Jens T. Kaiser

Crystal structures of transcription factor NusG in light of its nucleic acid- and protein-binding activities

Microbial transcription modulator NusG interacts with RNA polymerase and termination factor ρ, displaying striking functional homology to eukaryotic Spt5. The protein is also a translational regulator. We have determined crystal structures of Aquifex aeolicus NusG showing a modular design: an N‐terminal RNP‐like domain, a C‐terminal element with a KOW sequence motif and a species‐specific immunoglobulin‐like fold. The structures reveal bona fide nucleic acid binding sites, and nucleic acid binding activities can be detected for NusG from three organisms and for the KOW element alone. A conserved KOW domain is defined as a new class of nucleic acid binding folds. This module is a close structural homolog of tudor protein–protein interaction motifs. Putative protein binding sites for the RNP and KOW domains can be deduced, which differ from the areas implicated in nucleic acid interactions. The results strongly argue that both protein and nucleic acid contacts are important for NusGs functions and that the factor can act as an adaptor mediating indirect protein–nucleic acid associations.

Crystal structure of a DNA-dependent RNA polymerase (DNA primase)

Primases are essential components of the DNA replication apparatus in every organism. They catalyze the synthesis of oligoribonucleotides on single-stranded DNA, which subsequently serve as primers for the replicative DNA polymerases. In contrast to bacterial primases, the archaeal enzymes are closely related to their eukaryotic counterparts. We have solved the crystal structure of the catalytic primase subunit from the hyperthermophilic archaeon Pyrococcus furiosus at 2.3 Å resolution by multiwavelength anomalous dispersion methods. The structure shows a two-domain arrangement with a novel zinc knuckle motif located in the primase (prim) domain. In this first structure of a complete protein of the archaeal/eukaryotic primase family, the arrangement of the catalytically active residues resembles the active sites of various DNA polymerases that are unrelated in fold.

The Structural Basis of Riboflavin Binding to Schizosaccharomyces pombe 6,7-Dimethyl-8- ribityllumazine Synthase

Riboflavin is an essential cofactor in all organisms. Its direct biosynthetic precursor, 6,7-dimethyl-8-ribityllumazine, is synthesised by the enzyme 6,7-dimethyl-8-ribityllumazine synthase. Recently, we have found that the enzyme from Schizosaccharomyces pombe binds riboflavin, the final product of the pathway with a relatively high affinity with a KD of 1.2 microM. Here, we report on the crystal structure of lumazine synthase from S. pombe with bound riboflavin and compare the binding mode with those of the substrate analogue inhibitor 5-nitro-6-(D-ribitylamino)-2,4(1H,3H)-pyrimidinedione and of the product analogue 6-carboxyethyl-7-oxo-8-ribityllumazine. In all complexes the pyrimidinedione moieties of each respective ligand bind in a very similar orientation. Binding of riboflavin additionally involves a stacking interaction of the dimethylbenzene moiety with the side-chain of His94, a highly conserved residue in all lumazine synthases. The enzyme from Bacillus subtilis showed a KD of at least 1 mM whereas the very homologous enzyme from Saccharomyces cerevisiae had a comparable KD of 3.9 microM. Structural comparison of the S. cerevisiae, the S. pombe, and the mutant enzymes suggests that fine tuning of affinity is achieved by influencing this stacking interaction.

Binding and Docking of Synthetic Heterotrimeric Collagen Type IV Peptides with α1β1 Integrin

Collagen type IV, whose major and ubiquitous form consists of one 2 and two 1 chains, 2] forms a network that determines the biomechanical stability and macromolecular organization of the basement membrane and provides a scaffold into which other constituents of the tissue are incorporated . This collagen benzotriazol (HOAT) as activating reagents and the coupling times were extended to 3 ±4 h to avoid incomplete peptide bond formation. After the entire biotin-tagged and farnesylated peptide 23 had been assembled on the polymeric support, the seven Aloc groups present were removed simultaneously by treatment with Pd[PPh3]4 in the presence of piperidine for four hours. Removal of the catalyst was achieved by simple washing, which rendered the troublesome purification of the unmasked oligolysine peptide unnecessary. Finally, fully unmasked lipidated K-Ras peptide 2 was released from the solid support by treatment with 1% TFA in the presence of 2% TES. Under these conditions both O-trityl groups present in 24 were removed as well and the farnesyl group remained unattacked. Purification of the target peptide was readily achieved by means of HPLC on an RP-C18 column to yield the desired biotin-tagged and lipidated oligolysine peptide 2 (Figure 1) in high purity and with 11% overall yield.

X-ray structure of isoaspartyl dipeptidase from E.coli: a dinuclear zinc peptidase evolved from amidohydrolases.

L-aspartyl and L-asparaginyl residues in proteins spontaneously undergo intra-residue rearrangements forming isoaspartyl/beta-aspartyl residues linked through their side-chain beta-carboxyl group with the following amino acid. In order to avoid accumulation of isoaspartyl dipeptides left over from protein degradation, some bacteria have developed specialized isoaspartyl/beta-aspartyl zinc dipeptidases sequentially unrelated to other peptidases, which also poorly degrade alpha-aspartyl dipeptides. We have expressed and crystallized the 390 amino acid residue isoaspartyl dipeptidase (IadA) from E.coli, and have determined its crystal structure in the absence and presence of the phosphinic inhibitor Asp-Psi[PO(2)CH(2)]-LeuOH. This structure reveals an octameric particle of 422 symmetry, with each polypeptide chain organized in a (alphabeta)(8) TIM-like barrel catalytic domain attached to a U-shaped beta-sandwich domain. At the C termini of the beta-strands of the beta-barrel, the two catalytic zinc ions are surrounded by four His, a bridging carbamylated Lys and an Asp residue, which seems to act as a proton shuttle. A large beta-hairpin loop protruding from the (alphabeta)(8) barrel is disordered in the free peptidase, but forms a flap that stoppers the barrel entrance to the active center upon binding of the dipeptide mimic. This isoaspartyl dipeptidase shows strong topological homology with the alpha-subunit of the binickel-containing ureases, the dinuclear zinc dihydroorotases, hydantoinases and phosphotriesterases, and the mononuclear adenosine and cytosine deaminases, which all are catalyzing hydrolytic reactions at carbon or phosphorous centers. Thus, nature has adapted an existing fold with catalytic tools suitable for hydrolysis of amide bonds to the binding requirements of a peptidase.

Synthesis of New N‐(5‐Oxo‐2,5‐dihydro)pyrrol‐3‐yl Glycines and N‐(5‐Oxo‐2,5‐dihydro)pyrrol‐3‐yl Glycines Esters

Abstract Following our efforts towards the synthesis of new potential inhibitors of Xanthine Dehydrogenase (XDH), we describe here a general method for the preparation of N-(5-oxo-2,5-dihydro)pyrrol-3-yl glycines and N-(5-oxo-2,5-dihydro)pyrrol-3-yl glycine esters from glycine ethyl ester hydrochloride and various 4-hydroxy-5-oxo-2,5-dihydro-1H-pyrrole-3-carboxylic acid esters and carbonitriles.

Potassium cyanate as an amino-dehydroxylating agent: Synthesis of aminooxypyrrole mono, dicarboxylic acid esters, and carbonitrile

In the vast area of heterocyclic chemistry, the search for new small polyfunctionalized heterocyclic rings constitutes a key step towards the synthesis of larger systems. Amongst these intermediate heterocycles, ortho amino esters are electrophilic and nucleophilic centre containing reagents that have been widely used for further cyclization reactions such as the annelation of a pyridine, pyrimidine, diazepine, benzox-