The Crystal Structure of Human Tyrosyl-DNA Phosphodiesterase, Tdp1

AbstractTyrosyl-DNA phosphodiesterase (Tdp1) catalyzes the hydrolysis of a phosphodiester bond between a tyrosine residue and a DNA 3′ phosphate. The enzyme appears to be responsible for repairing the unique protein-DNA linkage that occurs when eukaryotic topoisomerase I becomes stalled on the DNA in the cell. The 1.69 Å crystal structure reveals that human Tdp1 is a monomer composed of two similar domains that are related by a pseudo-2-fold axis of symmetry. Each domain contributes conserved histidine, lysine, and asparagine residues to form a single active site. The structure of Tdp1 confirms that the protein has many similarities to the members of the phospholipase D (PLD) superfamily and indicates a similar catalytic mechanism. The structure also suggests how the unusual protein-DNA substrate binds and provides insights about the nature of the substrate in vivo

Engineering of microheterogeneity-resistant p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens

AbstractBy site-directed mutagenesis, Cys-116 was converted to Ser-116 in p-hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas fluorescens. In contrast to wild-type enzyme, the C116S mutant is no longer susceptible to oxidation by hydrogen peroxide and shows no reactivity towards 5,5'-dithiobis(2-nitrobenzoate). Crystals of the C116S mutant are isomorphous with the crystal form of wild-type enzyme. A difference electron density confirms the mutation made

Structure of cephalosporin acylase in complex with glutaryl-7-aminocephalosporanic acid and glutarate: insight into the basis of its substrate specificity

AbstractBackground: Semisynthetic cephalosporins are primarily synthesized from 7-aminocephalosporanic acid (7-ACA), which is obtained by environmentally toxic chemical deacylation of cephalosporin C (CPC). Thus, the enzymatic conversion of CPC to 7-ACA by cephalosporin acylase (CA) would be of great interest. However, CAs use glutaryl-7-ACA (GL-7-ACA) as a primary substrate and the enzyme has low turnover rates for CPC.Results: The binary complex structures of CA with GL-7-ACA and glutarate (the side-chain of GL-7-ACA) show extensive interactions between the glutaryl moiety of GL-7-ACA and the seven residues that form the side-chain pocket. These interactions explain why the D-α-aminoadipyl side-chain of CPC yields a poorer substrate than GL-7-ACA.Conclusions: This understanding of the nature of substrate specificity may be useful in the design of an enzyme with an improved performance for the conversion of CPC to 7-ACA. Additionally, the catalytic mechanism of the deacylation reaction was revealed by the ligand bound structures

In search of new lead compounds for trypanosomiasis drug design: A protein structure-based linked-fragment approach

A modular method for pursuing structure-based inhibitor design in the framework of a design cycle is presented. The approach entails four stages: (1) a design pathway is defined in the three-dimensional structure of a target protein; (2) this pathway is divided into subregions; (3) complementary building blocks, also called fragments, are designed in each subregion; complementarity is defined in terms of shape, hydrophobicity, hydrogen bond properties and electrostatics; and (4) fragments from different subregions are linked into potential lead compounds. Stages (3) and (4) are qualitatively guided by force-field calculations. In addition, the designed fragments serve as entries for retrieving existing compounds from chemical databases. This linked-fragment approach has been applied in the design of potentially selective inhibitors of triosephosphate isomerase from Trypanosoma brucei, the causative agent of sleeping sickness

Prediction of the Occurrence of the ADP-binding βαβ-fold in Proteins, Using an Amino Acid Sequence Fingerprint

An amino acid sequence "fingerprint” has been derived that can be used to test if a particular sequence will fold into a βαβ-unit with ADP-binding properties. It was deduced from a careful analysis of the known three-dimensional structures of ADP-binding βαβ-folds. This fingerprint is in fact a set of 11 rules describing the type of amino acid that should occur at a specific position in a peptide fragment. The total length of this fingerprint varies between 29 and 31 residues. By checking against all possible sequences in a database, it appeared that every peptide, which exactly follows this fingerprint, does indeed fold into an ADP-binding βαβ-unit.

