Most of the non-lysosomal proteolysis that occurs in eukaryotic cells is performed by a nonspecific and abundant barrel-shaped complex called the 20S proteasome. Substrates access the active sites, which are sequestered in an internal chamber, by traversing a narrow opening (alpha-annulus) that is blocked in the unliganded 20S proteasome by amino-terminal sequences of alpha-subunits. Peptide products probably exit the 20S proteasome through the same opening. 11S regulators (also called PA26 (ref. 4), PA28 (ref. 5) and REG) are heptamers that stimulate 20S proteasome peptidase activity in vitro and may facilitate product release in vivo. Here we report the co-crystal structure of yeast 20S proteasome with the 11S regulator from Trypanosoma brucei (PA26). PA26 carboxy-terminal tails provide binding affinity by inserting into pockets on the 20S proteasome, and PA26 activation loops induce conformational changes in alpha-subunits that open the gate separating the proteasome interior from the intracellular environment. The reduction in processivity expected for an open conformation of the exit gate may explain the role of 11S regulators in the production of ligands for major histocompatibility complex class I molecules.

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Poster Sessions illuminate probable mechanisms of catalysis and suggest how GlpD shuttles electrons into the respiratory pathway. Homologs of GlpD are found in practically all organisms, from prokaryotes to humans, with over 45% consensus protein sequences, signifying that these structural results on the prokaryotic enzyme may readily be applied to the eukaryotic GlpD enzymes. The capBCADE genes are responsible for synthesis and transport of the poly-gamma-D-glutamic acid capsule known to protect Bacillus anthracis from phagocytic killing during infection. Here, we present the crystal structures of CapD with and without the dipeptide alpha-L-Glu-L-Glu in the active site of the enzyme. CapD shares the structure of N-terminal nucleotide hydrolases and in particular Escherichia coli and Helicobacter pylori gamma-glutamyltranspeptidases. Unlike bacterial gammaglutamyltranspeptidases, CapD displays transpeptidation activity and its structure reveals a wide open active site for poly-gammaglutamate binding and processing. Based on sequence and structure comparison, we propose that Pro427, Gly428, Gly429 contribute to CapD activity by activating Thr352 and stabilizing an oxyanion hole via classical main chain amides hydrogen bonds. Adenosine kinase (AK) is a key enzyme in the purine salvage pathway of Mycobacterium tuberculosis, an intracellular pathogen that causes tuberculosis (TB). Mtb AK, a unique bacterial adenosine kinase catalyzes the phosphorylation of adenosine to adenosine monophosphate and is involved in the bioactivation of some nucleoside analogs that have demonstrated selective activity against M. tuberculosis. The mechanism of action of these adenosine analogs is likely to be different from those of current TB treatments; therefore, specific activation of nucleoside analogs by Mtb AK may prove to be a novel therapeutic intervention for TB, particularly for multidrug resistant TB. Specific inhibition of this key enzyme in the purine salvage pathway may also be exploited for therapeutic development. The crystal structures of the enzyme in complex with adenosine or one of three selected nucleoside analogs have been determined at 1.9 Å resolution with R factor of 0.19 and Rfree of 0.25. The structure reveals a tightly associated homo-dimer, which is different from the known human and T. gondii AK, but rather resembles the structure of ribokinases. The monomer consists of a small domain that is responsible for the dimer formation and a large catalytic domain. The active sites reveal the protein-ligand interaction and significant structural differences between the human and Mtb AK. The structural information provides the structural basis for the specific activation of nucleoside analogs by Mtb AK and should aid in the design of more potent and selective antimycobacterial agents. We thank SBC-CAT and NE-CAT at APS for access to beam lines 19-BM and 8-BM. This research is supported in part by NIH grants, AI55344 to RL.

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