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Abstract: Insect juvenile hormones (JHs) are a family of sesquiterpenoid molecules that are secreted into the haemolymph. JHs have multiple roles in insect development, metamorphosis and sexual maturation. A number of pesticides work by chemically mimicking JHs, thus preventing insects from developing and reproducing normally. The haemolymph levels of JH are governed by the rates of its biosynthesis and degradation. One enzyme involved in JH catabolism is JH diol kinase (JHDK), which uses ATP (or GTP) to phosphorylate JH diol to JH diol phosphate, which can be excreted. The X-ray structure of JHDK from the silkworm Bombyx mori has been determined at a resolution of 2.0 Å with an R factor of 19.0% and an Rfree of 24.8%. The structure possesses three EF-hand motifs which are occupied by calcium ions. This is in contrast to the recently reported structure of the JHDK-like-2 protein from B. mori (PDB entry 6kth), which possessed only one calcium ion. Since JHDK is known to be inhibited by calcium ions, it is likely that our structure represents the calcium-inhibited form of the enzyme. The electrostatic surface of the protein suggests a binding site for the triphosphate of ATP close to the N-terminal end of the molecule in a cavity between the N- and C-terminal domains. Superposition with a number of calcium-activated photoproteins suggests that there may be parallels between the binding of JH diol to JHDK and the binding of luciferin to aequorin.

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Abstract: Two of the world's most neglected tropical diseases, human African trypanosomiasis (HAT) and Chagas disease, are caused by protozoan parasites of the genus Trypanosoma. These organisms possess specialized metabolic pathways, frequently distinct from those in humans, which have potential to be exploited as novel drug targets. This study elucidates the structure and function of L-threonine-3-dehydrogenase (TDH) from T. brucei, the causative pathogen of HAT. TDH is a key enzyme in the metabolism of L-threonine, and an inhibitor of TDH has been shown to have trypanocidal activity in the procyclic form of T. brucei. TDH is a nonfunctional pseudogene in humans, suggesting that it may be possible to rationally design safe and specific therapies for trypanosomiasis by targeting this parasite enzyme. As an initial step, the TDH gene from T. brucei was expressed and the three-dimensional structure of the enzyme was solved by X-ray crystallography. In multiple crystallographic structures, T. brucei TDH is revealed to be a dimeric short-chain dehydrogenase that displays a considerable degree of conformational variation in its ligand-binding regions. Geometric simulations of the structure have provided insight into the dynamic behaviour of this enzyme. Furthermore, structures of TDH bound to its natural substrates and known inhibitors have been determined, giving an indication of the mechanism of catalysis of the enzyme. Collectively, these results provide vital details for future drug design to target TDH or related enzymes.

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Abstract: A number of X-ray analyses of an enzyme involved in a key early stage of tetrapyrrole biosynthesis are reported. Two structures of human 5-aminolaevulinate dehydratase (ALAD), native and recombinant, have been determined at 2.8 Å resolution, showing that the enzyme adopts an octameric quaternary structure in accord with previously published analyses of the enzyme from a range of other species. However, this is in contrast to the finding that a disease-related F12L mutant of the human enzyme uniquely forms hexamers [Breinig et al. (2003[Breinig, S., Kervinen, J., Stith, L., Wasson, A. S., Fairman, R., Wlodawer, A., Zdanov, A. & Jaffe, E. K. (2003). Nature Struct. Biol. 10, 757-763.]), Nature Struct. Biol. 10, 757–763]. Monomers of all ALADs adopt the TIM-barrel fold; the subunit conformation that assembles into the octamer includes the N-terminal tail of one monomer curled around the (α/β)8 barrel of a neighbouring monomer. Both crystal forms of the human enzyme possess two monomers per asymmetric unit, termed A and B. In the native enzyme there are a number of distinct structural differences between the A and B monomers, with the latter exhibiting greater disorder in a number of loop regions and in the active site. In contrast, the second monomer of the recombinant enzyme appears to be better defined and the active site of both monomers clearly possesses a zinc ion which is bound by three conserved cysteine residues. In native human ALAD, the A monomer also has a ligand resembling the substrate ALA which is covalently bound by a Schiff base to one of the active-site lysines (Lys252) and is held in place by an ordered active-site loop. In contrast, these features of the active-site structure are disordered or absent in the B subunit of the native human enzyme. The octameric structure of the zinc-dependent ALAD from the hyperthermophile Pyrobaculum calidifontis is also reported at a somewhat lower resolution of 3.5 Å. Finally, the details are presented of a high-resolution structure of the Escherichia coli ALAD enzyme co-crystallized with a noncovalently bound moiety of the product, porphobilinogen (PBG). This structure reveals that the pyrrole side-chain amino group is datively bound to the active-site zinc ion and that the PBG carboxylates interact with the enzyme via hydrogen bonds and salt bridges with invariant residues. A number of hydrogen-bond interactions that were previously observed in the structure of yeast ALAD with a cyclic intermediate resembling the product PBG appear to be weaker in the new structure, suggesting that these interactions are only optimal in the transition state.

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Abstract: Potato cathepsin D inhibitor (PDI) is a glycoprotein of 188 amino acids which can inhibit both the aspartic protease cathepsin D and the serine protease trypsin. Here we report the first X-ray structure of PDI at a resolution of 2.1 Å showing that PDI adopts a β-trefoil fold, which is typical of the Kunitz-family protease inhibitors, with the inhibitory loops protruding from the core. Possible reactive-site loops including one involving a unique disulphide and another involving a protruding 310 helix are identified and docking studies indicate the mode of action of this unusual bi-functional inhibitor.

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Abstract: The protein calexcitin was originally identified in molluscan photoreceptor neurons as a 20 kDa molecule which was up-regulated and phosphorylated following a Pavlovian conditioning protocol. Subsequent studies showed that calexcitin regulates the voltage-dependent potassium channel and the calcium-dependent potassium channel as well as causing the release of calcium ions from the endoplasmic reticulum (ER) by binding to the ryanodine receptor. A crystal structure of calexcitin from the squid Loligo pealei showed that the fold is similar to that of another signalling protein, calmodulin, the N- and C-terminal domains of which are known to separate upon calcium binding, allowing interactions with the target protein. Phosphorylation of calexcitin causes it to translocate to the cell membrane, where its effects on membrane excitability are exerted and, accordingly, L. pealei calexcitin contains two protein kinase C phosphorylation sites (Thr61 and Thr188). Thr-to-Asp mutations which mimic phosphorylation of the protein were introduced and crystal structures of the corresponding single and double mutants were determined, which suggest that the C-terminal phosphorylation site (Thr188) exerts the greatest effects on the protein structure. Extensive NMR studies were also conducted, which demonstrate that the wild-type protein predominantly adopts a more open conformation in solution than the crystallographic studies have indicated and, accordingly, normal-mode dynamic simulations suggest that it has considerably greater capacity for flexible motion than the X-ray studies had suggested. Like calmodulin, calexcitin consists of four EF-hand motifs, although only the first three EF-hands of calexcitin are involved in binding calcium ions; the C-terminal EF-hand lacks the appropriate amino acids. Hence, calexcitin possesses two functional EF-hands in close proximity in its N-terminal domain and one functional calcium site in its C-terminal domain. There is evidence that the protein has two markedly different affinities for calcium ions, the weaker of which is most likely to be associated with binding of calcium ions to the protein during neuronal excitation. In the current study, site-directed mutagenesis has been used to abolish each of the three calcium-binding sites of calexcitin, and these experiments suggest that it is the single calcium-binding site in the C-terminal domain of the protein which is likely to have a sensory role in the neuron.