Active site geometry and substrate recognition of the molybdenum hydroxylase quinoline 2-oxidoreductase.

The soil bacterium Pseudomonas putida 86 uses quinoline as a sole source of carbon and energy. Quinoline 2-oxidoreductase (Qor) catalyzes the first metabolic step converting quinoline to 2-oxo-1,2-dihydroquinoline. Qor is a member of the molybdenum hydroxylases. The molybdenum ion is coordinated by two ene-dithiolate sulfur atoms, two oxo-ligands, and a catalytically crucial sulfido-ligand, whose position in the active site was controversial. The 1.8 A resolution crystal structure of Qor indicates that the sulfido-ligand occupies the equatorial position at the molybdenum ion. The structural comparison of Qor with the allopurinol-inhibited xanthine dehydrogenase from Rhodobacter capsulatus allows direct insight into the mechanism of substrate recognition and the identification of putative catalytic residues. The active site protein variants QorE743V and QorE743D were analyzed to assess the catalytic role of E743.

Crystal structures of the antitermination factor NusB from Thermotoga maritima and implications for RNA binding.

NusB is a prokaryotic transcription factor involved in antitermination processes, during which it interacts with the boxA portion of the mRNA nut site. Previous studies have shown that NusB exhibits an all-helical fold, and that the protein from Escherichia coli forms monomers, while Mycobacterium tuberculosis NusB is a dimer. The functional significance of NusB dimerization is unknown. We have determined five crystal structures of NusB from Thermotoga maritima. In three crystal forms the protein appeared monomeric, whereas the two other crystal forms contained assemblies, which resembled the M. tuberculosis dimers. In solution, T. maritima NusB could be cross-linked as dimers, but it migrated as a monomer in gel-filtration analyses, suggesting a monomer/dimer equilibrium with a preference for the monomer. Binding to boxA-like RNA sequences could be detected by gel-shift analyses and UV-induced cross-linking. An N-terminal arginine-rich sequence is a probable RNA binding site of the protein, exhibiting aromatic residues as potential stacking partners for the RNA bases. Anions located in various structures support the assignment of this RNA binding site. The proposed RNA binding region is hidden in the subunit interface of dimeric NusB proteins, such as NusB from M. tuberculosis, suggesting that such dimers have to undergo a considerable conformational change or dissociate for engagement with RNA. Therefore, in certain organisms, dimerization may be employed to package NusB in an inactive form until recruitment into antitermination complexes.

Computational studies of the resistance patterns of mutant HIV-1 aspartic proteases towards ABT-538 (ritonavir) and design of new derivatives.

Kinetic characterization and cross resistance pattern studies of HIV-1 aspartic protase (PR) inhibitors have shown that some mutations cause considerable reduction in inhibition efficiency. We have performed a computational study of the binding of ABT-538 (ritonavir) with wild type (wt) PR and 12 model mutant structures (R8Q, V321, M461, V82A, V82F, V821, I84V, M46I/V82F, M46I/I84V, V32I/I84V, V82F/I84V and V32I/K45I/F53L/A71V/I84V/L89M (6X)) for which inhibition data are available. Our computational studies indicate a significant correlation between computed complexation energies of ABT-538 with the modeled mutant enzyme structures and the corresponding experimental inhibition constants. By evaluating non-bonding interaction energies between the inhibitor and the mutant enzymes, we have carried out a mechanistic analysis to ascertain the reasons underlying the decrease in binding affinities. This analysis indicated that several residues in addition to the mutated residues contribute to the loss of binding. Taking these considerations into account, a number of new derivatives of ABT-538 were designed, so as to increase van der Waal's and hydrogen bonding interactions with selected mutants. A significant improvement in calculated complexation energies towards both mutant and wt PR structures was obtained for several of the redesigned analogues.

Structural basis for the interaction of Escherichia coli NusA with protein N of phage lambda.

The C terminus of transcription factor NusA from Escherichia coli comprises two repeat units, which bind during antitermination to protein N from phage lambda. To delineate the structural basis of the NusA-lambdaN interaction, we attempted to crystallize the NusA C-terminal repeats in complex with a lambdaN peptide (residues 34-47). The two NusA domains became proteolytically separated during crystallization, and crystals contained two copies of the first repeat unit in contact with a single lambdaN fragment. The NusA modules employ identical regions to contact the peptide but approach the ligand from opposite sides. In contrast to the alpha-helical conformation of the lambdaN N terminus in complex with boxB RNA, residues 34-40 of lambdaN remain extended upon interaction with NusA. Mutational analyses indicated that only one of the observed NusA-lambdaN interaction modes is biologically significant, supporting an equimolar ratio of NusA and lambdaN in antitermination complexes. Solution studies indicated that additional interactions are fostered by the second NusA repeat unit, consistent with known compensatory mutations in NusA and lambdaN. Contrary to the RNA polymerase alpha subunit, lambdaN binding does not stimulate RNA interaction of NusA. The results demonstrate that lambdaN serves as a scaffold to closely oppose NusA and the mRNA in antitermination complexes.