A new native EcHsp31 structure suggests a key role of structural flexibility for chaperone function

Heat shock proteins and proteases play a crucial role in cell survival under conditions of environmental stress. The heat shock protein Hsp31, produced by gene hchA at elevated temperatures in Escherichia coli, is a homodimeric protein consisting of a large A domain and a smaller P domain connected by a linker. Two catalytic triads are present per dimer, with the Cys and His contributed by the A domain and an Asp by the P domain. A new crystal Form II confirms the dimer and catalytic triad arrangement seen in the earlier crystal Form I. In addition, several loops exhibit increased flexibility compared to the previous Hsp31 dimer structure. In particular, loops D2 and D3 are intriguing because their mobility leads to the exposure of a sizable hydrophobic patch made up by surface areas of both subunits near the dimer interface. The residues creating this hydrophobic surface are completely conserved in the Hsp31 family. At the same time, access to the catalytic triad is increased. These observations lead to the hypothesis for the functioning of Hsp31 wherein loops D2 and D3 play a key role: first, at elevated temperatures, by becoming mobile and uncovering a large hydrophobic area that helps in binding to client proteins, and second, by removing the client protein from the hydrophobic patch when the temperature decreases and the loops adopt their low-temperature positions at the Hsp31 surface. The proposed mode of action of flexible loops in the functioning of Hsp31 may be a general principle employed by other chaperones

Structure of Bovine Pancreatic Phospholipase A2 at 1.7Å Resolution

The crystal structure of bovine pancreatic phospholipase A2 has been refined to 1.7 Å resolution. The starting model for this refinement was the previously published structure at a resolution of 2.4 Å. This model was adjusted to the multiple isomorphous replacement map with Diamond’s real space refinement program and subsequently refined using Agarwal’s least-squares method. The final crystallographic R-factor is 17.1% and the estimated root-mean-square error in the positional parameters is 0.12 Å. The refined model allowed a detailed survey of the hydrogen-bonding pattern in the molecule. The essential calcium ion is located in the active site and is stabilized by one carboxyl group as well as by a peptide loop with many residues unvaried in all known phospholipase A2 sequences. Five of the oxygen ligands octahedrally surround the ion. The sixth octahedral position is shared between one of the carboxylate oxygens of Asp49 and a water molecule. The entrance to the active site is surrounded by residues involved in the binding of micelle substrates. The N-terminal region plays an important role here. Its α-NH3+ group is buried and interacts with Gln4, the carbonyl oxygen of Asn71 and a fully enclosed water molecule, which provides a link between the N terminus and several active site residues. A total of 106 water molecules was located in the final structure, most of them in a two-layer shell around the protein molecule. The mobility in the structure was derived from the individual atomic temperature factors. Minimum mobility is found for the main chain atoms in the central part of the two long α-helices. The active site is rather rigid.

Crystallographic Refinement by Incorporation of Molecular Dynamics: Thermostable Serine Protease Thermitase Complexed with Eglin c

In order to investigate the principles of protein thermostability, the crystal structure of thermitase from Thermoactinomyces vulgaris, a thermostable member of the subtilisin family of serine proteases, has been determined in a complex with eglin c. Eglin c is a serine protease inhibitor from the leech Hirudo medicinalis. After data collection with a television area-detector diffractometer and initial structure solution by molecular-replacement methods, crystallographic refinement proceeded with incorporation of molecular-dynamics techniques. It appeared that this refinement procedure has a large convergence radius with movements of more than 5 Å for many atoms. Two procedures for the crystallographic molecular-dynamics refinement have been tested. They differed mainly in time span and weight on the X-ray 'energy'. The best results were obtained with a procedure which allowed the molecular-dynamics technique to search a large area in conformational space by having less weight on the X-ray restraints and allowing more time. The use of molecular-dynamics refinement considerably simplified the laborious and difficult task of fitting the model in its electron density during the refinement process. The final crystallographic R factor is 17.9% at 2.2 Å resolution.

Structure of Porcine Pancreatic Phospholipase A2 at 2.6 Å Resolution and Comparison with Bovine Phospholipase A2

The previously published three-dimensional structure of porcine pancreatic prophospholipase A2 at 3 Å resolution was found to be incompatible with the structures of bovine phospholipase A2 and bovine prophospholipase A2. This was unexpected because of the very homologous amino acid sequences of these enzymes. Therefore, the crystal structure of the porcine enzyme was redetermined using molecular replacement methods with bovine phospholipase as the parent model. The structure was crystallographically refined at 2.6 Å resolution by fast Fourier transform and restrained least-squares procedures to an R-factor of 0.241. The crystals appeared to contain phospholipase A2 and not prophospholipase A2. Apparently the protein is slowly converted under the crystallization conditions employed. Our investigation shows that, in contrast to the previous report, the three-dimensional structure of porcine phospholipase A2 is very similar to that of bovine phospholipase A2, including the active site. Smaller differences were observed in some residues involved in the binding of aggregated substrates. However, an appreciable conformational difference is in the loop 59 to 70, where a single substitution at position 63 (bovine Val → porcine Phe) causes a complete rearrangement of the peptide chain. In addition to the calcium ion in the active site, a second calcium ion is present in the crystals; this is located on a crystallographic 2-fold axis and stabilizes the interaction between two neighbouring molecules