Svetlana E. Sedelnikova

Molecular basis of triclosan activity.

Triclosan (5-chloro-2-(2,4-dichlorophenoxy) phenol) has been used for more than 30 years as a general antibacterial and antifungal agent, and is found in formulations as diverse as toothpastes, cosmetics, antiseptic soaps, carpets, plastic kitchenware and toys. It has recently been suggested that triclosan blocks lipid biosynthesis by specifically inhibiting the enzyme enoyl-acyl carrier protein reductase (ENR). We have carried out a structural analysis and inhibition experiments on a complex of ENR from the bacterium Escherichia coli with triclosan and NAD+. We find that triclosan acts as a site-directed, very potent inhibitor of the enzyme by mimicking its natural substrate.

The structure of Pyrococcus furiosus glutamate dehydrogenase reveals a key role for ion-pair networks in maintaining enzyme stability at extreme temperatures

BACKGROUND The hyperthermophile Pyrococcus furiosus is one of the most thermostable organisms known, with an optimum growth temperature of 100 degrees C. The proteins from this organism display extreme thermostability. We have undertaken the structure determination of glutamate dehydrogenase from P. furiosus in order to gain further insights into the relationship between molecular structure and thermal stability. RESULTS The structure of P. furiosus glutamate dehydrogenase, a homohexameric enzyme, has been determined at 2.2 A resolution and compared with the structure of glutamate dehydrogenase from the mesophile Clostridium symbiosum. CONCLUSIONS Comparison of the structures of these two enzymes has revealed one major difference: the structure of the hyperthermophilic enzyme contains a striking series of ion-pair networks on the surface of the protein subunits and buried at both interdomain and intersubunit interfaces. We propose that the formation of such extended networks may represent a major stabilizing feature associated with the adaptation of enzymes to extreme temperatures.

A mechanism of drug action revealed by structural studies of enoyl reductase.

Enoyl reductase (ENR), an enzyme involved in fatty acid biosynthesis, is the target for antibacterial diazaborines and the front-line antituberculosis drug isoniazid. Analysis of the structures of complexes of Escherichia coli ENR with nicotinamide adenine dinucleotide and either thienodiazaborine or benzodiazaborine revealed the formation of a covalent bond between the 2′ hydroxyl of the nicotinamide ribose and a boron atom in the drugs to generate a tight, noncovalently bound bisubstrate analog. This analysis has implications for the structure-based design of inhibitors of ENR, and similarities to other oxidoreductases suggest that mimicking this molecular linkage may have generic applications in other areas of medicinal chemistry.

Crystal structure of DNA recombination protein RuvA and a model for its binding to the Holliday junction.

The Escherichia coli DNA binding protein RuvA acts in concert with the helicase RuvB to drive branch migration of Holliday intermediates during recombination and DNA repair. The atomic structure of RuvA was determined at a resolution of 1.9 angstroms. Four monomers of RuvA are related by fourfold symmetry in a manner reminiscent of a four-petaled flower. The four DNA duplex arms of a Holliday junction can be modeled in a square planar configuration and docked into grooves on the concave surface of the protein around a central pin that may facilitate strand separation during the migration reaction. The model presented reveals how a RuvAB-junction complex may also accommodate the resolvase RuvC.

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.

Crystal structure of the Lrp‐like transcriptional regulator from the archaeon Pyrococcus furiosus

The LrpA protein from the hyperthermophilic archaeon Pyrococcus furiosus belongs to the Lrp/AsnC family of transcriptional regulatory proteins, of which the Escherichia coli leucine‐responsive regulatory protein is the archetype. Its crystal structure has been determined at 2.9 Å resolution and is the first for a member of the Lrp/AsnC family, as well as one of the first for a transcriptional regulator from a hyperthermophile. The structure consists of an N‐terminal domain containing a helix–turn–helix (HtH) DNA‐binding motif, and a C‐terminal domain of mixed α/β character reminiscent of a number of RNA‐ and DNA‐binding domains. Pyrococcus furiosus LrpA forms a homodimer mainly through interactions between the antiparallel β‐sheets of the C‐terminal domain, and further interactions lead to octamer formation. The LrpA structure suggests how the protein might bind and possibly distort its DNA substrate through use of its HtH motifs and control gene expression. A possible location for an effector binding site is proposed by using sequence comparisons with other members of the family coupled to mutational analysis.

