable for X-ray crystallography and their crystallographic data.