Backbone Traces Research Articles

Three-dimensional structure of the complex of guanylate kinase from yeast with its substrate GMP

The enzyme guanylate kinase was isolated from baker's yeast and crystallized as a complex with its substrate GMP. The crystal structure was solved by multiple isomorphous replacement, solvent-flattening, restrained least-squares refinement, and simulated annealing. The current R-factor is 28.9% at a resolution of 2.0 Å. The model is given as a backbone tracing, the GMP binding site is shown in atomic detail. In its major domain (residues 1 to 32 and 82 to 186), the chain fold is closely similar to the adenylate kinases, while the minor domain (residues 33 to 81) differs grossly from the 3-helix fold of the adenylate kinases. Structural homology and mechanistical similarity allow us to assign the AMP site of the adenylate kinases on the basis of the GMP site.

Crystal structure of muconolactone isomerase at 3.3 Å resolution

The crystal structure of muconolactone isomerase from Pseudomonas putida, a unique molecule with ten 96 amino acid subunits and 5-fold, and 2-fold symmetries, has been solved at 3.3 Å resolution. The non-crystallographic symmetries were used to refine the initial single isomorphous replacement phases and produce an interpretable 10-fold averaged map. The backbone trace is complete and confirmed by the amino acid sequence fit. Each subunit is composed of a body with two α-helices and an antiparallel twisted β-sheet of four strands, and an extended arm. The helices and the sheet fold to form a two-layered structure with an enclosed hydrophobic core and a partially formed putative active site pocket. The C-terminal arm of another subunit related by a local dyad symmetry extends over the core to complete this pocket. The decameric protein is almost spherical, with the helices forming the external coat. There is a large hydrophilic cavity in the center with open ends along the 5-fold axis. Molecular interactions between subunits are extensive. Each subunit contacts four neighbors and loses nearly 40% of its solvent contact area on oligomerization.

Structure of the complex of adenylate kinase from Escherichia coli with the inhibitor P 1,P 5-di(adenosine-5′-)pentaphosphate

The enzyme adenylate kinase was isolated from Escherichia coli and crystallized together with a substrate-mimicking inhibitor. The crystal structure was solved using the multiple isomorphous replacement method at a resolution of 4.5 Å (1 Å = 0.1 nm). There are two enzyme-inhibitor complex molecules in the asymmetric unit, the relative positions of which are given. The resolution was extended to 2.3 Å starting with the model of the homologous enzyme from yeast and using constrained-restrained and restrained refinements together with the known non-crystallographic symmetry. The final R-factor is 35.9%. The corresponding model is given as backbone tracing. The structure will be used for protein-engineering studies.

The 3 A resolution structure of a D-galactose-binding protein for transport and chemotaxis in Escherichia coli.

X-ray diffraction studies of a D-galactose-binding protein essential for transport and chemotaxis in Escherichia coli have yielded a model of the polypeptide chain backbone. An initial polyalanine backbone trace was obtained at 3.2 A resolution by the molecular replacement technique, using a polyalanine search model derived from the refined structure of the L-arabinose-binding protein. Concurrently, a 3 A resolution electron-density map of the D-galactose receptor was determined from multiple isomorphous replacement (MIR) phases. The properly transformed initial polyalanine model superimposed on the MIR electron-density map proved to be an excellent guide in obtaining a final trace. The few changes made in the polyalanine model to improve the fit to the density were confined primarily to the COOH-terminal peptide and some loops connecting the elements of the secondary structure. Despite the lack of significant sequence homology, the overall course of the polypeptide backbone of the D-galactose-binding protein is remarkably similar to that of the L-arabinose-binding protein, the first structure in a series to be solved from this family of binding proteins. Both structures are elongated (axial ratios of 2:1) and composed of two globular domains. For both proteins, the arrangements of the elements of the secondary structure in both domains are identical; both lobes contain a core of beta-pleated sheet with a pair of helices on either side of the plane of the sheet. The four major hydrophobic clusters that stabilize the structure of the L-arabinose-binding protein are also present in the D-galactose-binding protein.