Structural insight into gene transcriptional regulation and effector binding by the Lrp/AsnC family

The Lrp/AsnC family of transcriptional regulatory proteins is found in both archaea and bacteria. Members of the family influence cellular metabolism in both a global (Lrp) and specific (AsnC) manner, often in response to exogenous amino acid effectors. In the present study we have determined both the first bacterial and the highest resolution structures for members of the family. Escherichia coli AsnC is a specific gene regulator whose activity is triggered by asparagine binding. Bacillus subtilis LrpC is a global regulator involved in chromosome condensation. Our AsnC-asparagine structure is the first for a regulator–effector complex and is revealed as an octameric disc. Key ligand recognition residues are identified together with a route for ligand access. The LrpC structure reveals a stable octamer supportive of a topological role in dynamic DNA packaging. The structures yield significant clues to the functionality of Lrp/AsnC-type regulators with respect to ligand binding and oligomerization states as well as to their role in specific and global DNA regulation.

A Burkholderia pseudomallei toxin inhibits helicase activity of translation factor eIF4A.

A toxin associated with a disease often observed in Vietnam veterans is identified and characterized. The structure of BPSL1549, a protein of unknown function from Burkholderia pseudomallei, reveals a similarity to Escherichia coli cytotoxic necrotizing factor 1. We found that BPSL1549 acted as a potent cytotoxin against eukaryotic cells and was lethal when administered to mice. Expression levels of bpsl1549 correlate with conditions expected to promote or suppress pathogenicity. BPSL1549 promotes deamidation of glutamine-339 of the translation initiation factor eIF4A, abolishing its helicase activity and inhibiting translation. We propose to name BPSL1549 Burkholderia lethal factor 1.

Analysis of the structure, substrate specificity, and mechanism of squash glycerol-3-phosphate (1)-acyltransferase.

BACKGROUND Glycerol-3-phosphate (1)-acyltransferase(G3PAT) catalyzes the incorporation of an acyl group from either acyl-acyl carrier proteins (acylACPs) or acyl-CoAs into the sn-1 position of glycerol 3-phosphate to yield 1-acylglycerol-3-phosphate. G3PATs can either be selective, preferentially using the unsaturated fatty acid, oleate (C18:1), as the acyl donor, or nonselective, using either oleate or the saturated fatty acid, palmitate (C16:0), at comparable rates. The differential substrate specificity for saturated versus unsaturated fatty acids seen within this enzyme family has been implicated in the sensitivity of plants to chilling temperatures. RESULTS The three-dimensional structure of recombinant G3PAT from squash chloroplast has been determined to 1.9 A resolution by X-ray crystallography using the technique of multiple isomorphous replacement and provides the first representative structure of an enzyme of this class. CONCLUSIONS The tertiary structure of G3PAT comprises two domains, the larger of which, domain II, features an extensive cleft lined by hydrophobic residues and contains at one end a cluster of positively charged residues flanked by a H(X)(4)D motif, which is conserved amongst many glycerolipid acyltransferases. We predict that these hydrophobic and positively charged residues represent the binding sites for the fatty acyl substrate and the phosphate moiety of the glycerol 3-phosphate, respectively, and that the H(X)(4)D motif is a critical component of the enzymes catalytic machinery.

Analysis of the structure and substrate binding of Phormidium lapideum alanine dehydrogenase.

The structure of the hexameric L-alanine dehydrogenase from Phormidium lapideum reveals that the subunit is constructed from two domains, each having the common dinucleotide binding fold. Despite there being no sequence similarity, the fold of alanine dehydrogenase is closely related to that of the family of D-2-hydroxyacid dehydrogenases, with a similar location of the active site, suggesting that these enzymes are related by divergent evolution. L-alanine dehydrogenase and the 2-hydroxyacid dehydrogenases also use equivalent functional groups to promote substrate recognition and catalysis. However, they are arranged differently on the enzyme surface, which has the effect of directing opposite faces of the keto acid to the dinucleotide in each case, forcing a change in absolute configuration of the product.